Synthetic signal peptides for directing secretion of heterologous proteins in yeast - Patents.com

Abstract

Provided herein are signal peptides that direct the secretion of expressed payload proteins in yeast. Methods of using the signal peptides for therapeutic and non-therapeutic applications are also provided. Compositions comprising yeast containing the signal peptides, and methods of using the yeast containing the signal peptides for therapeutic and non-therapeutic applications are also provided. Methods of designing and producing the disclosed signal peptides are also provided.

Description

[Technical field]

[0001] The present disclosure relates generally to signal peptides, and more specifically to synthetic signal peptides that increase secretion of recombinant proteins. [Background technology]

[0002] Yeast is routinely used as a host to produce proteins for research, therapeutic, and industrial purposes. Once produced, proteins typically translocate into the endoplasmic reticulum (ER), then are transported to the Golgi, and then secreted into the extracellular space. Movement along this secretory pathway is facilitated by a signal peptide, which usually contains about 16-30 amino acids and is fused to the N-terminus of the protein. However, despite considerable efforts to genetically optimize the synthesis of recombinant proteins by yeast, optimization of the chaperone pathway used by the synthesized proteins to reach the extracellular space has been relatively scarce and rarely successful. Thus, the production capacity of yeast remains too small to be used for industrial-type applications and is therefore limited to small-scale processes.

[0003] The most common signal peptide currently in use is the α-mating factor proprotein signal peptide α-MF from Saccharomyces cerevisiae. Its performance varies widely depending on the payload protein. Only time-consuming and resource-intensive direct experimental evaluation provides an assessment of its performance for any particular payload protein. Thus, effectively producing recombinant proteins in yeast increases the unpredictability and challenge, since α-MF is usually implemented as-is not only in S. cerevisiae but also in orthologous yeast strains. Several efforts have been made to optimize secretion, but most, if not all, rely on either empirical design or directed evolution, which are laborious, small-scale methods and require the native signal peptide as a starting template. Thus, there is a need to design a system that not only increases the secretion of recombinant proteins produced in yeast, but also applies across multiple yeast species. Summary of the Invention

[0004] In some embodiments, a pre-protein signal peptide is provided, in some embodiments, the pre-protein signal peptide comprises an amino acid sequence selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula IX, and Formula XIII.

[0005] In certain embodiments, Formula I, as described herein, is represented by: 1 -(A 2 ) w -A 3 -(A 4 ) x -(A 5 ) y -A 6 -A 7 -A 8 -A 9 -A 10 -(A 11 ) z (Formula I).

[0006] In certain embodiments, formula II is represented by the following, as described herein: 1 -(B 2 ) u -(B 3 ) v -(B 4 ) w -(B 5 ) x -(B 6 ) y -B 7 -B 8 -B 9 -B 10 -(B 11 ) z (Formula II).

[0007] In certain embodiments, formula III is represented by the following, as described herein: 1 -(C 2 ) r -(C 3 ) t -(C 4 ) u -[(C 5 ) v -(C 6 ) w ] x -(C 7 ) y -(C 8 ) z -C 9 -C 10 -C 11 -[C 12 -C 13 ] a (Formula III).

[0008] In certain embodiments, formula IV is represented by the following, as described herein: 1 -(D 2 ) q -(D 3 ) r -(D 4 ) t -(D 5 ) u -[(D 6 ) v -(D 7 ) x -(D 8) w -(D 9 ) y ] z -D 10 -D 11 -D 12 -[D 13 -D 14 ] a (Formula IV).

[0009] In certain embodiments, formula V is represented by the following, as described herein: 1 -[(E 2 ) i -(E 3 ) j -(E 4 ) q ] r -(E 5 ) t -(E 6 ) u -(E 7 ) v -[(E 8 ) w -(E 9 ) x ] y -(E 10 ) z -E 11 -E 12 -E 13 -[E 14 -E 15 ] a (Formula V).

[0010] In certain embodiments, formula IX is represented by the following, as described herein: 1 -(F 2 ) v -(F 3 ) w -[(F 4 ) x -(F 5 ) y ] z -F 6 -F 7 -F 8 -[F 9 -F 10 ] a (Formula IX).

[0011] In certain embodiments, formula XIII is represented by the following, as described herein: 1 -(L 2 ) x -[(L 3 ) a -(L 4 ) a ] y -[(L 5 ) a -(L 6 ) a -(L 7 ) a ] z -(L 8 ) a -(L 9 ) a -(L 10 ) a -(L 11 ) a -(L 12 ) a (Formula XIII).

[0012] In some embodiments, a pre-protein signal peptide is provided, in some embodiments, the pre-protein signal peptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73.

[0013] In some embodiments, a proprotein signal peptide is provided, in some embodiments, the proprotein signal peptide comprises an amino acid sequence selected from the group consisting of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, and Formula XV.

[0014] In certain embodiments, formula VI is represented by the following, as described herein: 1 -G 2 -G 3 -G 4 -G 5 -G 6 -G 7-G 8 -G 9 -G 10 -G 11 -G 12 -G 13 -G 14 -G 15 -G 16 -G 17 -G 18 -G 19 -G 20 -G 21 -G 22 -G 23 -G 24 -G 25 (Formula VI).

[0015] In certain embodiments, formula VII is represented by the following, as described herein: 1 ) m -(H 2 ) m -(H 3 ) m -(H 4 ) m -(H 5 ) m -(H 6 ) m -(H 7 ) m -(H 8 ) m -(H 9 ) m -(H 10 ) m -(H 11 ) m -(H 12 ) m -(H 13 ) m -(H 14 ) m -(H 15 ) m -(H 16 ) m -(H 17 ) m -(H 18 ) m -(H 19 ) m -(H 20 ) m -(H 21 ) m -(H 22 )m -(H 23 ) m -(H 24 ) m -(H 25 ) m -(H 26 ) m -(H 27 ) m -(H 28 ) m -(H 29 ) m -(H 30 ) m -(H 31 ) m -(H 32 ) m -(H 33 ) m -(H 34 ) m -(H 35 ) m -(H 36 ) m -H 37 -H 38 -H 39 -H 40 (Formula VII).

[0016] In certain embodiments, formula VIII is represented by the following, as described herein: 1 ) m -(I 2 ) m -(I 3 ) m -(I 4 ) m -(I 5 ) m -(I 6 ) m -(I 7 ) x -(I 8 ) m -(I 9 ) m -(I 10 ) m -(I 11 ) x -(I 12 ) m -(I 13 ) x -(I 14 ) x -(I15 ) m -(I 16 ) x -(I 17 ) m -I 18 -I 19 -I 20 -I 21 -I 22 -I 23 (Formula VIII).

[0017] In certain embodiments, formula X is represented by the following, as described herein: 1 ) z -(J 2 ) z -(J 3 ) z -(J 4 ) z -(J 5 ) z -(J 6 ) z -(J 7 ) z -(J 8 ) z -(J 9 ) z -(J 10 ) z -(J 11 ) z -(J 12 ) z -(J 13 ) z -(J 14 ) z -(J 15 ) z -(J 16 ) z -(J 17 ) z -(J 18 ) z -(J 19 ) z -(J 20 ) z -(J 21 ) z -J 22 -J 23 -J 24 -J 25 (Formula X).

[0018] In certain embodiments, formula XI is represented by the following, as described herein: 1 ) b -(K 2 ) b -(K 3 ) b -(K 4 ) b -(K 5 ) b -(K 6 ) b -(K 7 ) b -(K 8 ) b -(K 9 ) b -(K 10 ) b -(K 11 ) b -(K 12 ) b -(K 13 ) b -(K 14 ) b -(K 15 ) b -(K 16 ) b -(K 17 ) b -(K 18 ) b -(K 19 ) b -(K 20 ) b -(K 21 ) b -(K 22 ) b -(K 23 ) b -(K 24 ) b -(K 25 ) b -(K 26 ) b -(K 27 ) b -(K 28 ) b -(K 29 ) b -(K 30 ) b -(K 31 ) b -(K 32 ) b -(K33 ) b -(K 34 ) b -(K 35 ) b -(K 36 ) b -(K 37 ) b -(K 38 ) b -(K 39 ) b -(K 40 ) b -(K 41 ) b -(K 42 ) b -(K 43 ) b -(K 44 ) b -(K 45 ) b -(K 46 ) b -(K 47 ) b -(K 48 ) b -(K 49 ) b -(K 50 ) b -(K 51 ) b -(K 52 ) b -(K 53 ) b -(K 54 ) b -(K 55 ) b -(K 56 ) b -(K 57 ) b -(K 58 ) b -(K 59 ) b -(K 60 ) b -(K 61 ) b -(K 62 ) b -(K 63 ) b -(K 64 ) b -(K 65 ) b -(K 66) b -(K 67 ) b -(K 68 ) b -(K 69 ) b -(K 70 ) b -(K 71 ) b -(K 72 ) b -(K 73 ) b -(K 74 ) b -(K 75 ) b -(K 76 ) b -(K 77 ) b -(K 78 ) b -(K 79 ) b -(K 80 ) b -(K 81 ) b -(K 82 ) b -(K 83 ) b -(K 84 ) b -(K 85 ) b -(K 86 ) b -(K 87 ) b -(K 88 ) b -K 89 -K 89 -K 89 -K 89 -K 89 (Formula XI).

[0019] In certain embodiments, formula XIV is represented by the following, as described herein: 1 ) b -(M 2 ) b -(M 3 ) b -(M 4 ) b -(M 5 ) b -(M 6 )b -(M 7 ) b -(M 8 ) b -(M 9 ) b -(M 10 ) b -(M 11 ) b -(M 12 ) b -(M 13 ) b -(M 14 ) b -(M 15 ) b -(M 16 ) b -(M 17 ) b -(M 18 ) b -(M 19 ) b -(M 20 ) b -(M 21 ) b -(M 22 ) b -(M 23 ) b -(M 24 ) b -(M 25 ) b -(M 26 ) b -(M 27 ) b -(M 28 ) b -(M 29 ) b -(M 30 ) b -(M 31 ) b -(M 32 ) b -(M 33 ) b -(M 34 ) b -(M 35 ) b -(M 36 ) b -(M 37 ) b -(M 38 ) b -(M 39 ) b-(M 40 ) b -(M 41 ) b -(M 42 ) b -(M 43 ) b -(M 44 ) b -(M 45 ) b -(M 46 ) b -(M 47 ) b -(M 48 ) b -(M 49 ) b -(M 50 ) b -(M 51 ) b -(M 52 ) b -(M 53 ) b -(M 54 ) b -(M 55 ) b -(M 56 ) b -(M 57 ) b -(M 58 ) b -(M 59 ) b -(M 60 ) b -(M 61 ) b -(M 62 ) b -(M 63 ) b -(M 64 ) b -(M 65 ) b -(M 66 ) b -(M 67 ) c -(M 68 ) c -(M 69 ) c -(M 70 ) c (Formula XIV).

[0020] In certain embodiments, formula XV is represented by the following, as described herein: 1 ) b -(N 2 ) b -(N 3 ) b -(N 4 ) b -(N 5 ) b -(N 6 ) b -(N 7 ) b -(N 8 ) b -(N 9 ) b -(N 10 ) b -(N 11 ) b -(N 12 ) b -(N 13 ) b -(N 14 ) b -(N 15 ) b -(N 16 ) b -(N 17 ) b -(N 18 ) b -(N 19 ) b -(N 20 ) b -(N 21 ) b -(N 22 ) b -(N 23 ) b -(N 24 ) b -(N 25 ) b -(N 26 ) b -(N 27 ) b -(N 28 ) b -(N 29 ) b -(N 30 ) b -(N 31 ) b -(N 32 ) b -(N33 ) b -(N 34 ) b -(N 35 ) b -(N 36 ) b -(N 37 ) b -(N 38 ) b -(N 39 ) b -(N 40 ) b -(N 41 ) b -(N 42 ) b -(N 43 ) b -(N 44 ) b -(N 45 ) b -(N 46 ) b -(N 47 ) b -(N 48 ) b -(N 49 ) b -(N 50 ) b -(N 51 ) b -(N 52 ) b -(N 53 ) b -(N 54 ) b -(N 55 ) b -(N 56 ) b -(N 57 ) b -(N 58 ) b -(N 59 ) b -(N 60 ) b -(N 61 ) b -(N 62 ) b -(N 63 ) b -(N 64 ) b -(N 65 ) b -(N 66) b -(N 67 ) c -(N 68 ) c -(N 69 ) c -(N 70 ) c -(N 71 ) c (Formula XV).

[0021] In some embodiments, a proprotein signal peptide is provided, in which the proprotein signal peptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75.

[0022] In some embodiments, a preprotein plus proprotein signal peptide is provided, in some embodiments, the preprotein plus proprotein signal peptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:30.

[0023] In some embodiments, a polypeptide is provided. In some embodiments, the recombinant polypeptide is 1 ) n -(Y 1 ) m -Z 1 wherein X 1 is the preprotein signal peptide, and Y 1 is the proprotein signal peptide, and Z 1 is a payload protein, n is 0 or 1, m is 0 or 1, and n and m cannot be 0 simultaneously.

[0024] In some embodiments, a yeast is provided. In some embodiments, the yeast comprises a heterologous nucleic acid molecule encoding a polypeptide having a formula of (X1)n-(Y1)m-Z1, where X1 is a preprotein signal peptide provided herein, Y1 is a proprotein signal peptide provided herein, Z1 is a payload protein, n is 0 or 1, m is 0 or 1, and n and m cannot simultaneously be 0.

[0025] In some embodiments, methods for producing a payload protein are provided, hi some embodiments, the methods include transfecting yeast with a nucleic acid encoding a recombinant polypeptide provided herein, producing an engineered yeast, culturing the engineered yeast in an environment effective for growth of the engineered yeast, and inducing secretion of the payload protein by the engineered yeast.

[0026] In some embodiments, methods are provided for treating a disease or condition in a subject in need thereof, hi some embodiments, the methods comprise administering to the subject a therapeutically effective amount of a yeast provided herein. [Brief description of the drawings]

[0027] The foregoing and other features of the present disclosure will become more apparent from the following detailed description of several embodiments, which proceeds with reference to the accompanying drawings.

[0028] [Figure 1] Provided are four recombinant polypeptide constructs representing combinations of synthetic preprotein signal (sPre), synthetic proprotein signal (sPro), and native preprotein signal (nPre) peptides that may be utilized in accordance with the methods disclosed herein to increase secretion of payload proteins. [Diagram 2]1 provides a Western blot showing the amount of maltose binding protein (MBP) in cell-free supernatants secreted by wild-type and engineered K. lactis yeasts. [Figure 3A] 1 shows a graph of MBP accumulation by engineered K. lactis yeast (expressing the synthetic signal peptide synKlac-v1) versus wild-type K. lactis yeast over time. [Figure 3B] Accumulation of MBP by wild type versus engineered K. lactis yeast (expressing the synthetic signal peptide synKlac-v1) is shown graphically as a function of yeast growth (optical density). [Figure 4] FIG. 1 is a graph of MBP RNA expression in wild-type K. lactis yeast versus engineered K. lactis yeast (expressing the synthetic signal peptide synKlac-v1). [Diagram 5] 1 is a graph of normalized TNF-α levels produced by wild-type K. lactis yeast versus engineered K. lactis yeast (expressing the synthetic signal peptide synKlac-v1). [Figure 6] FIG. 1 is a graph of normalized phytase levels produced by wild-type P. pastoris (native signal peptide (PHO1, α-MF) versus engineered P. pastoris yeasts (expressing synthetic signal peptides synPichia-v1 or synPichia-v4). [Figure 7] Normalized insulin production by wild-type vs. engineered S. cerevisiae yeast (expressing the synthetic signal peptide synScer-v5) is reported. Insulin was quantified using ELISA and data was normalized to insulin mRNA levels for each variant tested. Figure 7A reports a comparison between yeast utilizing the synScer-v5 signal peptide and yeast utilizing the α-MF signal peptide. Figure 7B reports a comparison between yeast utilizing the synScer-v5 signal peptide and yeast expressing optYAP. [Figure 8]Figure 8A reports the normalized enzyme activity of purified invertase extracts produced by wild-type S. boulardii yeast versus purified invertase extracts produced by engineered S. boulardii yeast (expressing the synthetic signal peptide synScer-v1). Figure 8A reports the invertase activity from invertase purified from culture medium. Figure 8B reports the invertase activity from invertase purified from periplasmic extracts. [Figure 9] The activity of the invertase produced by the engineered S. boulardii yeast is reported compared to the activity of a commercial invertase at various pH levels. Figure 9A reports data from the engineered S. boulardii yeast. Figure 9B reports data from the commercial invertase. [Figure 10] FIG. 13 graphically depicts the change in glucose levels over time as an indirect measure of invertase activity produced in wild-type versus S. boulardii engineered to express invertase with the synthetic signal peptide synScer-v1. [Figure 11] 13A-13C graphically depict the amount of yeast in various gastrointestinal tissues of mice orally administered engineered S. boulardii yeast. [Figure 12] 1 shows a graph of the enzyme activity of invertase produced by wild-type S. boulardii versus invertase produced by engineered S. boulardii yeast (expressing the synthetic signal peptide synScer-v1). [Figure 13] Normalized IGF-1 production by wild-type S. boulardii versus engineered S. boulardii yeast (expressing synthetic signal peptides synScer-v1, synScer-v3, or synScer-v5) is shown graphically. [Figure 14] Normalized lysozyme production by wild-type S. boulardii versus engineered S. boulardii yeast (expressing synthetic signal peptides synScer-v4 or synScer-v5) is shown graphically. [Figure 15] Schematic showing survival of S. boulardii engineered to express a payload protein (mCherry) through the upper gastrointestinal tract of mice over time. [Figure 16] 13 graphically depicts sucrase activity per CFU in lyophilized S. boulardii yeast engineered to express sucrase fused to the synthetic signal peptide synScer-v1. [Figure 17] 1 shows a graph of the activity of sucrase as a function of pH expressed by S. boulardii yeast engineered to express sucrase fused to the synthetic signal peptide synScer-v1. [Figure 18] 13 graphically illustrates the loss of sucrase activity in the presence of glucose in S. boulardii yeast engineered to express sucrase fused to the synthetic signal peptide synScer-v1, compared to sucrase expressed in wild-type S. boulardii. [Figure 19] FIG. 13 graphically depicts persistence over time in gastrointestinal tissues by S. boulardii yeast engineered to express sucrase fused to the synthetic signal peptide synScer-v1. [Figure 20] FIG. 13 graphically depicts glucose excursion time curves of sucrose-challenged mice administered B. boulardii yeast engineered to express sucrase fused to the synthetic signal peptide synScer-v1. [Figure 21] 20 AUC data presented in bar graph format. [Figure 22] A variety of recombinant polypeptide constructs are provided that represent various combinations of synthetic and natural pre- and pro-protein signal peptides that can be utilized in accordance with the methods disclosed herein to improve the secretion efficiency of invertase proteins. [Figure 23]We report a comparison between normalized invertase production by S. boulardii engineered to express recombinant polypeptides containing the native or S. cerevisiae signal (SBsyn-Scerv1) versus normalized invertase production by S. boulardii engineered to express recombinant polypeptides containing various synthetic signal peptides derived from S. boulardii (SBsyn-Sbouv2, SBsyn-Sbouv3, SBsyn-Sbouv4). [Figure 24] Provided are various recombinant polypeptide constructs representing various combinations of synthetic and natural pre- and pro-protein signal peptides that can be utilized in accordance with the methods disclosed herein to improve the efficiency of secretion of lysozyme proteins. [Diagram 25] We report a comparison between normalized lysozyme production by S. boulardii engineered to express recombinant polypeptides containing the chicken lysozyme signal sequence versus normalized lysozyme production by S. boulardii engineered to express recombinant polypeptides containing various synthetic signal peptides derived from S. boulardii (SBsyn-Sbouv). [Figure 26] Recombinant polypeptide constructs are provided that represent combinations of synthetic pre- and pro-protein signal peptides that can be utilized in accordance with the methods disclosed herein to improve the efficiency of secretion of beta-galactosidase protein. [Figure 27] 13 graphically depicts normalized beta-galactosidase production by S. boulardii engineered to express a recombinant polypeptide containing a synthetic signal peptide from S. boulardii (SBsyn-Sbouv2). [Figure 28] A variety of recombinant polypeptide constructs are provided that represent various combinations of synthetic and natural pre- and pro-protein signal peptides that may be utilized in accordance with the methods disclosed herein to improve the efficiency of secretion of anti-TNFα proteins. [Figure 29]FIG. 13 graphically depicts normalized anti-TNFα activity production by S. boulardii engineered to express a recombinant polypeptide containing a synthetic signal peptide from S. boulardii (SBsyn-Sbouv1 and SBsyn-Sbouv2). [Diagram 30]

[0043] Figure 30 graphically illustrates the use of S. boulardii cells to secrete anti-TNFα antibody fragments. Figure 30A reports the secretion of monovalent anti-TNFα antibody fragments. Figure 30B reports the secretion of bivalent anti-TNFα antibody fragments. [Diagram 31] We compare secretion of invertase by S. boulardii cells transiently expressing the Sbouv2-invertase polypeptide with secretion of invertase by S. boulardii cells engineered by integrating into the S. boulardii genome a copy of a construct containing the Sbouv2 synthetic signal peptide fused to invertase for stable and reliable expression of invertase. [Diagram 32] A variety of recombinant polypeptide constructs are provided that represent various combinations of synthetic and natural pre- and pro-protein signal peptides that may be utilized in accordance with the methods disclosed herein to improve the efficiency of secretion of LCRF proteins. [Diagram 33] 13 graphically depicts normalized LCRF production by S. boulardii engineered to express a recombinant fusion protein containing a synthetic signal peptide from S. boulardii. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present disclosure presents a solution to the aforementioned problems by providing new synthetic signal peptides that direct the secretion of expressed proteins or peptides in yeast. The disclosed signal peptides overcome the performance variability challenges posed by previously characterized native signal peptides and can be used to facilitate the production and secretion of any protein or peptide from yeast.

[0030] The disclosed synthetic preprotein (sPre) and proprotein (sPro) signal peptides increase the secretion of any recombinant protein in yeast. Increased secretion can be advantageously achieved using synthetic preprotein signal peptides alone, synthetic proprotein signal peptides alone, or both. In any embodiment, a synthetic preprotein signal peptide can be used in combination with a natural proprotein (nPro) signal peptide or sPro signal peptide. Similarly, in any embodiment, a synthetic proprotein signal peptide can be used in combination with a natural preprotein (nPre) signal peptide or sPre signal peptide. Using a synthetic proprotein signal peptide with a synthetic preprotein signal peptide can further improve the secretion of a payload protein, for example, by facilitating Golgi transport. Advantageously, the signal peptides disclosed herein have been generated and optimized to promote the secretion of any payload protein from yeast. The use of the disclosed synthetic preprotein and proprotein signal peptides can be used to achieve increased secretion of any desired payload into any yeast-compatible environment, such as therapeutics, agriculture, or food.

[0031] Before the compositions and methods are described, it should be understood that the scope of the present invention is not limited to the specific processes, compositions, or methodologies described herein, as these may vary. It should also be understood that the terms used herein are for the purpose of describing specific versions or embodiments only, and are not intended to limit the scope of the present invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the methods and systems disclosed herein, the preferred methods, devices, and materials are described herein.

[0032] definition The following explanations of terms and methods are provided to better describe the present disclosure and to guide those of skill in the art in the practice of the present disclosure.

[0033] As used herein, "comprising" means "including," and the singular forms "a" or "an" or "the" include plural references unless the context clearly dictates otherwise. For example, a reference to "comprising a therapeutic agent" includes one or more such therapeutic agents. The term "or" refers to a single element of the listed alternative elements, unless the context clearly dictates otherwise. For example, the phrase "A or B" refers to A alone or B alone. The phrase "A, B, or a combination thereof" refers to A alone, B alone, or a combination of A and B. Similarly, "one or more of A and B" refers to a combination of A, B, or both A and B. The phrase "A and B" refers to a combination of A and B. Additionally, the various elements, features, and steps discussed herein, as well as other known equivalents to each of such elements, features, and steps, can be mixed and matched by those of skill in the art to perform methods in accordance with the principles described herein. Of the various elements, features, and steps, some may be specifically included while other elements may be specifically excluded in particular examples.

[0034] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Similar or equivalent methods and materials to those described herein can be used to practice or test this disclosure, but suitable methods and materials are described below. Materials, methods, and examples are illustrative only and are not intended to be limiting. All references cited herein are incorporated by reference in their entirety.

[0035] In some examples, numbers expressing properties such as amounts of ingredients, molecular weights, reaction conditions, etc., used to describe and claim certain embodiments should be understood as being modified in some cases by the term "about" or approximately. For example, "about" or "approximately" may indicate a variation of + / - 5% of the value being stated. Thus, in some embodiments, the numerical parameters set forth herein are approximations that may vary depending on the desired properties of the particular embodiment. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of some examples are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as practicable. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range.

[0036] In order to facilitate review of the various embodiments of the disclosure, the following explanations of specific terminology are provided:

[0037] As used herein, yeast refers to a microscopic fungus that consists of cells that reproduce by budding and can convert sugars into alcohol and carbon dioxide. As disclosed herein, yeast can be genetically modified to induce expression of a heterologous payload protein. As used herein, "genetically modified" or any grammatical variation thereof refers to the practice of introducing a nucleic acid or nucleic acid molecule that encodes and promotes expression of a recombinant protein into a yeast cell. The nucleic acid may be introduced transiently or the nucleic acid may be integrated into the genome of the yeast for stable expression. As used herein, the terms "nucleic acid" and "nucleic acid molecule" can be used interchangeably. A nucleic acid or nucleic acid molecule can be of any length. A nucleic acid can be DNA, mRNA, tRNA, or rRNA. A nucleic acid or nucleic acid molecule is composed of nucleotide monomers, with each triplet (codon) of monomers encoding either a triplet of RNA nucleotide monomers (if the nucleic acid is DNA) or an amino acid (if the nucleic acid is RNA). The DNA also includes one or more promoter regions that indicate where transcription of the DNA should begin. The mRNA also contains a ribosome binding site, which indicates where translation of the mRNA should begin, as well as one or more stop codons, which indicate where translation of the mRNA should end. Introduction of a nucleic acid or nucleic acid molecule into a yeast cell can be accomplished by any method known in the art. Such methods are described in more detail below.

[0038] In any embodiment or aspect disclosed herein, the nucleic acid encoding the recombinant polypeptide disclosed herein can be introduced into the yeast cell using any method known to those skilled in the art for such introduction. Such methods include transfection, transformation, transduction, infection (e.g., viral transduction), injection, microinjection, gene gun, nucleofection, nanoparticle bombardment, transformation, conjugation, by application of the nucleic acid in a gel, oil, or cream, by electroporation, using lipid-based transfection reagents, or by any other suitable transfection method. Those skilled in the art will easily understand and adapt such methods using readily identifiable literature sources.

[0039] As used herein, the terms "transformation" and "transfection" are intended to refer to a variety of art-recognized techniques for introducing exogenous nucleic acid into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran mediated transfection, lipofection (e.g., using commercially available reagents such as, for example, LIPOFECTIN® (Invitrogen Corp., San Diego, CA), LIPOFECTAMINE® (Invitrogen), FUGENE® (Roche Applied Science, Basel, Switzerland), JETPEI™ (Polyplus-transfection Inc., New York, NY), EFFECTENE® (Qiagen, Valencia, CA), DREAMFECT™ (OZ Biosciences, France), etc.), or electroporation (e.g., in vivo electroporation). Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.

[0040] Methods and materials for non-viral delivery of nucleic acid to cells further include biolistic methods, virosomes, liposomes, immunoliposomes, polycations or lipid-nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., TRANSFECTAM™ and LIPOFECTIN™). Cationic and neutral lipids suitable for efficient receptor-recognition lipofection of polynucleotides include those disclosed in WO91 / 17424 and WO91 / 16024.

[0041] The methods described herein include producing a recombinant polypeptide in a yeast host. As used herein, heterologous or recombinant describes a protein or nucleic acid that is not naturally found in or produced by the host yeast. As used herein, a "recombinant polypeptide" includes a payload protein and a synthetic signal peptide fused directly or indirectly thereto. As used herein, "recombinant polypeptide" and "recombinant fusion protein" may be used interchangeably in reference to a polypeptide that includes at least a first and a second component (e.g., a synthetic signal peptide and a payload protein). As used herein, a signal peptide is any protein or peptide fused directly or indirectly to the N-terminus of a payload protein that facilitates the extracellular secretion of the payload protein after it is produced. A signal peptide may include one or more of a preprotein signal peptide and a proprotein signal peptide.

[0042] Without wishing to be bound by theory, it is believed that the synthetic preprotein signal peptides disclosed herein facilitate efficient translocation of proteins from the ribosome to the endoplasmic reticulum, and the synthetic proprotein signal peptides disclosed herein facilitate transport of proteins from the ER to the Golgi apparatus for eventual secretion. Proprotein signal peptides are known to regulate various types of cellular processes, including promoting proper protein folding, such as trafficking and localization, hierarchical organization and oligomerization, and regulating protein activity function. Furthermore, the inclusion of a proprotein signal peptide can enrich for the amount of a protein in a particular cellular localization. For example, the inclusion of a proprotein sequence peptide in a protein of interest can enrich for the amount of the protein of interest in a yeast subplasmod. The effect of the preprotein signal peptides, proprotein signal peptides, or combinations thereof described herein in relation to facilitating translocation is target dependent. Without wishing to be bound by theory, in some embodiments, a preprotein signal peptide without a proprotein signal peptide will facilitate more efficient translocation and secretion. In some embodiments, a proprotein signal peptide without a preprotein signal peptide will facilitate more efficient translocation and secretion. In some embodiments, the inclusion of both a pre-protein and a pro-protein signal peptide promotes more efficient secretion.

[0043] The chemical structures of peptides are described herein by a series of one-letter amino acid abbreviations, or "amino acid sequences," or "sequences," which are conventional and known to those of skill in the art. Although reference sequences are explicitly disclosed, in any aspect and embodiment, the reference sequences may be modified to include conservative amino acid substitutions, as well as variants and fragments, while maintaining the properties and functionality of the reference sequence.

[0044] The methods disclosed herein utilize synthetic signal peptides to increase extracellular secretion of payload proteins by yeast. As used herein, "synthetic signal peptide" refers to a recombinantly produced signal peptide whose sequence is generated as provided herein. A recombinantly produced signal peptide may be referred to as a "synthetic signal peptide" or simply a "signal peptide". Signal peptides include one or more of synthetic preprotein (sPre) signal peptides and synthetic proprotein (sPro) signal peptides. As highlighted above, the term synthetic in this context refers to a recombinantly produced preprotein signal peptide or proprotein signal peptide whose sequence is generated as provided herein. The pre-signal peptides and pro-signal peptides may then be referred to as "synthetic" preprotein signal peptides or proprotein signal peptides, or simply preprotein peptides or proprotein signal peptides. In embodiments where a natural preprotein signal peptide or preprotein signal peptide is utilized or referred to, the peptide is so indicated. In the context of this application, the term "natural" refers to a pre-signal peptide or pro-signal peptide whose sequence is adopted, in whole or in part, from a pre-signal peptide sequence or a pro-signal peptide sequence known at the time of this application. In other words, a "natural" signal peptide is not generated using the formula or method provided herein. However, it should be understood that a synthetic signal peptide may include a synthetic pre-protein signal peptide fused with a natural proprotein signal peptide (sPre-nPro signal peptide). In another example, a synthetic signal peptide may include a natural pre-protein signal peptide fused to a synthetic proprotein signal peptide (nPre-sPro signal peptide). In yet another example, a synthetic signal peptide includes a synthetic pre-protein signal peptide and does not include a proprotein signal peptide. Similarly, a synthetic signal peptide may include a synthetic proprotein signal peptide but not a preprotein signal peptide.

[0045] Pre-protein signal peptides (synthetic or natural) contain 10-50 amino acids that are added either directly to the N-terminus of a payload protein or indirectly to the N-terminus of a payload protein via one or more of a Kex protease (KR) site, a Ste13 cleavage site, and a spacer therebetween.

[0046] A proprotein signal peptide comprises 10-200 amino acids that are either directly added to the N-terminus of a payload protein or indirectly added to the N-terminus of a payload protein via one or more of a KR site, a Ste13 cleavage site, and a spacer therebetween. Many proteins are naturally expressed containing a proprotein signal peptide, but as described, these native proprotein signal peptides often lack the activity to generate sufficient secretion of the payload protein. The various synthetic signal peptides described herein can be used as replacements for all or part of the native signal peptide.

[0047] The pre-protein signal peptide and / or pro-protein signal peptide, whether synthetic or natural, can be attached to adjacent amino acids via a linkage to the N-terminal amino acid of the adjacent amino acid, for example, by a peptide bond, a dipeptide spacer, or a membrane-binding / lipophilic alpha-helical peptide signal peptide (e.g., MISTIC, represented by the amino acid sequence FCTFFEKHHRKWDILLEKSTGVMEA or SEQ ID NO: 26).

[0048] As used herein, "hydropathy index" or "HP index" refers to the "intrinsic" hydrophobicity / hydrophilicity of amino acid side chains in peptides / proteins as defined in Kovacs JM, Mant CT, Hodges RS. Determination of intrinsic hydrophilicity / hydrophobicity of amino acid side chains in peptides in the absence of nearest-neighbor or conformational effects. Biopolymers.2006;84(3):283-97.doi:10.1002 / bip.20417. PMID:16315143;PMCID:PMC2744689, which is incorporated herein by reference in its entirety. The hydrophobicity / hydrophilicity value is determined via synthetic peptides, and the HP index value is calculated as the difference in RP-HPLC retention time between amino acid X at position i and amino acid Gly at position i+1. Thus, amino acids that are more hydrophobic than glycine have a positive HP index value, and amino acids that are more hydrophilic than glycine have a negative HP index value, with glycine having a value of 0. See Table 1 below for values ​​that correspond to those utilized in this application. [Table 1]

[0049] As used herein, "helicity" refers to the nonpolar phase helical tendency of each guest "X" residue in the experimental KKAAAXAAAAAXAAWAAXAAAKKKK (SEQ ID NO: 84)-amide peptide, as outlined in Deber CM, Wang C, Liu LP, Prior AS, Agrawal S, Muskat BL, Cuticchia AJ. TM Finder: a prediction program for transmembrane protein segments using a combination of hydrophobicity and nonpolar phase helicity scales. Protein Sci. 2001 Jan; 10(1): 212-9. doi: 10.1110 / ps.30301. PMID: 11266608; PMCID: PMC2249854, which is incorporated herein by reference in its entirety. The helicity values ​​of each amino acid are shown in Table 2 below. [Table 2]

[0050] As used herein, "payload protein" or "protein of interest" refers to a protein that is produced by a host and escorted into the extracellular space through the secretory pathway, facilitated by the presence of a synthetic signal peptide. Upon secretion into the extracellular space, all, some, or none of the synthetic signal peptide may be fused to the payload protein. Optionally, the payload protein that is still partially or completely attached to the synthetic signal peptide may be further processed, e.g., to remove the remaining signal peptide. The payload protein may be any protein, known or unknown, such as, for example, an enzyme, an enzyme inhibitor, a growth factor, a hormone, an antibody, an antigen, a vaccine, a therapeutic agent, or any combination thereof. More specific examples follow herein below.

[0051] The compositions disclosed herein can be provided to a subject in a variety of ways, including through administration of the composition to the subject. As used herein, administering or administering means providing a composition to a subject or providing a composition to a subject. Oral administration, as used herein, refers to delivery of an active agent via the mouth. Topical administration, as used herein, refers to delivery of an active agent to a body surface, such as the skin, mucous membrane (e.g., nasal membrane, vaginal membrane, oral membrane, etc.).

[0052] The payload proteins secreted by the various genetically modified yeasts disclosed herein, interchangeably referred to as "engineered yeasts," can be provided to a subject as a pharmaceutical composition. Additionally or alternatively, the engineered yeast itself can be provided to a subject as a pharmaceutical composition.

[0053] Various compositions disclosed herein may be useful for treating many diseases, such as cancer. As used herein, cancer refers to a condition characterized by uncontrolled cell proliferation. Examples of cancer include, but are not limited to, squamous cell carcinoma, small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, lung squamous cell carcinoma, gastrointestinal cancer, Hodgkin's lymphoma and non-Hodgkin's lymphoma, pancreatic cancer, glioblastoma, cervical cancer, colon cancer, colorectal cancer, endometrial or uterine cancer, kidney cancer such as renal cell carcinoma and Wilms' tumor, basal cell carcinoma, melanoma, prostate cancer, and esophageal cancer. In some embodiments, the disease or condition may include, but is not limited to, an infection, an autoimmune disease, an enzyme deficiency (including primary (congenital) enzyme deficiency and enzyme deficiency secondary to a functional bowel disorder), diabetes, obesity, a metabolic disorder, intestinal bacterial overgrowth, intestinal infection, bacterial vaginosis, short bowel syndrome, inflammatory bowel disease, irritable bowel syndrome, small bowel syndrome, celiac disease, gluten intolerance, colitis, peptic ulcer, gastritis, polyps, hemorrhoids, cirrhosis, or cancer.

[0054] The various compositions disclosed herein may include one or more drugs, biologics, or active agents, which are used interchangeably herein to refer to chemical compounds or compounds that induce a desired pharmacological or physiological effect, including therapeutically active, prophylactically active, or cosmetically active agents. "Drugs," "biologics," and "active agents" include any pharma- ceutically acceptable, pharmacologically active derivatives and analogs of those drugs, biologics, and active agents specifically mentioned herein, including, but not limited to, salts, esters, amides, prodrugs, active metabolites, inclusion complexes, analogs, and the like. Suitable drugs, biologics, and active agents include, but are not limited to, anti-alcohol agents, amino acids, ammonia detoxifiers, anabolic agents, analgesics, analgesics, androgens, anesthetics, appetite suppressant compounds, anorectics, antagonists, anti-allergy agents, anti-amebic agents, anti-anemic agents, anti-anginal agents, anti-anxiety agents, anti-arthritic agents, anti-atherosclerotic agents, antibacterial agents; anti-cancer agents, including anti-neoplastic agents, and anti-cancer supplementary potentiating agents; anticholinergic agents, anti-gallstone forming agents, anticoagulants, anti-coccidiolytic agents, anti-convulsants, anti-depressants, anti-diabetic agents, antidiarrheals, anti-diuretics, antidotes, anti-dyskinetics agents, antiemetics, anti-epileptic agents, anti-estrogens, anti-fibrinolytic agents, anti-fungal agents, agents), antiglaucoma agents, antihemophilic agents, antihemorrhagic agents, antihistamines, antilipidemic agents, antihyperlipoproteinemic agents, antihypertensive agents, antihypotensive agents; anti-infective agents, e.g. antibiotics and antivirals; anti-inflammatory agents, both steroidal and non-steroidal;Antikeratotic agents, antimalarials, antimicrobial agents, antimigraine agents, antimitotic agents, antifungal agents, antiemetic agents, antineoplastic agents, antineutropenic agents, antiobsessional agents, antiparasitic agents, antiparkinsonian agents, antipneumocystic agents, antiproliferative agents, antiprostatic hyperplasia agents, antiprotozoal agents, antipruritic agents, antipsoriatic agents, antipsychotic agents, antipyretics, antispasmodics, antirheumatic agents, schistosomicidal agents, antiseborrheic agents, antispasmodics, antithrombotic agents, antituberculous agents, antitussive agents, antiulcer agents, antiurolithic agents, agents), antivirals, gastroesophageal reflux disease drugs, anxiolytics, appetite suppressants, ADD and ADHD drugs, bacteriostatic and fungicidal agents, benign prostatic hyperplasia treatments, blood sugar regulators, bone resorption inhibitors, bronchodilators; carbonic anhydrase inhibitors;cardiovascular agents including antianginals, antiarrhythmics, beta blockers, calcium channel blockers, cardiac depressants, cardiovascular medications, cardioprotectants, and inotropes;central nervous system (CNS) acting agents, central nervous system stimulants, choleretics, cholinergics, cholinergic agonists, cholinesterase inactivators, anticoccidials, cognitive aids and cognition enhancers;cold medicines including decongestants;depressants, diagnostic aids, diuretics, dopamine agonists, ectoparasiticides, emetics;enzymes that inhibit the formation of plaque, tartar or caries;enzyme inhibitors, estrogens, fibrinolytics, fluoride anticaries / anti-caries agents, free oxygen radical scavengers, gastrointestinal motility enhancers agents), genetic materials, glucocorticoids, gonad-stimulating ingredients, hemostatic agents, herbal medicines, histamine H2 receptor antagonists, hormones, hormone decomposing agents (hormonolytics), hypnotics, cholesterol lowering agents, hypoglycemic agents, lipid lowering agents, antihypertensive agents, immune agents, immune stimulants, immune suppressants, impotence treatment adjuvants, inhibitors, keratolytic agents, leukotriene inhibitors, liver damage treatment; metal chelating agents such as ethylenediaminetetraacetic acid, tetrasodium salt;Mitotic inhibitors, mood regulators, mucolytics, mucosal protectants, muscle relaxants, mydriatics, opiate antagonists, neuroleptics, neuromuscular blockers, neuroprotectants, nicotine, NMDA antagonists, non-hormonal sterol derivatives; nutritional agents such as vitamins, essential amino acids and fatty acids; ophthalmic agents such as anti-glaucoma agents; oxytocin agents, analgesics, parasympatholytic agents, peptide drugs, plasminogen activators, platelet activating factor antagonists, platelet aggregation inhibitors, post-stroke and head injury treatments, potentiators, progestins, prostaglandins, prostate growth inhibitors, proteolytic enzymes as wound cleansers, prothyrotropin agents. agents), psychostimulants, psychotropic agents, radiopharmaceuticals, modulators, relaxants, redistributing agents, scabicides, sclerosants, sedatives, sedative-hypnotics, selective adenosine A1 antagonists, serotonin antagonists, serotonin inhibitors, serotonin receptor antagonists; steroids including progestogens, estrogens, corticosteroids, androgens and anabolic agents; smoking cessation agents, stimulants, depressants, sympathomimetics, synergists, thyroid hormones, antithyroid agents, thyroid hormone mimetics, tranquilizers, dental desensitizers; tooth whitening agents such as peroxides, metal chlorites, perborates, percarbonates, peroxyacids, and combinations thereof, unstable angina agents, uricosurics, vasoconstrictors, vasodilators including general coronary, peripheral and cerebral vasculature, wound medicines; wound healing agents, xanthine oxidase inhibitors, and the like;

[0055] Antibiotics refer to chemicals that can treat bacterial infections by inhibiting the growth of bacteria and other microorganisms or by destroying existing colonies.

[0056] Anti-inflammatory refers to active agents that reduce inflammation and swelling.

[0057] A chemotherapeutic agent refers to a chemical substance that has therapeutic utility in the treatment of diseases characterized by abnormal cell proliferation. Such diseases include tumors, neoplasms, and cancer. In one example, a chemotherapeutic agent is a radioactive compound. In one example, a chemotherapeutic agent is a biologic, such as a monoclonal antibody. Chemotherapy refers to the use of a chemotherapeutic agent.

[0058] Radiation therapy refers to the use of directed gamma or beta rays to induce sufficient damage to cells so as to limit or completely destroy their ability to function normally.

[0059] Various compositions disclosed herein may contain an effective amount of a drug, biologic, or active agent. An effective amount refers to an amount of a drug, biologic, or active agent (alone or together with one or more other active agents) sufficient to induce a desired response, such as preventing, treating, reducing, and / or ameliorating a condition. An effective amount of an active agent, alone or in combination with one or more other active agents, can be determined in many different ways, such as by assaying for a reduction in one or more signs or symptoms associated with a condition in a subject, or by measuring the levels of one or more molecules associated with the condition being treated.

[0060] Various compositions disclosed herein may contain various pharma- ceutically acceptable excipients.As used herein, pH adjuster or regulator refers to a compound or buffer used to achieve desired pH control in formulations.Exemplary pH regulators include acids (e.g., acetic acid, adipic acid, carbonic acid, citric acid, fumaric acid, phosphoric acid, sorbic acid, succinic acid, tartaric acid), bases (e.g., magnesium oxide, tribasic potassium phosphate), and their pharma-ceutically acceptable salts.

[0061] The pharma- ceutically acceptable carrier useful in the present disclosure is a carrier that is conventionally known in the art.The nature of the carrier can depend on the specific mode of administration adopted.For example, for oral application, usually includes pharma- ceutical and physiologically acceptable fluid such as water, physiological saline, balanced salt solution, aqueous dextrose solution, glycerol, etc. as vehicle.In addition to biologically neutral carrier, oral composition may also contain auxiliary substances such as wetting or emulsifying agents, preservatives, and pH buffering agents.

[0062] Antioxidants refer to compounds that inhibit oxidation or reactions promoted by oxygen or peroxides.

[0063] Mucoadhesive material refers to a material that adheres strongly to mucosa upon hydration without additional adhesive material and remains attached to the tissue in vivo.

[0064] Synthetic Signal Peptides In some embodiments, a synthetic signal peptide is provided that increases secretion of a payload protein from yeast. In some embodiments, the synthetic signal peptide comprises one or more of a synthetic preprotein signal peptide and a proprotein signal peptide, as described above. In any embodiment, a natural preprotein signal peptide or a proprotein signal peptide may be combined with the synthetic signal peptide, provided that at least one of the preprotein signal peptide and the proprotein signal peptide is synthetic. In some embodiments, a recombinant polypeptide is provided that comprises a synthetic signal peptide and a payload protein, and the synthetic signal peptide is fused, directly or indirectly, to the payload protein. In some embodiments, the synthetic signal peptide is fused directly to a protein of interest. In some embodiments, the synthetic signal peptide and the protein of interest are linked via a peptide linker. Suitable peptide linkers are known in the art, and any such linker may be utilized. In some embodiments, the linker is a flexible peptide linker. In some embodiments, the linker is a non-cleavable peptide linker. In some embodiments, the linker is a cleavable peptide linker. In some embodiments, the recombinant polypeptide comprises a synthetic preprotein signal peptide and a payload protein. For example, Figure 1 shows a construct representing a recombinant polypeptide comprising a synthetic signal peptide added to the N-terminus of a payload protein, where the synthetic signal peptide comprises only a synthetic preprotein signal peptide (sPre signal peptide, labeled A). In some embodiments, the recombinant polypeptide comprises a synthetic proprotein signal peptide and a payload protein. For example, Figure 1 shows a construct representing a recombinant polypeptide comprising a synthetic signal peptide added to the N-terminus of a payload protein, where the synthetic signal peptide comprises only a synthetic proprotein signal peptide (sPro signal peptide, labeled B). In some embodiments, the recombinant polypeptide comprises a synthetic preprotein signal peptide, a synthetic proprotein signal peptide, and a payload protein.For example, FIG. 1 shows a construct representing a recombinant polypeptide comprising a synthetic signal peptide added to the N-terminus of a payload protein, the synthetic signal peptide comprising both a synthetic preprotein signal peptide and a synthetic proprotein signal peptide (sPre-sPro signal peptide, labeled C). The preprotein signal peptide is added to the N-terminus of a proprotein signal peptide, which is added to the N-terminus of a payload protein. In some embodiments, the recombinant polypeptide comprises a native preprotein signal peptide, a synthetic proprotein signal peptide, and a payload protein. For example, FIG. 1 shows a construct representing a recombinant polypeptide comprising a synthetic signal peptide comprising a native preprotein signal peptide fused to a synthetic proprotein signal peptide (nPre-sPro signal peptide, labeled D). In some embodiments, the recombinant polypeptide comprises a synthetic preprotein signal peptide, a native proprotein signal peptide, and a payload protein.

[0065] Table 3 below lists various amino acid sequences referred to herein. In Table 3, the amino acids included in parentheses are optional. When multiple amino acids are included in parentheses, it should be understood that any one of the amino acids can be added or removed without adding the other amino acid. The sequences EEGEPK (SEQ ID NO: 78) and DVVYPK (SEQ ID NO: 79) are spacers, and DKREEGPK (SEQ ID NO: 80), KREEGPK (SEQ ID NO: 81), DKREKRE (SEQ ID NO: 82), and DKR (SEQ ID NO: 83) are Kex protease sites. [Table 3] TIFF2024511941000005.tif173141

[0066] In addition to the Kex protease sites listed in the examples above, the pre- and pro-protein signal peptides of the present disclosure may also optionally contain a KEX2 cleavage site, such as that provided by the amino acid sequence NVISKR (SEQ ID NO: 68), or the amino acid sequence SDVTKR (SEQ ID NO: 69). In any embodiment, the sequence of SEQ ID NO: 68 may be added to the C-terminus or N-terminus of any pre- or pro-protein signal peptide provided herein. Thus, in some embodiments, the pre-protein signal peptide is as provided. In some embodiments, the pro-protein signal peptide is as provided. In any embodiment, the sequence of SEQ ID NO: 69 may be added to the C-terminus or N-terminus of any pre- or pro-protein signal peptide provided herein. Thus, in some embodiments, the pre-protein signal peptide is as provided. In some embodiments, the pro-protein signal peptide is as provided.

[0067] In some embodiments, the KEX2 cleavage site can be represented by the formula: X 4 X 3 X 2 X 1 B 1 B 2 (Formula XII) In the formula, i)X 1 , X 2 , and X 3 is not G, and ii) X 2 and X 3 is G and X 4 is A or X 5 If S, then X 1 is not S, and iii) X 3 is A and X 2 If S, then X 4 is not T, or iv) X 1 is not D, but B 1 and B. 2are each independently a basic amino acid. Details of formula XII are described in U.S. Patent No. 8,936,917, which is incorporated herein by reference in its entirety. Thus, in any embodiment, the sequence of formula XII can be added to the C-terminus or N-terminus of any preprotein or proprotein signal peptide provided herein. In some embodiments, the preprotein signal peptide is as provided. In some embodiments, the proprotein signal peptide is as provided.

[0068] Any synthetic preprotein or proprotein signal peptide can be combined with some or all of the known signal peptides. Examples of known signal peptides that can be combined with any of SEQ ID NOs: 1-25, 31-38, 55-58, and 70-75 in Table 3 to generate a synthetic signal peptide include, but are not limited to, HSp150, PHO5, SUC2, KILM1, GGP1, SUN, PLB, CRH, EXG, AGA2, HAS pre-pro, PIR1, XPR2 pre, XPR2 pre-pro, pGKL, SCW, and DSE.

[0069] One of ordinary skill in the art would be able to develop a nucleic acid that encodes the expression of any one of SEQ ID NOs: 1-38, 55-58, and 70-75. Table 4 below provides examples of nucleotide sequences that can be used to generate the synthetic peptides described in Table 3. It should be understood that the nucleic acid sequences provided in Table 4 are exemplary and are not intended to be limiting in any way. Due to the degenerate nature of codons, other nucleic acid molecules can be used. In some embodiments, the nucleic acid molecule is codon optimized for expression in a bacterial system. In some embodiments, the nucleic acid molecule is codon optimized for expression in a eukaryotic system or cell. [Table 4] TIFF2024511941000007.tif65162

[0070] The synthetic signal peptides disclosed herein are optimized for use in yeast and can be used to direct the expression of any protein. Specific examples of suitable yeast species are provided herein below to illustrate the specific synthetic signal peptides that have been developed.

[0071] As noted above, Table 3 discloses amino acid sequences, however, in any aspect and embodiment, any of the sequences in Table 3 may be modified with conservative amino acid substitutions to produce active variants that maintain the properties and functionality of the primary sequence. These conservative amino acid substitutions may be generally described by the following formulas, which encapsulate the consensus sequences as well as the variant sequences. Various formulas detailing the variant sequences are now described.

[0072] In some embodiments, a pre-protein signal peptide is provided, in some embodiments, the pre-protein signal peptide comprises an amino acid sequence selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula IX, and Formula XIII.

[0073] Variants of SEQ ID NO:1 (Formula I) In some embodiments, the preprotein signal peptide comprises an amino acid sequence represented by: A 1 -(A 2 ) w -A 3 -(A 4 ) x -(A 5 ) y -(A 6 )-(A 7 )-(A 8 )-(A 9 )-(A 10 )-(A 11 ) z (Formula I) During the ceremony, w and x are each independently 1, 2, 3, 4, or 5; y is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; z is 1, 2, or 3; A 1 is methionine, Each A 2 are independently a neutral or positively charged amino acid having a hydropathic index of less than about 1; Each A 3 , A 5 , A 8 , and A 10 are independently amino acids having a hydropathic index greater than -1, excluding W and C; Each A 4 are independently basic or neutral amino acids excluding P, W, M, and C; Each A 6 are, independently, amino acids having a hydropathic index greater than −1, excluding W, M, and C; Each A 7 are independently non-aromatic amino acids, excluding P, having a hydropathic index of less than about 1.9 and an isoelectric point of about 5.4 to about 7.5, inclusive; Each A 9 are, independently, amino acids having a hydropathic index greater than about -1.3, Each A 11 are independently neutral amino acids having a molecular weight less than about 133 g / mol.

[0074] In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, w is 3. In some embodiments, w is 4. In some embodiments, w is 5. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3. In some embodiments, x is 4. In some embodiments, x is 5. In some embodiments, y can be an integer selected from 2 to 18, 4 to 16, 6 to 14, 8 to 12, 7 to 11, and 8 to 10. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3. It should be understood that the values ​​of w, x, y, and z are each independently selected, and the value of any variable w, x, y, or z is independent of the values ​​selected for the other variables. 3 , A 5 , A 8 , and A 10 is each independently an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, P, E, Y, Q, and N. In some embodiments, each A 3 , A 5 , A 8 , and A 10 is each independently an amino acid selected from the group consisting of L, V, A, and I. In some embodiments, A 3 are each an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, P, E, Y, Q, and N. In some embodiments, A 3 is an amino acid selected from the group consisting of L, V, A, and I. In some embodiments, A 5 are each an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, P, E, Y, Q, and N. In some embodiments, A 5is an amino acid selected from the group consisting of L, V, A, and I. In some embodiments, A 8 are each an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, P, E, Y, Q, and N. In some embodiments, A 8 is an amino acid selected from the group consisting of L, V, A, and I. In some embodiments, A 10 are each an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, P, E, Y, Q, and N. In some embodiments, A 10 is an amino acid selected from the group consisting of L, V, A, and I. In some embodiments, each A 11 is independently an amino acid selected from the group consisting of N, S, T, C, A, V, G, I, L, and P. In some embodiments, each A 11 is independently an amino acid selected from the group consisting of A, L, and G. In some embodiments, each A 2 are independently amino acids selected from the group consisting of K, R, H and Q. In embodiments where any one of w, x, y, z is an integer greater than 1, each amino acid in the group described by w, x, y, z is independently selected from the group of disclosed amino acids and thus may be the same or different. For example, when w is 3 (A 2 ) w For this group, A 2 A 2 A 2 and each A 2 are independently neutral or positively charged amino acids having a hydropathic index of less than about 1. This meaning extends to all further formulas disclosed herein and below, unless expressly indicated otherwise.

[0075] In some embodiments, the sequence of SEQ ID NO:1 can be derived from Formula I as follows: w is 1, x is 2, y is 9, z is 2, and A 1 is methionine, and A 2 is K and A 3 is L and A4 The first and second examples are both S and A. 5 The nine examples are all L and A 6 is S and A 7 is S and A 8 is L and A 9 is V and A 10 is L and A 11 An example of both is A.

[0076] Variants of SEQ ID NOs: 4 to 7 (Formula II) In certain embodiments, the preprotein signal peptide comprises an amino acid sequence represented by: B 1 -(B 2 ) u -(B 3 ) v -(B 4 ) w -(B 5 ) x -(B 6 ) y -(B 7 )-(B 8 )-(B 9 )-(B 10 )-(B 11 ) z (Formula II) During the ceremony, u and w are each independently 0, 1, 2, or 3; v and z are each independently 1, 2, or 3; x is 0, 1, or 2; y is 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20; B 1 is methionine, Each B 2 , B 4 , B 6 , B 8 , and B 10 are each independently an amino acid having a hydropathic index greater than about -1, excluding W and C; Each B 3are independently positively charged or polar amino acids having a hydropathic index of less than about 1; Each B 5 is independently a polar amino acid having a hydropathic index of about -5 to about -0.5, excluding P, W, M, and C, or an amino acid having an isoelectric point of about 5 to 11; Each B 7 and B. 11 are each independently a neutral amino acid having a molecular weight less than about 133 g / mol; B 9 is an amino acid having a hydropathic index greater than about -1.3.

[0077] In some embodiments, u is 0. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3. In some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, w is 3. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, v is 3. In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3. In some embodiments, x is 0. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, y can be an integer selected from 2 to 18, 4 to 16, 6 to 14, 8 to 12, 7 to 11, and 8 to 10. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20. It should be understood that the values ​​of u, w, v, z, x, and y are each independently selected, and the value of any variable u, w, v, z, x, or y is independent of the values ​​selected for the other variables. In some embodiments, each B 2 , B 4 , B 6 , B8 , and B 10 is each independently an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, each B 2 , B 4 , B 6 , B 8 and B. 10 is each independently an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, each B 2 is independently an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, each B 2 is independently an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, each B 4 is independently an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, each B 4 is independently an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, each B 6 is independently an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, each B 6 is independently an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, B 8 is an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, B 8 is an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, B 10 is an amino acid selected from the group consisting of A, G, I, L, M, F, S, T, V, N, Q, E, P, and Y. In some embodiments, B 10 is an amino acid selected from the group consisting of L, V, A, F, and I. In some embodiments, each B 5is independently an amino acid selected from the group consisting of K, R, E, D, G, A, V, L, I, F, S, T, Y, N, and H. In some embodiments, each B 5 is independently an amino acid selected from the group consisting of K, R, E, and D. In some embodiments, each B 5 is independently an amino acid selected from the group consisting of G, A, V, L, I, F, S, T, Y, N, K, R, and H. In some embodiments, each B 7 and B. 11 is each independently an amino acid selected from the group consisting of A, S, G, and P. In some embodiments, B 7 is an amino acid selected from the group consisting of A, S, G, and P. In some embodiments, each B 11 is independently an amino acid selected from the group consisting of A, S, G, and P. In some embodiments, B 9 is an amino acid selected from the group consisting of A, C, G, I, L, M, F, S, T, W, Y, V, N, Q, D, E, and P. In some embodiments, each B 3 is each independently an amino acid selected from the group consisting of K, R, H and Q. As described herein, in embodiments where any one of u, w, v, z, x and y is an integer greater than 1, each amino acid in the group described by u, w, v, z, x and y is independently selected from the disclosed group of amino acids and thus may be the same or different.

[0078] In some embodiments, the sequence of SEQ ID NO:4 can be derived from Formula II as follows: u is 0, v is 1, w is 1, x is 1, y is 11, z is 3, and B 1 is methionine, B is 2 does not exist, B 3 is K and B 4 is L and B 5 is S, and 11 (11)B 6 The string of residues is: TLLLTLLLLLL;B 7is A and B 8 is L and B 9 is V 、 B 10 is L, and three (3)B 11 The string of residues is: AAS.

[0079] In some embodiments, the sequence of SEQ ID NO:5 can be derived from Formula II as follows: u is 1, v is 1, w is 1, x is 0, y is 11, z is 3, and B 1 is methionine, B is 2 is L and B 3 is K and B 4 is L and B 5 does not exist, and there are 11 (11)B 6 The string of residues is: LLLILLLLLLV;B 7 is S and B 8 is L and B 9 is V and B 10 is L, and three (3)B 11 The string of residues is: AAS.

[0080] In some embodiments, the sequence of SEQ ID NO:6 can be derived from Formula II as follows: u is 0, v is 1, w is 0, x is 0, y is 15, z is 3, and B 1 is methionine, B is 2 does not exist, B 3 is K and B 4 does not exist, B 5 does not exist, and all 15 (15)B 6 The residue is L and B 7 is A and B 8 is L and B 9 is V and B 10 is L, and three (3)B 11 The string of residues is: AAS.

[0081] In some embodiments, the sequence of SEQ ID NO:7 can be derived from Formula II as follows: u is 0, v is 1, w is 0, x is 0, y is 6, z is 3, and B 1 is methionine, B is 2 does not exist, B 3 is K and B 4 does not exist, B 5 does not exist, and all six (6)B 6 The residue is L and B 7 is S and B 8 is L and B 9 is V and B 10 is L, and three (3)B 11 The string of residues is: AAS.

[0082] Variant of SEQ ID NO: 9 (Formula III) In some embodiments, the preprotein signal peptide comprises an amino acid sequence represented by: C 1 -(C 2 ) r -(C 3 ) t -(C 4 ) u -[(C 5 ) v -(C 6 ) w ] x -(C 7 ) y -(C 8 ) z -(C 9 )-(C 10 )-(C 11 )-[(C 12 )-(C 13 )] a (Formula III) In the formula, C 2 ~C 13 has the properties set out in Table 5 below, [Table 5] In the formula, r is an integer selected from 1 to 3, t, u, y, and z are independently integers selected from 0 to 3, inclusive; each v and w is independently an integer selected from 0 to 2, inclusive; x is an integer selected from 2 to 10, inclusive; a is 0 or 1.

[0083] In some embodiments, C 1 is methionine. In some embodiments, each C 2 are amino acids having, independently, an isoelectric point of about 5.6 to about 10.8, a molecular weight of about 105 g / mol to about 175 g / mol, a hydropathic index of about -5.1 to about 0.6, and a helicity of about 0.8 to about 1. In some embodiments, each C 3 , C 5 , C 8 , and C 10 are each independently an amino acid having an isoelectric point of about 2.75 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 3 are independently amino acids having an isoelectric point of about 2.75 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 5 are independently amino acids having an isoelectric point of about 2.75 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 8 are independently amino acids having an isoelectric point of about 2.75 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 10is an amino acid having an isoelectric point of about 2.75 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 4 and C 7 are each independently an amino acid having an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 4 , independently, an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 7 , independently, an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 6 , C 9 , C 11 , and C 12 are each independently an amino acid having an isoelectric point of about 2.75 to about 9.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -4 to about 34, and a helicity of about 0.5 to about 1.3. 6 are each independently an amino acid having an isoelectric point of about 2.75 to about 9.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -4 to about 34, and a helicity of about 0.5 to about 1.3. 9 is an amino acid having an isoelectric point of about 2.75 to about 9.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -4 to about 34, and a helicity of about 0.5 to about 1.3. 11 is an amino acid having an isoelectric point of about 2.75 to about 9.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -4 to about 34, and a helicity of about 0.5 to about 1.3.12 is an amino acid having an isoelectric point of about 2.75 to about 9.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -4 to about 34, and a helicity of about 0.5 to about 1.3. 13 is an amino acid having an isoelectric point of about 5.6 to about 6.3, a molecular weight of about 105 g / mol to about 120 g / mol, a hydropathic index of about 0 to about 9.4, and a helicity of about 0.5 to about 1.1.

[0084] In some embodiments, r is 1. In some embodiments, r is 2 and in some embodiments, r is 3. In some embodiments, t is 0. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, u is 0. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3. In some embodiments, y is 0. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, z is 0. In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3. In some embodiments, v is 0. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, x can be an integer selected from 3 to 9, 4 to 8, 6 to 10, 8 to 10, 2 to 5, and 3 to 6. In some embodiments, x is 2. In some embodiments, x is 3. In some embodiments, x is 4. In some embodiments, x is 5. In some embodiments, x is 6. In some embodiments, x is 7. In some embodiments, x is 8. In some embodiments, x is 9. In some embodiments, x is 10. In some embodiments, a is 0 and [(C 12 )-(C 13 )] a In some embodiments, a is 1 and no residue given by [(C 12 )-(C 13 )] aIt should be understood that the values ​​of r, t, u, y, z, v, w, and x are each independently selected, and that the value of any variable r, t, u, y, z, v, w, or x is independent of the values ​​selected for the other variables. In some embodiments, each C 3 , C 5 , C 8 , and C 10 is each independently an amino acid selected from the group consisting of L, F, I, V, A, W, Y, T, Q, S, H, C, N, D, R, P, K, G, E, and M. In some embodiments, each C 3 , C 5 , C 8 , and C 10 is each independently an amino acid selected from the group consisting of L, F, I, V, and A. In some embodiments, each C 3 is independently an amino acid selected from the group consisting of L, F, I, V, A, W, Y, T, Q, S, H, C, N, D, R, P, K, G, E, and M. In some embodiments, each C 3 is independently an amino acid selected from the group consisting of L, F, I, V, and A. In some embodiments, each C 5 is independently an amino acid selected from the group consisting of L, F, I, V, A, W, Y, T, Q, S, H, C, N, D, R, P, K, G, E, and M. In some embodiments, each C 5 is independently an amino acid selected from the group consisting of L, F, I, V, and A. In some embodiments, each C 8 is independently an amino acid selected from the group consisting of L, F, I, V, A, W, Y, T, Q, S, H, C, N, D, R, P, K, G, E, and M. In some embodiments, each C 8 is independently an amino acid selected from the group consisting of L, F, I, V, and A. In some embodiments, C 10 is an amino acid selected from the group consisting of L, F, I, V, A, W, Y, T, Q, S, H, C, N, D, R, P, K, G, E, and M. In some embodiments, C 10is an amino acid selected from the group consisting of L, F, I, V, and A. In some embodiments, each C 6 , C 9 , C 11 , and C 12 is each independently an amino acid selected from the group consisting of A, S, V, G, I, L, F, C, T, K, P, Q, N, Y, E, D, M, and W. In some embodiments, each C 6 , C 9 , C 11 , and C 12 is each independently an amino acid selected from the group consisting of A and S. In some embodiments, each C 6 is independently an amino acid selected from the group consisting of A, S, V, G, I, L, F, C, T, K, P, Q, N, Y, E, D, M, and W. In some embodiments, each C 6 is independently an amino acid selected from the group consisting of A and S. In some embodiments, C 9 is an amino acid selected from the group consisting of A, S, V, G, I, L, F, C, T, K, P, Q, N, Y, E, D, M, and W. In some embodiments, C 9 is an amino acid selected from the group consisting of A and S. In some embodiments, C 11 is an amino acid selected from the group consisting of A, S, V, G, I, L, F, C, T, K, P, Q, N, Y, E, D, M, and W. In some embodiments, C 11 is an amino acid selected from the group consisting of A and S. In some embodiments, C 12 is an amino acid selected from the group consisting of A, S, V, G, I, L, F, C, T, K, P, Q, N, Y, E, D, M, and W. In some embodiments, C 12 is an amino acid selected from the group consisting of A and S. In some embodiments, each C 2 is independently an amino acid selected from the group consisting of K, R, H, S, and Q. In some embodiments, C 13 is an amino acid selected from the group consisting of P, T, and S. In some embodiments, each C 4 and C 7is each independently an amino acid selected from the group consisting of S, N, Q, R, T, K, A, Y, H, V, I, F, G, W, C, P, and L. In some embodiments, each C 4 and C 7 is each independently an amino acid selected from the group consisting of S, N, Q, R, T, K, A, and Y. In some embodiments, each C 4 is independently an amino acid selected from the group consisting of S, N, Q, R, T, K, A, Y, H, V, I, F, G, W, C, P, and L. In some embodiments, each C 4 is independently an amino acid selected from the group consisting of S, N, Q, R, T, K, A, and Y. In some embodiments, each C 7 is independently an amino acid selected from the group consisting of S, N, Q, R, T, K, A, Y, H, V, I, F, G, W, C, P, and L. In some embodiments, each C 7 is an amino acid independently selected from the group consisting of S, N, Q, R, T, K, A, and Y. As described herein, in embodiments where any one of r, t, u, y, z, v, w, and x is an integer greater than 1, each amino acid in the group described by r, t, u, y, z, v, w, and x is independently selected from the disclosed group of amino acids and thus may be the same or different.

[0085] Furthermore, [(C 5 ) v -(C 6 ) w ] x Considering the above, in embodiments where x is an integer greater than 1, the formula [(C 5 ) v -(C 6 ) w ] x is [(C 5 ) v -(C 6 ) w It should be understood that this does not indicate that (C 5 ) v -(C 6 ) wIf is expanded due to x exceeding 1, then C 5 Each instance of may be independently selected from the suitable amino acids detailed above, and similarly, C 6 Each instance of may be independently selected from the appropriate amino acids detailed above. For example, [(C 5 ) 1 -(C 6 ) 1 ] 2 Taking into account the assumption, the formula is the first and second C 5 are both L, and the first and second C 6 We can generate an array LALA, where both C and C are A. Similarly, 5 is L, and the first C 6 is A and the second is C 5 is V and the second C 6 In a further example, a LAVC can be produced in which [(C 5 ) 1 -(C 6 ) 1 ] 3 Considering the assumption, the formula is the first, second and third C 5 are all L, and the first, second and third C 6 We can generate an array LALALA in which all A's are in the first C 5 is L, and the first C 6 The first is A, the second is C 5 is V, the second is C 6 is C, and the third C 5 is H, and the third is C 6 For example, we can generate a LAVCHP where x is P. The same function of x also applies to the values ​​of v and w. For example, [(C 5 ) v -(C 6 ) w ] 2 Given this assumption, each instance of v and w may be an integer between 0 and 2, as described above. Thus, the first instance of v and the second instance of v may each be 1, or the first instance of v may be 1 and the second instance of v may be 2.

[0086] Thus, for example, the compound of formula III, C, in which x is 3 1 -(C 2 ) r -(C 3 ) t -(C 4 ) u -[(C 5 ) v -(C 6 ) w ] x -(C 7 ) y -(C 8 ) z -(C 9 )-(C 10 )-(C 11 )-[(C 12 )-(C 13 )] a When taking into account the above, it can be envisaged that the formula of formula III can be written as follows: C 1 -(C 2 ) r -(C 3 ) t -(C 4 ) u -(C 5 ) v -(C 6 ) w -(C 5 ) v -(C 6 ) w -(C 5 ) v -(C 6 ) w -(C 7 ) y -(C 8 ) z -(C 9 )-(C 10 )-(C 11 )-[(C 12 )-(C 13 )] a wherein each v and w is independently selected from 0, 1, or 2; 5 and C 6are independently selected from the suitable amino acids outlined above. This meaning extends to all further formulae disclosed herein and below, unless expressly indicated otherwise.

[0087] In some embodiments, the sequence of SEQ ID NO:9 can be derived from Formula III as follows: r is 1, t is 2, u is 2, v is 2, w is 2, x is 2, y is 2, z is 1, a is 1, and C 1 is methionine, and C 2 is K, and two (2)C 3 The string of residues is as follows: LS, two (2) C 4 The string of residues is as follows: SL, [(C 5 ) 2 -(C 6 ) 2 ] 2 A string of eight (8) residues given by: LLALLLAL, two (2) C 7 The string of residues is as follows: AS, C 8 is L and C 9 is A and C 10 is L and C 11 is A and C 12 exists, A, and C 13 exists and P.

[0088] Variant of SEQ ID NO: 12 (Formula IV)

[0089] In some embodiments, the preprotein signal peptide comprises an amino acid sequence represented by: D 1 -(D 2 ) q -(D 3 ) r -(D 4 ) t -(D 5 ) u -[(D 6 ) v -(D 7 )x -(D 8 ) w -(D 9 ) y ] z -(D 10 )-(D 11 )-(D 12 )-[(D 13 )-(D 14 )] a (Formula IV) In the formula, D 2 ~D 14 has the properties set forth in Table 6 below, [Table 6] q is an integer selected from 1, 2, or 3 (inclusive); r, t, and u are independently integers selected from 0, 1, 2, or 3, inclusive; each v, w, x, and y is independently an integer selected from 0, 1, or 2, inclusive; z is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10 (inclusive); a is 0 or 1.

[0090] In some embodiments, D 1 is methionine. In some embodiments, each D 2 are amino acids having, independently, an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 3 are independently amino acids having an isoelectric point of about 5 to about 10.8, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -4 to about 34, and a helicity of about 0.5 to about 1.3. 4 , D 9 , and D 11are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 4 are amino acids having, independently, an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 9 are amino acids having, independently, an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 11 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 5 are independently amino acids having an isoelectric point of about 3.2 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.75 to about 1.3. 6 are amino acids having, independently, an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 7 are independently amino acids having an isoelectric point of about 5.4 to about 6.1, a molecular weight of about 117 g / mol to about 205 g / mol, a hydropathic index of about 2.5 to about 34, and a helicity of about 1 to about 1.3. 8 , D 10 , D 12 , and D 13 are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 182 g / mol, a hydropathic index of about -5.1 to about 32, and a helicity of about 0.75 to about 1.3.8 are amino acids having, independently, an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 182 g / mol, a hydropathic index of about -5.1 to about 32, and a helicity of about 0.75 to about 1.3. 10 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 182 g / mol, a hydropathic index of about -5.1 to about 32, and a helicity of about 0.75 to about 1.3. 12 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 182 g / mol, a hydropathic index of about -5.1 to about 32, and a helicity of about 0.75 to about 1.3. 13 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 182 g / mol, a hydropathic index of about -5.1 to about 32, and a helicity of about 0.75 to about 1.3. 14 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 182 g / mol, a hydropathic index of about -5.1 to about 32, and a helicity of about 0.5 to about 1.3.

[0091] In some embodiments, q is 1. In some embodiments, q is 2. In some embodiments, q is 3. In some embodiments, r is 0. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, t is 0. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, u is 0. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3. In some embodiments, v is 0. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, x is 0. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, y is 0. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, z may be an integer selected from 3 to 9, 4 to 8, 6 to 10, 8 to 10, 2 to 5, or 3 to 6 (all inclusive). In some embodiments, z is 2. In some embodiments, z is 3. In some embodiments, z is 4. In some embodiments, z is 5. In some embodiments, z is 6. In some embodiments, z is 7. In some embodiments, z is 8. In some embodiments, z is 9. In some embodiments, z is 10. In some embodiments, a is 0 and [(D 13 )-(D 14 )] a In some embodiments, a is 1 and no residue given by [(D 13 )-(D 14 )] aIt should be understood that the values ​​of r, t, u, v, w, x, y, and z are each independently selected, and that the value of any variable r, t, u, v, w, x, y, or z is independent of the values ​​selected for other variables. In some embodiments, each D 2 is independently an amino acid selected from the group consisting of K and R. In some embodiments, each D 3 is independently an amino acid selected from the group consisting of F, L, I, W, V, M, Y, P, C, A, Q, and S. In some embodiments, each D 4 , D 9 and D. 11 is each independently an amino acid selected from the group consisting of L, I, F, W, V, M, Y, A, T, N, S, G, E, D, C, Q, R, H, P, and K. In some embodiments, each D 4 , D 9 and D. 11 is each independently an amino acid selected from the group consisting of L and I. In some embodiments, each D 4 is independently an amino acid selected from the group consisting of L, I, F, W, V, M, Y, A, T, N, S, G, E, D, C, Q, R, H, P, and K. In some embodiments, each D 4 is independently an amino acid selected from the group consisting of L and I. In some embodiments, each D 9 is independently an amino acid selected from the group consisting of L, I, F, W, V, M, Y, A, T, N, S, G, E, D, C, Q, R, H, P, and K. In some embodiments, each D 9 is independently an amino acid selected from the group consisting of L and I. In some embodiments, D 9 is an amino acid selected from the group consisting of L, I, F, W, V, M, Y, A, T, N, S, G, E, D, C, Q, R, H, P, and K. In some embodiments, D 9 is an amino acid selected from the group consisting of L and I. In some embodiments, D 11is an amino acid selected from the group consisting of L, I, F, W, V, M, Y, A, T, N, S, G, E, D, C, Q, R, H, P, and K. In some embodiments, D 11 is an amino acid selected from the group consisting of L and I. In some embodiments, each D 5 is independently an amino acid selected from the group consisting of S, N, Q, R, T, G, K, E, H, A, C, Y, V, W, I, F, and L. In some embodiments, each D 8 , D 10 , D 12 , and D 13 is each independently an amino acid selected from the group consisting of A, S, T, G, V, L, C, Y, K, I, F, Q, N, H, R, E, D, and M. In some embodiments, each D 8 , D 10 , D 12 , D 13 is each independently an amino acid selected from the group consisting of A and S. In some embodiments, each D 8 is independently an amino acid selected from the group consisting of A, S, T, G, V, L, C, Y, K, I, F, Q, N, H, R, E, D, and M. In some embodiments, each D 8 is independently an amino acid selected from the group consisting of A and S. In some embodiments, D 10 is an amino acid selected from the group consisting of A, S, T, G, V, L, C, Y, K, I, F, Q, N, H, R, E, D, and M. In some embodiments, D 10 is an amino acid selected from the group consisting of A and S. In some embodiments, D 12 is an amino acid selected from the group consisting of A, S, T, G, V, L, C, Y, K, I, F, Q, N, H, R, E, D, and M. In some embodiments, D 12 is an amino acid selected from the group consisting of A and S. In some embodiments, D 13 is an amino acid selected from the group consisting of A, S, T, G, V, L, C, Y, K, I, F, Q, N, H, R, E, D, and M. In some embodiments, D 13is an amino acid selected from the group consisting of A and S. In some embodiments, each D 7 is independently an amino acid selected from the group consisting of V, W, I, L, F, and T. In some embodiments, each D 6 is independently an amino acid selected from the group consisting of L, I, A, T, S, G, N, RK, Y, Q, C, H, W, and M. In some embodiments, each D 6 is independently an amino acid selected from the group consisting of L and I. In some embodiments, D 14 is an amino acid selected from the group consisting of P, Y, M, V, A, T, Q, S, N, G, I, E, D, L, F, R, K, and H. As described herein, in embodiments where any one of r, t, u, v, w, x, y, and z is an integer greater than 1, each amino acid in the group described by r, t, u, v, w, x, y, and z is independently selected from the disclosed group of amino acids and thus may be the same or different.

[0092] As outlined with respect to formula III, -[(D 6 ) v -(D 7 ) x -(D 8 ) w -(D 9 ) y ] z The portion of formula IV given by [(D 6 ) v -(D 7 ) x -(D 8 ) w -(D 9 ) y ] should not be interpreted as "z" repetitions of the formula, but rather, when expanded "z" times, each v, x, w, and y may be independently selected from the integers provided above, and each D 6 , D 7 , D 8 , and D 9 may be independently selected from the suitable amino acids provided above.

[0093] Thus, for example, the compound of formula D of formula IV, in which z is 3 1 -(D 2 ) q -(D 3 ) r -(D 4 ) t -(D 5 ) u -[(D 6 ) v -(D 7 ) x -(D 8 ) w -(D 9 ) y ] z -(D 10 )-(D 11 )-(D 12 )-[(D 13 )-(D 14 )] a When taking into account the above, the formula of formula IV can be envisaged to be written as follows: D 1 -(D 2 ) q -(D 3 ) r -(D 4 ) t -(D 5 ) u -(D 6 ) v -(D 7 ) x -(D 8 ) w -(D 9 ) y -(D 6 ) v -(D 7 ) x -(D 8 ) w -(D 9 ) y -(D 6 ) v -(D 7 ) x -(D 8 ) w -(D 9 ) y -(D 10 )-(D 11 )-(D 12 )-[(D13 )-(D 14 )] a wherein each v, x, w, and y is independently selected from 0, 1, or 2; 6 , D 7 , D 8 , and D 9 are independently selected from the suitable amino acids outlined above.

[0094] In some embodiments, the sequence of SEQ ID NO:12 can be derived from Formula IV as follows: q is 1, r is 1, t is 1, u is 2, v is 0 for all instances of z, x is 0 for all instances of z, w is 1, y is 1, z is 6, a is 1, and D 1 is methionine, D 2 is K and D 3 is F and D 4 is L and 2(2)D 5 The string of residues is: SL; D for all instances of z 6 does not exist, and for all instances of z, D 7 does not exist, and [(D 8 ) 1 -(D 9 ) 1 ] 6 A string of twelve (12) residues given by: LLALVAALALAL;D 10 is A and D 11 is L and D 12 is A and D 13 exists, A, and D 14 exists and is P.

[0095] Variants of SEQ ID NOs: 14, 15, and 16 (Formula V) In some embodiments, the preprotein signal peptide comprises an amino acid sequence represented by: E 1 -[(E 2 ) i -(E 3 )j -(E 4 ) q ] r -(E 5 ) t -(E 6 ) u -(E 7 ) v -[(E 8 ) w -(E 9 ) x ] y -(E 10 ) z -(E 11 )-(E 12 )-(E 13 )-[(E 14 )-(E 15 )] a Formula V In the formula, E 2 ~E 15 has the properties set forth in Table 7 below, [Table 7] wherein each i, j, q, w, x, and a is independently 0 or 1; r is an integer selected from 1, 2, or 3 (inclusive); t, u, v, and z are independently integers selected from 0, 1, 2, or 3, inclusive; y is an integer selected from 2, 3, 4, 5, 6, 7, 8, 9, or 10, inclusive.

[0096] In some embodiments, E 1 is methionine. In some embodiments, each E 2 are independently amino acids having an isoelectric point of about 3.2 to about 10.8, a molecular weight of about 105 g / mol to about 175 g / mol, a hydropathic index of about -4 to about 1, and a helicity of about 0.85 to about 1. In some embodiments, each E 3are amino acids having, independently, an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75.1 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. 4 are independently amino acids having an isoelectric point of about 5 to about 10.8, a molecular weight of about 105 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. 5 and E 8 are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. 5 are amino acids having, independently, an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. 8 are amino acids having, independently, an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. 6 are independently amino acids having an isoelectric point of about 5 to about 10.8, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. 7 are amino acids having, independently, an isoelectric point of about 5 to about 9.75, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -4 to about 33.5, and a helicity of about 0.79 to about 1.3. 9 , E 13 , and E 14are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. 9 are amino acids having, independently, an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. 13 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. 14 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 33.5, and a helicity of about 0.57 to about 1.3. 10 and E 12 are independently amino acids having an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 10 are, independently, amino acids having an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 12 is an amino acid having an isoelectric point of about 5 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 11 is an amino acid having an isoelectric point of about 5 to about 9.75, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -4 to about 33.5, and a helicity of about 0.79 to about 1.3. 15is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -4 to about 15.5, and a helicity of about 0.57 to about 1.2.

[0097] In some embodiments, i is 0. In some embodiments, i is 1. In some embodiments, j is 0. In some embodiments, j is 1. In some embodiments, q is 0. In some embodiments, q is 1. In some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, x is 0. In some embodiments, x is 1. In some embodiments, r is 1. In some embodiments, r is 2. In some embodiments, r is 3. In some embodiments, t is 0. In some embodiments, t is 1. In some embodiments, t is 2. In some embodiments, t is 3. In some embodiments, u is 0. In some embodiments, u is 1. In some embodiments, u is 2. In some embodiments, u is 3. In some embodiments, v is 0. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, v is 3. In some embodiments, z is 0. In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3. In some embodiments, y may be an integer selected from 3 to 9, 4 to 8, 6 to 10, 8 to 10, 2 to 5, or 3 to 6 (all inclusive). In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, y is 5. In some embodiments, y is 6. In some embodiments, y is 7. In some embodiments, y is 8. In some embodiments, y is 9. In some embodiments, y is 10. In some embodiments, a is 0 and [(E 14 )-(E15 )] a In some embodiments, a is 1 and no residue given by [(E 14 )-(E 15 )] a It should be understood that the values ​​of i, j, q, w, x, r, t, u, v, z, and y are each independently selected, and that the value of any variable i, j, q, w, x, r, t, u, v, z, or y is independent of the values ​​selected for the other variables. In some embodiments, each E 2 is independently an amino acid selected from the group consisting of K, R, S, Q, and E. In some embodiments, each E 3 is independently an amino acid selected from the group consisting of F, L, I, W, V, Y, P, A, T, Q, N, S, G, D, R, K, and H. In some embodiments, each E 3 is independently an amino acid selected from the group consisting of F, L, I, W, V, and Y. In some embodiments, each E 4 is independently an amino acid selected from the group consisting of K, R, H, S, C, P, Y, M, V, W, I, L, and F. In some embodiments, each E 4 may be independently K, R, H, and S. In some embodiments, each E 5 and E 8 is each independently an amino acid selected from the group consisting of L, I, F, V, C, A, Y, T, Q, N, S, K, H, W, G, D, M, P, E, and R. In some embodiments, each E 5 and E 8 is each independently an amino acid selected from the group consisting of L, I, F, V, and C. In some embodiments, each E 5 is independently an amino acid selected from the group consisting of L, I, F, V, C, A, Y, T, Q, N, S, K, H, W, G, D, M, P, E, and R. In some embodiments, each E 5 is independently an amino acid selected from the group consisting of L, I, F, V, and C. In some embodiments, each E 8is independently an amino acid selected from the group consisting of L, I, F, V, C, A, Y, T, Q, N, S, K, H, W, G, D, M, P, E, and R. In some embodiments, each E 8 is independently an amino acid selected from the group consisting of L, I, F, V, and C. In some embodiments, each E 6 is independently an amino acid selected from the group consisting of T, Q, S, A, C, R, K, H, P, V, W, I, F, and L. In some embodiments, each E 7 is independently an amino acid selected from the group consisting of S, G, K, A, C, Y, V, and W. In some embodiments, each E 9 , E 13 , and E 14 is each independently an amino acid selected from the group consisting of A, T, G, S, V, I, L, Y, W, F, C, Q, N, P, E, M, R, K, D, and H. In some embodiments, each E 9 , E 13 , and E 14 is each independently an amino acid selected from the group consisting of A, T, G, S, V, I, and L. In some embodiments, each E 9 is independently an amino acid selected from the group consisting of A, T, G, S, V, I, L, Y, W, F, C, Q, N, P, E, M, R, K, D, and H. In some embodiments, each E 9 is independently an amino acid selected from the group consisting of A, T, G, S, V, I, and L. In some embodiments, each E 10 and E 12 is independently an amino acid selected from the group consisting of L, F, I, V, C, Y, T, Q, N, S, K, H, M, G, A, W, D, P, E, and R. In some embodiments, each E 10 and E 12 is independently an amino acid selected from the group consisting of L, F, I, V, and C. In some embodiments, each E 10 is independently an amino acid selected from the group consisting of L, F, I, V, C, Y, T, Q, N, S, K, H, M, G, A, W, D, P, E, and R. In some embodiments, each E 10is independently an amino acid selected from the group consisting of L, F, I, V, and C. In some embodiments, E 12 is an amino acid selected from the group consisting of L, F, I, V, C, Y, T, Q, N, S, K, H, M, G, A, W, D, P, E, and R. In some embodiments, E 12 is an amino acid selected from the group consisting of L, F, I, V, and C. In some embodiments, E 13 is an amino acid selected from the group consisting of A, T, G, S, V, I, L, Y, W, F, C, Q, N, P, E, M, R, K, D, and H. In some embodiments, E 13 is an amino acid selected from the group consisting of A, T, G, S, V, I, and L. In some embodiments, E 14 is an amino acid selected from the group consisting of A, T, G, S, V, I, L, Y, W, F, C, Q, N, P, E, M, R, K, D, and H. In some embodiments, E 14 is an amino acid selected from the group consisting of A, T, G, S, V, I, and L. In some embodiments, each E 11 is independently an amino acid selected from the group consisting of V, W, I, C, L, A, T, S, and K. In some embodiments, each E 15 are amino acids independently selected from the group consisting of S, N, R, T, G, K, E, D, P, and Y. As described herein, in embodiments where any one of r, t, u, v, z, and y is an integer greater than 1, each amino acid in the group described by r, t, u, v, z, and y is independently selected from the disclosed group of amino acids, and thus may be the same or different.

[0098] As outlined with respect to formula III, [(E 8 ) w -(E 9 ) x ] y The part of formula V given by [(E 8 ) w -(E 9 ) x] should not be construed as "y" repetitions of E , but rather, when expanded "y" times, each w and x can be independently selected from the integers provided above, and each E 8 and E 9 can be independently selected from the suitable amino acids provided above. The same applies to [(E 2 ) i -(E 3 ) j -(E 4 ) q ] r It should be understood that the portion of formula V is given by:

[0099] Thus, for example, formula E of formula V where r is 2 and y is 2 1 -[(E 2 ) i -(E 3 ) j -(E 4 ) q ] r -(E 5 ) t -(E 6 ) u -(E 7 ) v -[(E 8 ) w -(E 9 ) x ] y -(E 10 ) z -(E 11 )-(E 12 )-(E 13 )-[(E 14 )-(E 15 )] a When taking into account the above, it can be assumed that the formula of formula V is written as follows: E 1 -(E 2 ) i -(E 3 ) j -(E 4 ) q -(E 2 ) i -(E 3 ) j -(E 4 ) q -(E5 ) t -(E 6 ) u -(E 7 ) v -(E 8 ) w -(E 9 ) x -(E 8 ) w -(E 9 ) x -(E 10 ) z -(E 11 )-(E 12 )-(E 13 )-[(E 14 )-(E 15 )] a wherein each i, j, q, w, and x is independently selected from 0 or 1; 2 , E 3 , E 4 , E 8 , and E 9 are independently selected from the suitable amino acids outlined above.

[0100] In some embodiments, the sequence of SEQ ID NO:14 can be derived from formula V as follows: i is 1, j is 1, q is 1, r is 1, t is 1, u is 2, v is 0, w is 1, x is 1, y is 5, z is 0, a is 1, and E 1 is methionine, E 2 is K and E 3 is F and E 4 is K and E 5 is L, and two (2)E 6 The string of residues is as follows: TL;E 7 does not exist, and [(E 8 ) 1 -(E 9 ) 1 ] 5 A string of ten (10) residues given by: LAALLALAAL; E 10 does not exist, E 11is V and E 12 is L and E 13 is A and E 14 exists, A, and E 15 exists and is S.

[0101] In some embodiments, the sequence of SEQ ID NO:15 can be derived from formula V as follows: i is 1, j is 1, q is 1, r is 1, t is 1, u is 2, v is 0, w is 1, x is 1, y is 4, z is 0, a is 1, and E 1 is methionine, E 2 is K and E 3 is F and E 4 is S and E 5 is S and two (2)E 6 The string of residues is as follows: IL;E 7 does not exist, and [(E 8 ) 1 -(E 9 ) 1 ] 4 A string of eight (8) residues given by: LLLALLAL; E 10 does not exist, E 11 is V and E 12 is L and E 13 is A and E 14 exists, A, and E 15 exists and is S.

[0102] In some embodiments, the sequence of SEQ ID NO:16 can be derived from formula V as follows: i is 1, j is 1, q is 1, r is 2, t is 1, u is 2, v is 0, w is 1, x is 1, y is 3, z is 0, a is 1, and E 1 is methionine, [(E 2 ) 1 -(E 3 ) 1 -(E 4 ) 1 ]2 A string of six (6) residues given by: KLLSLL; E 5 is A and two (2)E 6 The string of residues is as follows: LL;E 7 does not exist, and [(E 8 ) 1 -(E 9 ) 1 ] 3 A string of six (6) residues given by: LLLASL; E 10 does not exist, E 11 is V and E 12 is L and E 13 is A and E 14 exists, A, and E 15 exists and is S.

[0103] Variants of SEQ ID NOs: 31, 32, and 33 (Formula IX)

[0104] In some embodiments, the preprotein signal peptide comprises an amino acid sequence represented by: F 1 -(F 2 ) v -(F 3 ) w -[(F 4 ) x -(F 5 ) y ] z -(F 6 )-(F 7 )-(F 8 )-[(F 9 )-(F 10 )] a (Formula IX) In the formula, F 1 ~F 10 has the properties set forth in Table 8 below, [Table 8] wherein v and w are independently an integer selected from 0, 1, 2, or 3 (inclusive); x and y are independently selected from 0, 1, 2, 3, or 4; z is an integer selected from 1, 2, 3, 4, 5, 6, 7, or 8 (inclusive); a is 0 or 1.

[0105] In some embodiments, F 1 is an amino acid having an isoelectric point of about 5.4 to about 11, a molecular weight of about 89 g / mol to about 175 g / mol, a hydropathic index of about -4 to about 31, and a helicity of about 0.9 to about 1.3. 2 are independently amino acids having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 3 and F 7 are each independently an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 3 are amino acids having, independently, an isoelectric point of about 3 to about 11, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 7 is an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 4 are independently amino acids having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 5 , F 6 , F 8 , and F 9are each independently an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 5 are amino acids having, independently, an isoelectric point of about 3 to about 11, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 6 is an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 8 is an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 9 is an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 10 is an amino acid having an isoelectric point of about 3 to about 11, a molecular weight of about 89 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3.

[0106] In some embodiments, v is 0. In some embodiments, v is 1. In some embodiments, v is 2. In some embodiments, v is 3. In some embodiments, w is 0. In some embodiments, w is 1. In some embodiments, w is 2. In some embodiments, w is 3. In some embodiments, x is 0. In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3. In some embodiments, x is 4. In some embodiments, y is 0. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, z may be an integer selected from 3 to 8, 4 to 8, 6 to 8, 2 to 5, or 3 to 6 (all inclusive). In some embodiments, z is 1. In some embodiments, z is 2. In some embodiments, z is 3. In some embodiments, z is 4. In some embodiments, z is 6. In some embodiments, z is 7. In some embodiments, z is 8. In some embodiments, a is 0 and [(F 9 )-(F 10 )] a In some embodiments, a is 1 and no residue given by [(F 9 )-(F 10 )] a It should be understood that the values ​​of v, w, x, y, and z are each independently selected, and that the value of any variable v, w, x, y, or z is independent of the values ​​selected for the other variables. 1 is an amino acid selected from the group consisting of M, F, L, A, S, or R. In some embodiments, each F 2 is independently an amino acid selected from the group consisting of K, R, H, S, G, N, Q, E, T, A, C, P, Y, V, W, I, L, and F. In some embodiments, each F2 is independently an amino acid selected from the group consisting of K, R, H, S, G, N, Q, E, T, and A. In some embodiments, each F 3 and F 7 is independently an amino acid selected from the group consisting of S, Q, R, T, K, H, I, F, L, P, N, G, E, D, A, Y, M, V, W, and C. In some embodiments, each F 3 and F 7 is independently an amino acid selected from the group consisting of S, Q, R, T, K, H, I, F, and L. In some embodiments, each F 4 is independently an amino acid selected from the group consisting of L, I, V, M, A, F, W, Y, P, C, T, Q, N, S, G, E, R, K, and H. In some embodiments, each F 4 is independently an amino acid selected from the group consisting of L, I, V, M, and A. In some embodiments, each F 5 , F 6 , F 8 , and F 9 is each independently an amino acid selected from the group consisting of A, C, G, S, V, L, T, F, Q, N, P, Y, E, K, H, W, I, M, and R. In some embodiments, each F 5 , F 6 , F 8 , and F 9 is each independently an amino acid selected from the group consisting of A, C, G, S, V, and L. In some embodiments, F 10 is an amino acid selected from the group consisting of P, C, Y, M, V, A, T, Q, S, N, W, G, I, E, L, F, R, K, and H. As described herein, in embodiments where any one of v, w, x, y, and z is an integer greater than 1, each amino acid in the group described by v, w, x, y, and z is independently selected from the disclosed group of amino acids and thus may be the same or different.

[0107] As outlined with respect to formula III, [(F 4 ) x -(F 5 )y ] z The moiety of formula IX given by [(F 4 ) x -(F 5 ) y ] should not be interpreted as "z" repetitions of the formula, but rather, when expanded "z" times, each x and y can be independently selected from the integers provided above, and each F 4 and F 5 may be independently selected from the suitable amino acids provided above.

[0108] Thus, for example, the compound of formula F of formula IX, in which z is 3 1 -(F 2 ) v -(F 3 ) w -[(F 4 ) x -(F 5 ) y ] z -(F 6 )-(F 7 )-(F 8 )-[(F 9 )-(F 10 )] a When taking into account the above, the formula of formula IX can be envisaged to be written as follows: F 1 -(F 2 ) v -(F 3 ) w -(F 4 ) x -(F 5 ) y -(F 4 ) x -(F 5 ) y -(F 4 ) x -(F 5 ) y -(F 6 )-(F 7 )-(F 8 )-[(F 9 )-(F 10 )] a wherein each x and y is independently selected from 0, 1, 2, 3, or 4;4 and F 5 are independently selected from the suitable amino acids outlined above.

[0109] In some embodiments, the sequence of SEQ ID NO:31 can be derived from Formula IX as follows: v is 3, w is 0, x is 1, y is 1, z is 6, a is 1, and F 1 is methionine, and three (3)F 2 The residue string is: KSS;F 3 does not exist, and [(F 4 ) 1 -(F 5 ) 1 ] 6 A string of twelve (12) residues given by: LLLLALLALAAL; F 6 is A and F 7 is S and F 8 is A and F 9 exists, is A, and is F 10 exists and is P.

[0110] In some embodiments, the sequence of SEQ ID NO:32 can be derived from Formula IX as follows: v is 2, w is 0, x is 1, y is 1, z is 6, a is 1, and F 1 is methionine and has two (2)F 2 The string of residues is: KS;F 3 does not exist, and [(F 4 ) 1 -(F 5 ) 1 ] 6 A string of twelve (12) residues given by: SLLLLLLALASL;F 6 is A and F 7 is L and F 8 is A and F 9 exists, is A, and is F 10 exists and is P.

[0111] In some embodiments, the sequence of SEQ ID NO:33 can be derived from Formula IX as follows: v is 3, w is 0, x is 1, y is 1, z is 7, a is 1, and F 1 is methionine, and three (3)F 2 The residue string is: KSS;F 3 does not exist, and [(F 4 ) 1 -(F 5 ) 1 ] 7 The string of fourteen (14) residues given by is: SLLLLALLALLAAL; F 6 is A and F 7 is S and F 8 is A and F 9 exists, is A, and is F 10 exists and is P.

[0112] Variants of SEQ ID NOs: 70, 71, 72, and 73 (Formula XIII) In some embodiments, the preprotein signal peptide comprises an amino acid sequence represented by: L 1 -(L 2 ) x -[(L 3 ) a -(L 4 ) a ] y -[(L 5 ) a -(L 6 ) a -(L 7 ) a ] z -(L 8 ) a -(L 9 ) a -(L 10 ) a -(L 11 ) a -(L 12 ) a (Formula XIII) In the formula, L2 ~L 12 has the properties set out in Table 9 below, [Table 9] During the ceremony, x is 1, 2, or 3; y is 1, 2, 3, or 4; z is 5, 6, 7, 8, 9, or 10; Each a is independently 0 or 1.

[0113] In some embodiments, L 1 is methionine. In some embodiments, each L 2 are independently amino acids having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 3 and L 6 are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 3 are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 6 are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 4 , L 7 , and L 9 are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 4are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 7 are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 9 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 5 , L 8 , L 10 , and L 11 are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 5 are each independently an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 8 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 10 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 11 is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3. 12is an amino acid having an isoelectric point of about 2.7 to about 10.8, a molecular weight of about 75 g / mol to about 205 g / mol, a hydropathic index of about -5.1 to about 34, and a helicity of about 0.5 to about 1.3.

[0114] In some embodiments, x is 1. In some embodiments, x is 2. In some embodiments, x is 3. In some embodiments, y is 1. In some embodiments, y is 2. In some embodiments, y is 3. In some embodiments, y is 4. In some embodiments, z is 5. In some embodiments, z is 6. In some embodiments, z is 7. In some embodiments, z is 8. In some embodiments, z is 9. In some embodiments, z is 10. In some embodiments, a is 0. In some embodiments, a is 1. It should be understood that the values ​​of any of the variables x, y, z, and a are each independently selected, and the value of any of the variables x, y, z, or a is independent of the values ​​selected for the other variables. In some embodiments, L 1 is methionine. In some embodiments, each L 2 is independently an amino acid selected from the group consisting of R, K, H, S, G, N, Q, D, T, A, C, P, Y, M, V, W, I, F, and L. In some embodiments, each L 2 is independently an amino acid selected from the group consisting of R, K, and H. In some embodiments, L 3 is absent. In some embodiments, L 3 In some embodiments, each L 3 is independently an amino acid selected from the group consisting of S, N, Q, R, T, K, P, G, E, H, D, A, C, Y, M, V, W, I, F, and L. In some embodiments, each L 3 is independently an amino acid selected from the group consisting of S, N, Q, R, T, K, and P. In some embodiments, L 4 is absent. In some embodiments, L 4In some embodiments, each L 4 is independently an amino acid selected from the group consisting of L, F, I, W, V, T, M, Y, P, C, A, Q, N, S, G, E, D, R, K, and H. In some embodiments, each L 4 is independently an amino acid selected from the group consisting of L, F, I, W, V, and T. In some embodiments, L 5 is absent. In some embodiments, L 5 In some embodiments, each L 5 is independently an amino acid selected from the group consisting of A, T, G, S, C, P, I, L, F, R, V, Q, Y, K, N, E, D, H, M, and W. In some embodiments, each L 5 is independently an amino acid selected from the group consisting of A, T, G, and S. In some embodiments, L 6 is absent. In some embodiments, L 6 In some embodiments, each L 6 is independently an amino acid selected from the group consisting of S, N, Q, R, T, K, P, G, E, H, D, A, C, Y, M, V, W, I, F, and L. In some embodiments, each L 6 is independently an amino acid selected from the group consisting of S, N, Q, R, T, K, and P. In some embodiments, L 7 is absent. In some embodiments, L 7 In some embodiments, each L 7 is independently an amino acid selected from the group consisting of L, F, I, W, V, T, M, Y, P, C, A, Q, N, S, G, E, D, R, K, and H. In some embodiments, each L 7 is independently an amino acid selected from the group consisting of L, F, I, W, V, and T. In some embodiments, L 8 is absent. In some embodiments, L 8 In some embodiments, L 8is an amino acid selected from the group consisting of A, T, G, S, C, P, I, L, F, R, V, Q, Y, K, N, E, D, H, M, and W. In some embodiments, L 8 is an amino acid selected from the group consisting of A, T, G, and S. In some embodiments, L 9 is absent. In some embodiments, L 9 In some embodiments, L 9 is an amino acid selected from the group consisting of L, F, I, W, V, T, M, Y, P, C, A, Q, N, S, G, E, D, R, K, and H. In some embodiments, L 9 is an amino acid selected from the group consisting of L, F, I, W, V, and T. In some embodiments, L 10 is absent. In some embodiments, L 10 In some embodiments, L 10 is an amino acid selected from the group consisting of A, T, G, S, C, P, I, L, F, R, V, Q, Y, K, N, E, D, H, M, and W. In some embodiments, L 10 is an amino acid selected from the group consisting of A, T, G, and S. In some embodiments, L 11 is absent. In some embodiments, L 11 In some embodiments, L 11 is an amino acid selected from the group consisting of A, T, G, S, C, P, I, L, F, R, V, Q, Y, K, N, E, D, H, M, and W. In some embodiments, L 11 is an amino acid selected from the group consisting of A, T, G, and S. In some embodiments, L 12 is absent. In some embodiments, L 12 In some embodiments, L 12 is an amino acid selected from the group consisting of P, T, S, D, C, Y, M, V, A, Q, N, W, G, I, E, L, F, R, K, and H. In some embodiments, L 12is an amino acid selected from the group consisting of P, T, S, and D. As described herein, in embodiments where any one of x, y, and z is an integer greater than 1, each amino acid in the group described by x, y, and z is independently selected from the disclosed group of amino acids, and thus may be the same or different.

[0115] As outlined with respect to formula III, [(L 5 ) a -(L 6 ) a -(L 7 ) a ] z The moiety of formula XIII given by [(L 5 ) a -(L 6 ) a -(L 7 ) a ] should not be interpreted as "z" repetitions of ], but rather, when expanded "z" times, each a may be independently selected from the integers provided above, and each L 5 , L 6 , and L 7 may be independently selected from the suitable amino acids provided above.

[0116] Thus, for example, the compound of formula L of formula XIII, in which z is 5, 1 -(L 2 ) x -[(L 3 ) a -(L 4 ) a ] y -[(L 5 ) a -(L 6 ) a -(L 7 ) a ] z -(L 8 ) a -(L 9 ) a -(L 10 ) a -(L 11 ) a -(L 12 ) aWhen taking into account the above, the formula of formula XIII can be envisaged to be written as follows: L 1 -(L 2 ) x -[(L 3 ) a -(L 4 ) a ] y -(L 5 ) a -(L 6 ) a -(L 7 ) a -(L 5 ) a -(L 6 ) a -(L 7 ) a -(L 5 ) a -(L 6 ) a -(L 7 ) a -(L 5 ) a -(L 6 ) a -(L 7 ) a -(L 5 ) a -(L 6 ) a -(L 7 ) a -(L 8 ) a -(L 9 ) a -(L 10 ) a -(L 11 ) a -(L 12 ) a wherein each a is independently 0 or 1; 5 , L 6 , and L 7 are independently selected from suitable amino acids as outlined above.

[0117] In some embodiments, the sequence of SEQ ID NO:70 can be derived from Formula XIII as follows: x is 1, y is 2, z is 6, and L1 is methionine, L 2 is R, and [(L 3 ) a -(L 4 ) a ] 2 All four instances of "a" in [(L 3 ) 1 -(L 4 ) 1 ] 2 A string of four (4) residues given by: SLSL; all (L 5 ) a For all (L 6 ) a For all (L 7 ) a For "a", "a" is 1, and [(L 5 ) 1 -(L 7 ) 1 ] 6 A string of twelve (12) residues given by: ALLLLLALLASL;L 6 does not exist, L 8 exists, A, and L 9 exists and is L, and L 10 exists, A, and L 11 exists, A, and L 12 exists and is P.

[0118] In some embodiments, the sequence of SEQ ID NO:71 can be derived from Formula XIII as follows: x is 1, y is 2, z is 6, and L 1 is methionine, L 2 is R, and [(L 3 ) a -(L 4 ) a ] 2 All four instances of "a" in [(L 3 ) 1 -(L 4 ) 1 ] 2A string of four (4) residues given by: LSLS; all (L 5 ) a For all (L 6 ) a For all (L 7 ) a For "a", "a" is 1, and [(L 5 ) 1 -(L 7 ) 1 ] 6 A string of twelve (12) residues given by: LLLLLLALLASL;L 6 does not exist, L 8 exists, A, and L 9 exists and is L, and L 10 exists, A, and L 11 exists, A, and L 12 exists and is P.

[0119] In some embodiments, the sequence of SEQ ID NO:72 can be derived from Formula XIII as follows: x is 1, y is 2, z is 6, and L 1 is methionine, L 2 is R, and [(L 3 ) a -(L 4 ) a ] 2 All four instances of "a" in [(L 3 ) 1 -(L 4 ) 1 ] 2 A string of four (4) residues given by: LSSL; all (L 5 ) a For all (L 6 ) a For all (L 7 ) a For "a", "a" is 1, and [(L 5 ) 1 -(L 7 )1 ] 6 The string of twelve (12) residues given by: LLGLLLALAASL; 6 does not exist, L 8 exists, A, and L 9 exists and is L, and L 10 exists, A, and L 11 exists, A, and L 12 exists and is P.

[0120] In some embodiments, the sequence of SEQ ID NO:73 can be derived from Formula XIII as follows: x is 1, y is 1, z is 7, and L 1 is methionine, L 2 is R, and [(L 3 ) a -(L 4 ) a ] 2 Both instances of "a" in [(L 3 ) 1 -(L 4 ) 1 ] 1 A string of two (2) residues given by: LS; all (L 5 ) a For all (L 6 ) a For all (L 7 ) a For "a", "a" is 1, and [(L 5 ) 1 -(L 7 ) 1 ] 7 The string of fourteen (14) residues given by: LLLALLALLALASL; 6 does not exist, L 8 exists, A, and L 9 exists and is L, and L 10 exists, A, and L 11 exists, A, and L 12 exists and is P.

[0121] Variant of SEQ ID NO: 21 (Formula VI) In some embodiments, the proprotein signal peptide comprises an amino acid sequence represented by: G 1 -G 2 -G 3 -G 4 -G 5 -G 6 -G 7 -G 8 -G 9 -G 10 -G 11 -G 12 -G 13 -G 14 -G 15 -G 16 -G 17 -G 18 -G 19 -G 20 -G 21 -G 22 -G 23 -G 24 -G 25 (Formula VI) Table 10 below describes the various substitutions that can be made, with preferred amino acids underlined. [Table 10]

[0122] In some embodiments, G 1 is an amino acid selected from the group consisting of I, L, F, V, A, N, S, D, R, and K. In some embodiments, G 2 is an amino acid selected from the group consisting of P, S, N, G, and E. In some embodiments, G 3 is an amino acid selected from the group consisting of L, F, I, V, Y, A, S, R, and H. In some embodiments, G 4 is an amino acid selected from the group consisting of V, M, P, Y, A, T, S, N, K, and H. In some embodiments, G 5is an amino acid selected from the group consisting of A, G, R, Y, K, D, M, V, W, I, and L. In some embodiments, G 6 is an amino acid selected from the group consisting of N, R, and K. In some embodiments, G 7 is an amino acid selected from the group consisting of V, P, A, T, Q, G, E, D, R, and K. In some embodiments, G 8 is an amino acid selected from the group consisting of P, Y, T, Q, S, N, W, F, R, K, and H. In some embodiments, G 9 is an amino acid selected from the group consisting of F, L, A, Q, N, S, E, G, D, and H. In some embodiments, G 10 is an amino acid selected from the group consisting of H, S, N, D, Q, E, T, Y, M, V, I, and L. In some embodiments, G 11 is an amino acid selected from the group consisting of S, R, T, G, K, E, D, and P. In some embodiments, G 12 is an amino acid selected from the group consisting of D, E, Q, N, A, and V. In some embodiments, G 13 is an amino acid selected from the group consisting of N, S, E, D, T, H, K, A, and P. In some embodiments, G 14 is an amino acid selected from the group consisting of G, S, N, H, E, C, Y, L, and F. In some embodiments, G 15 is an amino acid selected from the group consisting of S, T, and H. In some embodiments, G 16 is an amino acid selected from the group consisting of E, D, Q, N, S, T, K, and A. In some embodiments, G 17 is an amino acid selected from the group consisting of W, N, D, and R. In some embodiments, G 18 is an amino acid selected from the group consisting of L and F. In some embodiments, G 19 is an amino acid selected from the group consisting of Y, V, A, Q, N, S, E, D, L, R, K, and H. In some embodiments, G 20is an amino acid selected from the group consisting of K, R, S, and I. In some embodiments, G 21 is R. In some embodiments, G 22 is an amino acid selected from the group consisting of D, E, N, S, T, G, A, Y, and L. In some embodiments, G 23 is an amino acid selected from the group consisting of V, P, Y, I, A, E, K, F, T, S, G, D, M, and N. In some embodiments, G 23 is an amino acid selected from the group consisting of V, P, Y, I, A, E, and K. In some embodiments, G 24 is an amino acid selected from the group consisting of V, P, Y, I, A, E, K, F, T, S, G, D, M, and N. In some embodiments, G 24 is an amino acid selected from the group consisting of V, P, Y, I, A, E, and K. In some embodiments, G 25 is an amino acid selected from the group consisting of Y, P, A, T, Q, S, E, F, and H.

[0123] In some embodiments, the proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 21 (IPLVANVSFNSDNGSQWLYKRDVVY).

[0124] Variants of SEQ ID NOs: 22, 23, and 24 (Formula VII and Formula VIII) In some embodiments, the proprotein signal peptide comprises an amino acid sequence represented by: (H 1 ) m -(H 2 ) m -(H 3 ) m -(H 4 ) m -(H 5 ) m -(H 6 ) m -(H 7 ) m -(H 8 ) m -(H 9 )m -(H 10 ) m -(H 11 ) m -(H 12 ) m -(H 13 ) m -(H 14 ) m -(H 15 ) m -(H 16 ) m -(H 17 ) m -(H 18 ) m -(H 19 ) m -(H 20 ) m -(H 21 ) m -(H 22 ) m -(H 23 ) m -(H 24 ) m -(H 25 ) m -(H 26 ) m -(H 27 ) m -(H 28 ) m -(H 29 ) m -(H 30 ) m -(H 31 ) m -(H 32 ) m -(H 33 ) m -(H 34 ) m -(H 35 ) m -(H 36 ) m -H 37 -H 38 -H 39 -H 40 (Formula VII) wherein each m is independently 0, 1, or 2. Table 11 below describes the various amino acids that may be used at each position, with preferred amino acids underlined. [Table 11]

[0125] In some embodiments, amino acid position H 1 ~H 36 may be omitted or may be repeated up to one extra time (i.e., included 0-2 times), with each repeat being independently selected from the amino acids shown. 1 ~H 36 It should be understood that the omission or repetition of is independent of the omission or repetition of any amino acid at another position. In some embodiments, the minimum length of the sequence generated by Formula VII is fourteen (14) amino acids.

[0126] In some embodiments, each H 1 is independently absent. 1 is independently an amino acid selected from the group consisting of E, D, S, L, G, Q, and A. In some embodiments, each H 1 is independently an amino acid selected from the group consisting of E, D, and S. In some embodiments, each H 2 is independently absent. 2 is independently an amino acid selected from the group consisting of P, S, R, T, N, G, D, K, and A. In some embodiments, each H 2 is independently an amino acid selected from the group consisting of P, S, and R. In some embodiments, each H 3 is independently absent. 3 is independently an amino acid selected from the group consisting of W and Y. In some embodiments, each H 4 is independently absent. 4 is independently an amino acid selected from the group consisting of S, N, A, P, and V. In some embodiments, each H 5 is independently absent. 5is independently an amino acid selected from the group consisting of T, Q, A, E, F, and S. In some embodiments, each H 5 is independently T. In some embodiments, each H 6 is independently absent. 6 is independently an amino acid selected from the group consisting of L, F, and I. In some embodiments, each H 7 is independently absent. 7 is independently an amino acid selected from the group consisting of F, V, M, T, S, and K. In some embodiments, each H 8 is independently absent. 8 is independently an amino acid selected from the group consisting of V, P, I, A, S, and K. In some embodiments, each H 9 is independently absent. 9 is independently an amino acid selected from the group consisting of T, G, V, W, and A. In some embodiments, each H 9 is independently an amino acid selected from the group consisting of T, G, and V. In some embodiments, each H 10 is independently absent. 10 is independently an amino acid selected from the group consisting of R, H, S, G, N, E, T, and V. In some embodiments, each H 11 is independently absent. 11 is independently an amino acid selected from the group consisting of S, G, D, A, and M. In some embodiments, each H 12 is independently absent. 12 is independently an amino acid selected from the group consisting of T, S, E, G, D, K, and H. In some embodiments, each H 13 is independently absent. 13is independently an amino acid selected from the group consisting of L, M, Y, N, S, D, and K. In some embodiments, each H 14 is independently absent. 14 is independently an amino acid selected from the group consisting of D, Q, N, S, K, and C. In some embodiments, each H 15 is independently absent. 15 is independently an amino acid selected from the group consisting of E, S, D, L, and G. In some embodiments, each H 15 is independently an amino acid selected from the group consisting of E and S. In some embodiments, each H 16 is independently absent. 16 is independently an amino acid selected from the group consisting of I, L, V, M, A, and T. In some embodiments, each H 17 is independently absent. 17 is independently an amino acid selected from the group consisting of T, G, V, W, and A. In some embodiments, each H 17 is independently an amino acid selected from the group consisting of T, G, and V. In some embodiments, each H 18 is independently absent. 18 is independently an amino acid selected from the group consisting of D, E, S, T, K, and G. In some embodiments, each H 19 is independently absent. 19 is independently an amino acid selected from the group consisting of Y, F, and L. In some embodiments, each H 20 is independently absent. 20 is independently an amino acid selected from the group consisting of N, Q, S, T, R, and F. In some embodiments, each H 21 is independently absent. 21is independently an amino acid selected from the group consisting of S, K, T, A, Y, M, and F. In some embodiments, each H 21 is independently an amino acid selected from the group consisting of S and K. In some embodiments, each H 22 is independently absent. 22 is independently an amino acid selected from the group consisting of T, Q, S, D, C, V, and L. In some embodiments, each H 23 is independently absent. 23 is independently an amino acid selected from the group consisting of G, S, K, N, H, D, W, and L. In some embodiments, each H 24 is independently absent. In some embodiments, each H 24 is independently an amino acid selected from the group consisting of I, L, V, P, N, and E. In some embodiments, each H 25 is independently absent. 25 is independently an amino acid selected from the group consisting of A, T, G, R, Y, L, F, and E. In some embodiments, each H 25 is independently A. In some embodiments, each H 26 is independently absent. 26 is independently an amino acid selected from the group consisting of V, I, F, M, L, A, and T. In some embodiments, each H 26 is independently an amino acid selected from the group consisting of V, I, and F. In some embodiments, each H 27 is independently absent. 27 is independently an amino acid selected from the group consisting of D, E, Q, N, S, A, and I. In some embodiments, each H 28 is independently absent. 28 is independently an amino acid selected from the group consisting of P, S, R, T, N, G, D, K, and A. In some embodiments, each H 28is independently an amino acid selected from the group consisting of P, S, and R. In some embodiments, each H 29 is independently absent. 29 is independently an amino acid selected from the group consisting of E, D, T, A, Y, M, V, I, F, and L. In some embodiments, each H 30 is independently absent. 30 is independently an amino acid selected from the group consisting of T, Q, A, E, F, and S. In some embodiments, each H 30 is independently T. In some embodiments, each H 31 is independently absent. 31 is independently an amino acid selected from the group consisting of F, W, V, M, S, G, and R. In some embodiments, each H 32 is independently absent. 32 is independently an amino acid selected from the group consisting of H, S, E, G, and T. In some embodiments, each H 33 is independently absent. 33 is independently an amino acid selected from the group consisting of A, T, G, R, Y, L, F, and E. In some embodiments, each H 33 is independently A. In some embodiments, each H 34 is independently absent. 34 is independently an amino acid selected from the group consisting of S, K, T, A, Y, M, and F. In some embodiments, each H 34 is independently an amino acid selected from the group consisting of S and K. In some embodiments, each H 35 is independently absent. 35 is independently an amino acid selected from the group consisting of R, K, S, and Q. In some embodiments, each H 36 is independently absent. 36is independently an amino acid selected from the group consisting of H, R, S, T, A, V, W, and L. In some embodiments, H 37 is an amino acid selected from the group consisting of K, Q, D, A, and I. In some embodiments, H 38 is an amino acid selected from the group consisting of R, K, T, and F. In some embodiments, H 39 is an amino acid selected from the group consisting of D, N, S, T, K, A, Y, and L. In some embodiments, H 40 is an amino acid selected from the group consisting of V, I, F, M, L, A, and T. In some embodiments, H 40 is an amino acid selected from the group consisting of V, I, and F.

[0127] In some embodiments, the proprotein signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 23, and 24.

[0128] In some embodiments, the proprotein signal peptide comprises an amino acid sequence represented by: (I 1 ) m -(I 2 ) m -(I 3 ) m -(I 4 ) m -(I 5 ) m -(I 6 ) m -(I 7 ) x -(I 8 ) m -(I 9 ) m -(I 10 ) m -(I 11 ) x -(I 12 ) m -(I 13 ) x -(I 14 ) x -(I 15 ) m -(I16 ) x -(I 17 ) m -I 18 -I 19 -I 20 -I 21 -I 22 -I 23 (Formula VIII) wherein each m is independently 0, 1, or 2, and each x is independently 0, 1, 2, 3, or 4. Table 12 below describes the various amino acids that may be used at each position, with preferred amino acids underlined. [Table 12]

[0129] In some embodiments, amino acid position I 1 ~I 6 , I 8 , I 9 , I 12 , I 15 , and I 17 may be omitted or may be repeated up to one extra time (i.e., included 0-2 times), with each repeat independently selected from the amino acids shown. 7 , I 11 , I 13 , I 14 , and I 16 may be omitted or repeated up to three extra times (i.e., included 0-4 times), with each repeat independently selected from the amino acids shown. Furthermore, it should be understood that the omission or repeat of any amino acid position 1-9 and 11-17 is independent of the omission or repeat of any amino acid at another position. In some embodiments, the minimum length of a sequence generated using Formula VIII is 17 amino acids.

[0130] In some embodiments, each I 1 is independently absent. In some embodiments, each I 1is independently an amino acid selected from the group consisting of S, Q, E, A, I, G, V, R, T, and Y. In some embodiments, each I 1 is independently an amino acid selected from the group consisting of A, Q, and E. In some embodiments, each I 2 is independently absent. In some embodiments, each I 2 is independently an amino acid selected from the group consisting of T, S, E, R, P, V, I, and F. In some embodiments, each I 3 is independently absent. In some embodiments, each I 3 is independently L. In some embodiments, each I 4 is independently absent. In some embodiments, each I 4 is independently an amino acid selected from the group consisting of T, N, K, and M. In some embodiments, each I 5 is independently absent. In some embodiments, each I 5 is independently an amino acid selected from the group consisting of P, A, and D. In some embodiments, each I 6 is independently absent. In some embodiments, each I 6 is independently an amino acid selected from the group consisting of S, Q, E, A, I, G, V, R, T, and Y. In some embodiments, each I 6 is independently an amino acid selected from the group consisting of A, Q, and E. In some embodiments, each I 7 is independently absent. In some embodiments, each I 7 is independently an amino acid selected from the group consisting of T, S, K, H, Y, V, and F. In some embodiments, each I 8 is independently absent. In some embodiments, each I 8 is independently an amino acid selected from the group consisting of F, L, W, A, T, M, Y, and C. In some embodiments, each I 8 is independently an amino acid selected from the group consisting of F, L, W, A, and T. In some embodiments, each I 9is independently absent. In some embodiments, each I 9 is independently an amino acid selected from the group consisting of I, L, and V. In some embodiments, each I 10 is independently absent. In some embodiments, each I 10 is independently an amino acid selected from the group consisting of G, S, N, E, D, A, K, H, C, P, and F. In some embodiments, each I 10 is independently an amino acid selected from the group consisting of G and S. In some embodiments, each I 11 is independently absent. In some embodiments, each I 11 is independently an amino acid selected from the group consisting of I, L, V, A, T, and S. In some embodiments, each I 12 is independently absent. In some embodiments, each I 12 is independently an amino acid selected from the group consisting of T, N, A, E, and G. In some embodiments, each I 13 is independently absent. In some embodiments, each I 13 is independently an amino acid selected from the group consisting of E, Q, S, T, R, K, A, L, D, and F. In some embodiments, each I 13 is independently E. In some embodiments, each I 14 is independently absent. In some embodiments, each I 14 is independently an amino acid selected from the group consisting of T, S, Q, F, A, G, V, I, and L. In some embodiments, each I 14 is independently an amino acid selected from the group consisting of T and S. In some embodiments, each I 15 is independently absent. In some embodiments, each I 15 is independently an amino acid selected from the group consisting of F, L, W, A, T, M, Y, and C. In some embodiments, each I 15 is independently an amino acid selected from the group consisting of F, L, W, A, and T. In some embodiments, each I 16is independently absent. In some embodiments, each I 16 is independently an amino acid selected from the group consisting of G, S, N, E, D, A, K, H, C, P, and F. In some embodiments, each I 16 is independently an amino acid selected from the group consisting of G and S. In some embodiments, each I 17 is independently absent. In some embodiments, each I 17 is independently an amino acid selected from the group consisting of I, L, V, N, A, T, and S. In some embodiments, each I 17 is independently an amino acid selected from the group consisting of I, L, and V. In some embodiments, I 18 is an amino acid selected from the group consisting of R, K, Q, and A. In some embodiments, I 18 is R. In some embodiments, I 19 is an amino acid selected from the group consisting of H, R, S, N, T, A, V, and W. In some embodiments, I 20 is an amino acid selected from the group consisting of K, N, Q, D, E, A, and I. In some embodiments, I 21 is an amino acid selected from the group consisting of R, K, Q, and A. In some embodiments, I 21 is R. In some embodiments, I 22 is an amino acid selected from the group consisting of D, N, S, A, Y, and L. In some embodiments, I 23 is an amino acid selected from the group consisting of V, I, L, F, and A.

[0131] Variants of primary SEQ ID NOs: 34, 35, 36, 37, and 38 (Formula X)

[0132] In some embodiments, the proprotein signal peptide comprises an amino acid sequence represented by: (J 1 ) z -(J 2 ) z -(J 3 )z -(J 4 ) z -(J 5 ) z -(J 6 ) z -(J 7 ) z -(J 8 ) z -(J 9 ) z -(J 10 ) z -(J 11 ) z -(J 12 ) z -(J 13 ) z -(J 14 ) z -(J 15 ) z -(J 16 ) z -(J 17 ) z -(J 18 ) z -(J 19 ) z -(J 20 ) z -(J 21 ) z -J 22 -J 23 -J 24 -J 25 (Formula X) wherein each z is independently 0, 1, 2, 3, 4, or 5. Table 13 below describes the various amino acids that may be used at each position, with preferred amino acids underlined. [Table 13] TIFF2024511941000017.tif29160

[0133] In some embodiments, amino acid position J 1 ~J 21 may be omitted or may be repeated up to four extra times (i.e., from 0 to 5 times), with each repeat being independently selected from the amino acids shown. 1 ~J21 It should be understood that the omission or repetition of is independent of the omission or repetition of any amino acid at another position.

[0134] In some embodiments, each J 1 is independently absent. In some embodiments, each J 1 is independently an amino acid selected from the group consisting of H, K, G, A, P, F, and L. In some embodiments, each J 2 is independently absent. In some embodiments, each J 2 is independently an amino acid selected from the group consisting of D, E, N, G, P, H, T, R, K, and A. In some embodiments, each J 2 is independently an amino acid selected from the group consisting of D, E, N, G, and P. In some embodiments, each J 3 is independently absent. In some embodiments, each J 3 is independently an amino acid selected from the group consisting of G, A, P, V, and L. In some embodiments, each J 4 is independently absent. In some embodiments, each J 4 is independently an amino acid selected from the group consisting of F, I, P, A, S, E, D, R, and K. In some embodiments, each J 5 is independently absent. In some embodiments, each J 5 is independently an amino acid selected from the group consisting of S, R, T, G, K, E, D, and C. In some embodiments, each J 6 is independently absent. In some embodiments, each J 6 is independently an amino acid selected from the group consisting of T, S, A, D, and F. In some embodiments, each J 7 is independently absent. In some embodiments, each J 7 is independently an amino acid selected from the group consisting of D, E, N, G, P, H, T, R, K, and A. In some embodiments, each J 7is independently an amino acid selected from the group consisting of D, E, N, G, and P. In some embodiments, each J 8 is independently absent. In some embodiments, each J 8 is independently an amino acid selected from the group consisting of Y, C, A, W, I, S, E, D, F, L, R, and K. In some embodiments, each J 9 is independently absent. In some embodiments, each J 9 is independently an amino acid selected from the group consisting of H, K, N, D, G, T, A, C, Y, V, and L. In some embodiments, each J 10 is independently absent. In some embodiments, each J 10 is independently an amino acid selected from the group consisting of L, V, A, G, E, I, P, and R. In some embodiments, each J 10 is independently an amino acid selected from the group consisting of L, V, A, G, and E. In some embodiments, each J 11 is independently absent. In some embodiments, each J 11 is independently an amino acid selected from the group consisting of I, W, V, Y, P, T, N, S, R, and K. In some embodiments, each J 12 is independently absent. In some embodiments, each J 12 is independently an amino acid selected from the group consisting of A, G, Q, N, R, Y, E, D, and L. In some embodiments, each J 13 is independently absent. In some embodiments, each J 13 is independently an amino acid selected from the group consisting of I, L, W, V, M, Y, P, A, S, and G. In some embodiments, each J 14 is independently absent. In some embodiments, each J 14 is independently an amino acid selected from the group consisting of V, C, L, F, A, T, N, G, and R. In some embodiments, each J 15 is independently absent. In some embodiments, each J 15is independently an amino acid selected from the group consisting of G, S, R, K, A, T, H, E, W, L, and F. In some embodiments, each J 16 is independently absent. In some embodiments, each J 16 is independently an amino acid selected from the group consisting of D, E, Q, S, H, T, R, G, Y, V, F, and L. In some embodiments, each J 17 is independently absent. In some embodiments, each J 17 is independently an amino acid selected from the group consisting of E, S, G, Y, I, and L. In some embodiments, each J 18 is independently absent. In some embodiments, each J 18 is independently an amino acid selected from the group consisting of A, S, P, H, and V. In some embodiments, each J 19 is independently absent. In some embodiments, each J 19 is independently an amino acid selected from the group consisting of N, E, R, K, and A. In some embodiments, each J 20 is independently absent. In some embodiments, each J 20 is independently an amino acid selected from the group consisting of R, T, V, I, and L. In some embodiments, each J 20 is independently R. In some embodiments, each J 21 is independently absent. In some embodiments, each J 21 is independently an amino acid selected from the group consisting of L, V, A, G, E, I, P, and R. In some embodiments, each J 21 is independently an amino acid selected from the group consisting of L, V, A, G, and E. In some embodiments, each J 22 is independently absent. In some embodiments, J 22 is an amino acid selected from the group consisting of K, R, D, T, M, and W. In some embodiments, J 23 is an amino acid selected from the group consisting of R, T, V, I, and L. In some embodiments, J 24is an amino acid selected from the group consisting of S, N, G, E, D, P, and W. In some embodiments, J 25 is an amino acid selected from the group consisting of A, T, S, Y, M, V, and L.

[0135] In some embodiments, the proprotein signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 34, 35, 36, 37, and 38.

[0136] Variants of primary SEQ ID NOs: 34, 35, 36, 37, and 38 (Formula XI) In some embodiments, the proprotein signal peptide comprises an amino acid sequence represented by: (K 1 ) b -(K 2 ) b -(K 3 ) b -(K 4 ) b -(K 5 ) b -(K 6 ) b -(K 7 ) b -(K 8 ) b -(K 9 ) b -(K 10 ) b -(K 11 ) b -(K 12 ) b -(K 13 ) b -(K 14 ) b -(K 15 ) b -(K 16 ) b -(K 17 ) b -(K 18 ) b -(K 19 ) b -(K 20 ) b -(K 21 ) b -(K 22) b -(K 23 ) b -(K 24 ) b -(K 25 ) b -(K 26 ) b -(K 27 ) b -(K 28 ) b -(K 29 ) b -(K 30 ) b -(K 31 ) b -(K 32 ) b -(K 33 ) b -(K 34 ) b -(K 35 ) b -(K 36 ) b -(K 37 ) b -(K 38 ) b -(K 39 ) b -(K 40 ) b -(K 41 ) b -(K 42 ) b -(K 43 ) b -(K 44 ) b -(K 45 ) b -(K 46 ) b -(K 47 ) b -(K 48 ) b -(K 49 ) b -(K 50 ) b -(K 51 ) b -(K 52 ) b -(K 53 ) b -(K 54 ) b -(K 55 )b -(K 56 ) b -(K 57 ) b -(K 58 ) b -(K 59 ) b -(K 60 ) b -(K 61 ) b -(K 62 ) b -(K 63 ) b -(K 64 ) b -(K 65 ) b -(K 66 ) b -(K 67 ) b -(K 68 ) b -(K 69 ) b -(K 70 ) b -(K 71 ) b -(K 72 ) b -(K 73 ) b -(K 74 ) b -(K 75 ) b -(K 76 ) b -(K 77 ) b -(K 78 ) b -(K 79 ) b -(K 80 ) b -(K 81 ) b -(K 82 ) b -(K 83 ) b -(K 84 ) b -(K 85 ) b -(K 86 ) b -(K 87 ) b -(K 88 ) b-K 89 -K 89 -K 89 -K 89 -K 89 (Formula XI) wherein each b is independently 0, 1, 2, or 3. Table 14 below lists the various amino acids that may be used at each position, with preferred amino acids underlined. [Table 14] TIFF2024511941000019.tif235151TIFF2024511941000020.tif238152TIFF2024511941000021.tif237145TIFF2024511941000022.tif125153

[0137] In some embodiments, amino acid position K 1 ~K 88 may be omitted or may be repeated up to two extra times (i.e., 0 to 3 times), with each repeat being independently selected from the amino acids shown. 1 ~K 88 It should be understood that the omission or repetition of is independent of the omission or repetition of any amino acid at another position.

[0138] In some embodiments, each K 1 is independently absent. In some embodiments, each K 1 is independently an amino acid selected from the group consisting of S, G, D, A, C, P, and Y. In some embodiments, each K 2 is independently absent. In some embodiments, each K 2 is independently an amino acid selected from the group consisting of Q, S, E, T, R, K, G, A, Y, M, V, and I. In some embodiments, each K 3 is independently absent. In some embodiments, each K 3is independently an amino acid selected from the group consisting of G, S, N, T, Q, D, P, L, F, V, K, A, and C. In some embodiments, each K 3 is independently G. In some embodiments, each K 4 is independently absent. In some embodiments, each K 4 is independently an amino acid selected from the group consisting of R, G, N, D, A, P, Y, and L. In some embodiments, each K 5 is independently absent. In some embodiments, each K 5 is independently an amino acid selected from the group consisting of E, A, V, Q, G, Y, M, I, and L. In some embodiments, each K 5 is independently an amino acid selected from the group consisting of E, A, and V. In some embodiments, each K 6 is independently absent. In some embodiments, each K 6 is independently an amino acid selected from the group consisting of S, Q, R, T, D, G, E, A, and K. In some embodiments, each K 6 is independently an amino acid selected from the group consisting of S, Q, R, T, and D. In some embodiments, each K 7 is independently absent. In some embodiments, each K 7 is independently an amino acid selected from the group consisting of N, Q, R, H, K, A, I, F, and L. In some embodiments, each K 8 is independently absent. In some embodiments, each K 8 is independently an amino acid selected from the group consisting of A, T, Q, G, R, K, D, L, F, C, V, S, and H. In some embodiments, each K 8 is independently A. In some embodiments, each K 9 is not independently present. In some embodiments, each K 9 is independently an amino acid selected from the group consisting of G, S, N, T, Q, D, P, L, F, V, K, A, and C. In some embodiments, each K 9 is independently G. In some embodiments, each K10 is independently absent. In some embodiments, each K 10 is independently an amino acid selected from the group consisting of K, H, E, A, Y, L, and F. In some embodiments, each K 11 is independently absent. In some embodiments, each K 11 is independently an amino acid selected from the group consisting of S, T, K, E, A, C, W, F, and L. In some embodiments, each K 12 is independently absent. In some embodiments, each K 12 is independently an amino acid selected from the group consisting of K, R, H, S, Q, D, E, and A. In some embodiments, each K 13 is independently absent. In some embodiments, each K 13 is independently an amino acid selected from the group consisting of G, S, T, E, P, W, R, N, and Q. In some embodiments, each K 13 is independently G. In some embodiments, each K 14 is independently absent. In some embodiments, each K 14 is independently an amino acid selected from the group consisting of D, Q, S, G, V, E, N, H, R, P, and F. In some embodiments, each K 14 is independently an amino acid selected from the group consisting of D, Q, S, G, and V. In some embodiments, each K 15 is independently absent. In some embodiments, each K 15 is independently an amino acid selected from the group consisting of C, A, M, V, S, E, G, I, F, and L. In some embodiments, each K 16 is independently absent. In some embodiments, each K 16 is independently an amino acid selected from the group consisting of R, K, S, Q, T, Y, N, V, I, L, and C. In some embodiments, each K 16 is independently an amino acid selected from the group consisting of R, K, S, Q, T, and Y. In some embodiments, each K 17is independently absent. In some embodiments, each K 17 is independently an amino acid selected from the group consisting of A, G, S, Q, Y, E, D, H, and I. In some embodiments, each K 18 is independently absent. In some embodiments, each K 18 is independently an amino acid selected from the group consisting of R, K, S, Q, T, Y, N, V, I, L, and C. In some embodiments, each K 18 is independently an amino acid selected from the group consisting of R, K, S, Q, T, and Y. In some embodiments, each K 19 is independently absent. In some embodiments, each K 19 is independently an amino acid selected from the group consisting of E, D, T, H, K, G, P, V, and L. In some embodiments, each K 20 is independently absent. In some embodiments, each K 20 is independently an amino acid selected from the group consisting of F, L, I, V, M, T, G, and R. In some embodiments, each K 21 is independently absent. In some embodiments, each K 21 is independently an amino acid selected from the group consisting of E, D, S, G, A, C, and P. In some embodiments, each K 22 is independently absent. In some embodiments, each K 22 is independently an amino acid selected from the group consisting of D, T, G, A, Y, N, S, C, P, W, and I. In some embodiments, each K 22 is independently an amino acid selected from the group consisting of D, T, G, A, and Y. In some embodiments, each K 23 is independently absent. In some embodiments, each K 23 is independently an amino acid selected from the group consisting of G, S, N, E, D, Y, and L. In some embodiments, each K 24 is independently absent. In some embodiments, each K 24is independently an amino acid selected from the group consisting of T, S, E, G, P, and I. In some embodiments, each K 25 is independently absent. In some embodiments, each K 25 is independently an amino acid selected from the group consisting of K, S, G, T, and L. In some embodiments, each K 26 is independently absent. In some embodiments, each K 26 is independently an amino acid selected from the group consisting of S, G, K, E, D, P, and F. In some embodiments, each K 27 is independently absent. In some embodiments, each K 27 is independently an amino acid selected from the group consisting of P, A, E, L, T, Q, S, G, K, Y, F, C, V, W, and R. In some embodiments, each K 27 is independently an amino acid selected from the group consisting of P and A. In some embodiments, each K 28 is not independently present. In some embodiments, each K 28 is independently an amino acid selected from the group consisting of E, D, Q, S, T, P, and L. In some embodiments, each K 29 is independently absent. In some embodiments, each K 29 is independently an amino acid selected from the group consisting of A, T, S, E, V, W, and I. In some embodiments, each K 30 is independently absent. In some embodiments, each K 30 is independently an amino acid selected from the group consisting of K, H, S, G, N, Q, P, and Y. In some embodiments, each K 31 is independently absent. In some embodiments, each K 31 is independently an amino acid selected from the group consisting of L, F, V, P, A, N, G, and H. In some embodiments, each K 32 is independently absent. In some embodiments, each K 32 is independently an amino acid selected from the group consisting of A, G, N, P, R, E, and K. In some embodiments, each K33 is independently absent. In some embodiments, each K 33 is independently an amino acid selected from the group consisting of R, S, N, A, P, Y, V, I, F, and G. In some embodiments, each K 33 is independently an amino acid selected from the group consisting of R and S. In some embodiments, each K 34 is independently absent. In some embodiments, each K 34 is independently an amino acid selected from the group consisting of E, S, T, V, I, H, A, P, F, and L. In some embodiments, each K 34 is independently an amino acid selected from the group consisting of E, S, T, V, and I. In some embodiments, each K 35 is independently absent. In some embodiments, each K 35 is independently an amino acid selected from the group consisting of A, T, Q, P, R, V, N, E, and L. In some embodiments, each K 35 is independently an amino acid selected from the group consisting of A, T, Q, P, and R. In some embodiments, each K 36 is independently absent. In some embodiments, each K 36 is independently an amino acid selected from the group consisting of R, K, H, G, Q, D, T, Y, and F. In some embodiments, each K 37 is independently absent. In some embodiments, each K 37 is independently an amino acid selected from the group consisting of D, E, N, T, C, Y, V, I, and L. In some embodiments, each K 38 is independently absent. In some embodiments, each K 38 is independently an amino acid selected from the group consisting of S, Q, R, T, D, G, E, A, and K. In some embodiments, each K 38 is independently an amino acid selected from the group consisting of S, Q, R, T, and D. In some embodiments, each K 39 is not independently present. In some embodiments, each K 39is independently an amino acid selected from the group consisting of K, S, G, Q, D, E, A, M, I, and L. In some embodiments, each K 40 is independently absent. In some embodiments, each K 40 is independently an amino acid selected from the group consisting of H, K, S, D, E, T, P, and L. In some embodiments, each K 41 is independently absent. In some embodiments, each K 41 is independently an amino acid selected from the group consisting of A, T, S, N, P, V, L, and F. In some embodiments, each K 42 is independently absent. In some embodiments, each K 42 is independently an amino acid selected from the group consisting of K, D, M, V, I, L, and F. In some embodiments, each K 43 is independently absent. In some embodiments, each K 43 are, independently, G, S, N, T, Q, D, P, L, F, V, K, A, and C. In some embodiments, each K 43 is independently G. In some embodiments, each K 44 is independently absent. In some embodiments, each K 44 is independently an amino acid selected from the group consisting of L, T, F, V, P, A, K, and I. In some embodiments, each K 44 is independently an amino acid selected from the group consisting of L and T. In some embodiments, each K 45 is independently absent. In some embodiments, each K 45 is independently an amino acid selected from the group consisting of G, S, K, N, T, Q, D, A, P, L, F, and V. In some embodiments, each K 45 is independently G. In some embodiments, each K 46 is independently absent. In some embodiments, each K 46 is independently an amino acid selected from the group consisting of L, F, Q, S, G, and D. In some embodiments, each K47 is independently absent. In some embodiments, each K 47 is independently an amino acid selected from the group consisting of S, R, E, A, P, V, W, and L. In some embodiments, each K 48 is independently absent. In some embodiments, each K 48 is independently an amino acid selected from the group consisting of A, S, V, G, Q, R, E, D, L, T, K, F, C, and H. In some embodiments, each K 48 is independently A. In some embodiments, each K 49 is independently absent. In some embodiments, each K 49 is independently an amino acid selected from the group consisting of E, S, T, R, G, A, P, and L. In some embodiments, each K 50 is independently absent. In some embodiments, each K 50 is independently an amino acid selected from the group consisting of S, N, R, A, P, and Y. In some embodiments, each K 51 is independently absent. In some embodiments, each K 51 is independently an amino acid selected from the group consisting of G, A, T, H, M, V, L, and F. In some embodiments, each K 52 is independently absent. In some embodiments, each K 52 is independently an amino acid selected from the group consisting of S, T, H, A, C, M, and L. In some embodiments, K 53 is independently absent. In some embodiments, each K 53 is independently an amino acid selected from the group consisting of G, S, T, E, P, W, R, N, and Q. In some embodiments, each K 53 is independently G. In some embodiments, each K 54 is independently absent. In some embodiments, each K 54 is independently an amino acid selected from the group consisting of S, H, Y, F, N, Q, R, T, G, and K. In some embodiments, K 54is independently S. In some embodiments, each K 55 is independently absent. In some embodiments, each K 55 is independently an amino acid selected from the group consisting of A, T, Q, E, M, V, I, L, and F. In some embodiments, each K 56 is independently absent. In some embodiments, each K 56 is independently an amino acid selected from the group consisting of S, N, E, A, P, F, and L. In some embodiments, each K 57 is independently absent. In some embodiments, each K 57 is independently an amino acid selected from the group consisting of D, S, R, K, A, V, W, I, and F. In some embodiments, each K 58 is independently absent. In some embodiments, each K 58 is independently an amino acid selected from the group consisting of K, S, G, D, T, L, R, E, Y, and N. In some embodiments, each K 58 is independently an amino acid selected from the group consisting of K, S, G, D, T, and L. In some embodiments, each K 59 is independently absent. In some embodiments, each K 59 is independently an amino acid selected from the group consisting of S, R, G, A, V, and F. In some embodiments, each K 60 is independently absent. In some embodiments, each K 60 is independently an amino acid selected from the group consisting of A, T, Q, G, R, K, D, L, F, C, V, S, and H. In some embodiments, each K 60 is independently A. In some embodiments, each K 61 is independently absent. In some embodiments, each K 61 is independently an amino acid selected from the group consisting of R, S, G, N, E, T, A, and V. In some embodiments, each K 62 is independently absent. In some embodiments, each K 62is independently an amino acid selected from the group consisting of E, S, T, V, I, H, A, P, F, and L. In some embodiments, each K 63 is independently absent. In some embodiments, each K 63 is independently an amino acid selected from the group consisting of A, G, S, Q, R, E, D, V, L, T, K, F, C, and H. In some embodiments, each K 63 is independently A. In some embodiments, each K 64 is independently absent. In some embodiments, each K 64 is independently an amino acid selected from the group consisting of E, A, V, Q, G, Y, M, I, and L. In some embodiments, each K 64 is independently an amino acid selected from the group consisting of E, A, and V. In some embodiments, each K 65 is independently absent. In some embodiments, each K 65 is independently an amino acid selected from the group consisting of G, S, T, E, P, W, R, N, and Q. In some embodiments, each K 65 is independently G. In some embodiments, each K 66 is independently absent. In some embodiments, each K 66 is independently an amino acid selected from the group consisting of A, G, P, M, N, V, and S. In some embodiments, each K 66 is independently an amino acid selected from the group consisting of A, G, P, and M. In some embodiments, each K 67 is independently absent. In some embodiments, each K 67 is independently an amino acid selected from the group consisting of T, Q, E, N, S, A, Y, V, W, and F. In some embodiments, each K 67 is independently an amino acid selected from the group consisting of T, Q, and E. In some embodiments, each K 68 is independently absent. In some embodiments, each K 68 is independently an amino acid selected from the group consisting of I, V, P, and A. In some embodiments, each K69 is independently absent. In some embodiments, each K 69 is independently an amino acid selected from the group consisting of D, Q, S, G, V, E, N, H, R, P, and F. In some embodiments, each K 69 is independently an amino acid selected from the group consisting of D, Q, S, G, and V. In some embodiments, each K 70 is independently absent. In some embodiments, each K 70 is independently an amino acid selected from the group consisting of G, S, R, N, T, Y, L, and F. In some embodiments, each K 71 is independently absent. In some embodiments, each K 71 is independently an amino acid selected from the group consisting of E, D, N, S, T, H, and Y. In some embodiments, each K 72 is independently absent. In some embodiments, each K 72 is independently an amino acid selected from the group consisting of L, I, W, V, A, T, S, E, R, and K. In some embodiments, each K 73 is independently absent. In some embodiments, each K 73 is independently an amino acid selected from the group consisting of G, S, K, A, C, F, N, T, Q, D, P, L, and V. In some embodiments, each K 73 is independently G. In some embodiments, each K 74 is independently absent. In some embodiments, each K 74 is independently an amino acid selected from the group consisting of A, S, N, P, K, V, I, and L. In some embodiments, each K 75 is independently absent. In some embodiments, each K 75 is independently an amino acid selected from the group consisting of P, A, E, L, T, Q, S, G, K, Y, F, C, V, W, and R. In some embodiments, each K 75 is independently an amino acid selected from the group consisting of P and A. In some embodiments, each K 76is independently absent. In some embodiments, each K 76 is independently an amino acid selected from the group consisting of L, T, F, V, P, A, K, and I. In some embodiments, each K 76 is independently an amino acid selected from the group consisting of L and T. In some embodiments, each K 77 is independently absent. In some embodiments, each K 77 is independently an amino acid selected from the group consisting of M, V, Y, L, A, N, E, and H. In some embodiments, each K 78 is independently absent. In some embodiments, each K 78 is independently an amino acid selected from the group consisting of D, T, G, A, Y, N, S, C, P, W, and I. In some embodiments, each K 78 is independently an amino acid selected from the group consisting of D, T, G, A, and Y. In some embodiments, each K 79 is independently absent. In some embodiments, each K 79 is independently an amino acid selected from the group consisting of A, S, V, G, Q, R, E, D, L, T, K, F, C, and H. In some embodiments, each K 79 is independently A. In some embodiments, each K 80 is independently absent. In some embodiments, each K 80 is independently an amino acid selected from the group consisting of K, R, S, A, P, V, I, and L. In some embodiments, each K 81 is independently absent. In some embodiments, each K 81 is independently an amino acid selected from the group consisting of F, L, V, A, T, S, E, D, R, and K. In some embodiments, each K 82 is independently absent. In some embodiments, each K 82 is independently an amino acid selected from the group consisting of L, F, M, A, N, G, and E. In some embodiments, each K 83 is independently absent. In some embodiments, each K 83is independently an amino acid selected from the group consisting of D, S, H, A, V, I, F, and L. In some embodiments, each K 84 is independently absent. In some embodiments, each K 84 is independently an amino acid selected from the group consisting of A, T, Q, S, R, V, L, G, H, F, K, D, and C. In some embodiments, each K 84 is independently A. In some embodiments, each K 85 is independently absent. In some embodiments, each K 85 is independently an amino acid selected from the group consisting of T, Q, E, N, S, A, Y, V, W, and F. In some embodiments, each K 85 is independently an amino acid selected from the group consisting of T, Q, and E. In some embodiments, each K 86 is independently absent. In some embodiments, each K 86 is independently an amino acid selected from the group consisting of A, P, R, Y, K, D, M, L, and F. In some embodiments, each K 87 is independently absent. In some embodiments, each K 87 is independently an amino acid selected from the group consisting of N, S, D, T, A, P, and L. In some embodiments, each K 88 is independently absent. In some embodiments, each K 88 is independently an amino acid selected from the group consisting of R, S, N, A, P, Y, V, I, F, and G. In some embodiments, each K 88 is independently an amino acid selected from the group consisting of R and S. In some embodiments, K 89 is an amino acid selected from the group consisting of K, R, H, G, E, T, Y, and I. In some embodiments, K 90 is an amino acid selected from the group consisting of R, S, G, N, Q, A, Y, and W. In some embodiments, K 90 is R. In some embodiments, K 91is an amino acid selected from the group consisting of V, I, and F. In some embodiments, K 92 is an amino acid selected from the group consisting of A, G, P, M, N, V, and S. In some embodiments, K 92 is an amino acid selected from the group consisting of A, G, P, and M. In some embodiments, K 93 is an amino acid selected from the group consisting of E, D, Q, S, R, K, M, and L.

[0139] Variant of SEQ ID NO: 74 (Formula XIV) In some embodiments, the proprotein signal peptide comprises an amino acid sequence represented by: (M 1 ) b -(M 2 ) b -(M 3 ) b -(M 4 ) b -(M 5 ) b -(M 6 ) b -(M 7 ) b -(M 8 ) b -(M 9 ) b -(M 10 ) b -(M 11 ) b -(M 12 ) b -(M 13 ) b -(M 14 ) b -(M 15 ) b -(M 16 ) b -(M 17 ) b -(M 18 ) b -(M 19 ) b -(M 20 ) b -(M 21 ) b -(M 22 )b -(M 23 ) b -(M 24 ) b -(M 25 ) b -(M 26 ) b -(M 27 ) b -(M 28 ) b -(M 29 ) b -(M 30 ) b -(M 31 ) b -(M 32 ) b -(M 33 ) b -(M 34 ) b -(M 35 ) b -(M 36 ) b -(M 37 ) b -(M 38 ) b -(M 39 ) b -(M 40 ) b -(M 41 ) b -(M 42 ) b -(M 43 ) b -(M 44 ) b -(M 45 ) b -(M 46 ) b -(M 47 ) b -(M 48 ) b -(M 49 ) b -(M 50 ) b -(M 51 ) b -(M 52 ) b -(M 53 ) b -(M 54 ) b -(M 55 ) b-(M 56 ) b -(M 57 ) b -(M 58 ) b -(M 59 ) b -(M 60 ) b -(M 61 ) b -(M 62 ) b -(M 63 ) b -(M 64 ) b -(M 65 ) b -(M 66 ) b -(M 67 ) c -(M 68 ) c -(M 69 ) c -(M 70 ) c (Formula XIV) wherein each b is independently 0, 1, 2, or 3, and each c is independently 1 or 2. Table 15 below lists the various amino acids that may be used at each position, with preferred amino acids underlined. [Table 15] TIFF2024511941000024.tif224162TIFF2024511941000025.tif228161TIFF20245119410 00026.tif241162TIFF2024511941000027.tif241161TIFF2024511941000028.tif218160

[0140] In some embodiments, amino acid position M 1 ~M 66 may be omitted or may be repeated up to two extra times (i.e., included 0-3 times), with each repeat being independently selected from the amino acids shown. 1 ~M 66It should be understood that the omission or repetition of is independent of the omission or repetition of any amino acid at another position. 67 ~M 70 may be repeated up to one additional time (i.e., 1-2 times), with each repeat being independently selected from the amino acids shown. 67 ~M 70 It should be understood that the repetition of is independent of the repetition of any amino acid at another position.

[0141] In some embodiments, each M 1 is independently absent. In some embodiments, each M 1 is independently an amino acid selected from the group consisting of A, T, C, S, Y, E, H, V, W, I, L, F, G, Q, N, P, R, K, D, and M. In some embodiments, each M 1 is independently A. In some embodiments, each M 2 is independently absent. In some embodiments, each M 2 is independently an amino acid selected from the group consisting of S, T, A, N, R, G, E, P, V, F, L, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M 2 is independently S. In some embodiments, each M 3 is independently absent. In some embodiments, each M 3 is independently an amino acid selected from the group consisting of G, S, R, A, T, Q, E, D, C, Y, I, L, and N. In some embodiments, each M 3 is independently G. In some embodiments, each M 4 is independently absent. In some embodiments, each M 4 is independently an amino acid selected from the group consisting of R, H, N, Q, E, A, Y, M, V, W, F, and L. In some embodiments, each M 4 is independently R. In some embodiments, each M 5is independently absent. In some embodiments, each M 5 is independently an amino acid selected from the group consisting of P, Y, A, T, Q, S, G, D, R, K, C, V, I, L, and H. In some embodiments, each M 5 is independently P. In some embodiments, each M 6 is independently absent. In some embodiments, each M 6 is independently an amino acid selected from the group consisting of T, Q, N, S, A, E, G, D, H, P, F, L, C, K, V, R, Y, I, M, and W. In some embodiments, each M 6 is independently T. In some embodiments, each M 7 is independently absent. In some embodiments, each M 7 is independently an amino acid selected from the group consisting of A, G, S, Q, N, K, D, T, C, Y, E, H, V, W, I, L, F, P, R, and M. In some embodiments, each M 7 is independently A. In some embodiments, each M 8 is independently absent. In some embodiments, each M 8 is independently an amino acid selected from the group consisting of T, Q, N, S, A, G, C, R, K, P, Y, M, V, I, L, F, E, W, D, and H. In some embodiments, each M 8 is independently T. In some embodiments, each M 9 is independently absent. In some embodiments, each M 9 is independently an amino acid selected from the group consisting of G, S, H, P, R, A, T, Q, E, D, C, Y, V, I, L, N, W, F, K, and M. In some embodiments, each M 9 is independently G. In some embodiments, each M 10 is independently absent. In some embodiments, each M 10 is independently an amino acid selected from the group consisting of Q, E, and W. In some embodiments, each M 11 is independently absent. In some embodiments, each M 11is independently an amino acid selected from the group consisting of V, I, L, F, C, A, and T. In some embodiments, each M 11 is independently an amino acid selected from the group consisting of V, I, and L. In some embodiments, each M 12 is independently absent. In some embodiments, each M 12 is independently an amino acid selected from the group consisting of S, G, A, N, Q, R, T, K, E, H, D, P, I, F, V, C, Y, L, M, and W. In some embodiments, each M 12 is independently S. In some embodiments, each M 13 is independently absent. In some embodiments, each M 13 is independently an amino acid selected from the group consisting of T, Q, N, S, D, P, F, A, E, G, H, L, C, K, V, R, Y, I, M, and W. In some embodiments, each M 13 is independently T. In some embodiments, each M 14 is independently absent. In some embodiments, each M 14 is independently an amino acid selected from the group consisting of L, F, I, V, M, Y, A, T, Q, N, S, D, K, P, E, R, H, G, and C. In some embodiments, each M 14 is independently L. In some embodiments, each M 15 is independently absent. In some embodiments, each M 15 is independently an amino acid selected from the group consisting of S, P, V, E, T, A, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M 15 is independently S. In some embodiments, each M 16 is independently absent. In some embodiments, each M 16 is independently an amino acid selected from the group consisting of T, S, A, E, G, C, R, P, Y, M, V, W, I, F, L, Q, N, D, H, and K. In some embodiments, each M 16 is independently T. In some embodiments, each M 17is independently absent. In some embodiments, each M 17 is independently an amino acid selected from the group consisting of D, E, Q, T, K, P, F, N, S, G, A, Y, R, and V. In some embodiments, each M 17 is independently D. In some embodiments, each M 18 is independently absent. In some embodiments, each M 18 is independently an amino acid selected from the group consisting of G, S, H, P, R, D, N, A, T, Q, E, C, Y, V, I, L, W, F, K, and M. In some embodiments, each M 18 is independently G. In some embodiments, each M 19 is independently absent. In some embodiments, each M 19 is independently an amino acid selected from the group consisting of T, P, F, S, A, E, G, C, R, Y, M, V, W, I, L, Q, N, D, H, and K. In some embodiments, each M 19 is independently T. In some embodiments, each M 20 is independently absent. In some embodiments, each M 20 is independently an amino acid selected from the group consisting of L, F, I, V, Y, A, T, Q, S, D, M, N, K, P, E, R, H, G, and C. In some embodiments, each M 20 is independently L. In some embodiments, each M 21 is independently absent. In some embodiments, each M 21 is independently an amino acid selected from the group consisting of F, L, W, Y, and P. In some embodiments, each M 21 is independently F. In some embodiments, each M 22 is independently absent. In some embodiments, each M 22 is independently an amino acid selected from the group consisting of P, K, Y, A, T, Q, S, G, D, R, C, V, I, L, and H. In some embodiments, each M 22 is independently P. In some embodiments, each M 23is independently absent. In some embodiments, each M 23 is independently an amino acid selected from the group consisting of T, P, F, S, A, E, G, C, R, Y, M, V, W, I, L, Q, N, D, H, and K. In some embodiments, each M 23 is independently T. In some embodiments, each M 24 is independently absent. In some embodiments, each M 24 is independently an amino acid selected from the group consisting of S, T, A, N, R, G, E, P, V, F, L, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M 24 is independently S. In some embodiments, each M 25 is independently absent. In some embodiments, each M 25 is independently an amino acid selected from the group consisting of F, W, Y, and P. In some embodiments, each M 25 is independently F. In some embodiments, each M 26 is independently absent. In some embodiments, each M 26 is independently an amino acid selected from the group consisting of T, P, F, Q, N, S, A, E, G, D, K, Y, C, V, I, L, and H. In some embodiments, each M 26 is independently T. In some embodiments, each M 27 is independently absent. In some embodiments, each M 27 is independently an amino acid selected from the group consisting of D, E, Q, N, S, T, R, K, G, A, Y, P, V, and F. In some embodiments, each M 27 is independently D. In some embodiments, each M 28 is independently absent. In some embodiments, each M 28 is independently an amino acid selected from the group consisting of T, Q, N, S, A, G, C, R, K, P, Y, M, V, I, L, F, E, W, D, and H. In some embodiments, each M 28 is independently T. In some embodiments, each M 29is independently absent. In some embodiments, each M 29 is independently an amino acid selected from the group consisting of S, T, E, A, P, V, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M 29 is independently S. In some embodiments, each M 30 is independently absent. In some embodiments, each M 30 is independently an amino acid selected from the group consisting of D, Q, N, H, K, G, C, and Y. In some embodiments, each M 31 is independently absent. In some embodiments, each M 31 is independently an amino acid selected from the group consisting of F, L, W, Y, and P. In some embodiments, each M 31 is independently F. In some embodiments, each M 32 is independently absent. In some embodiments, each M 32 is independently an amino acid selected from the group consisting of S, T, E, A, P, V, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M 32 is independently S. In some embodiments, each M 33 is independently absent. In some embodiments, each M 33 is independently an amino acid selected from the group consisting of A, G, S, Q, N, K, D, T, C, Y, E, H, V, W, I, L, F, P, R, and M. In some embodiments, each M 33 is independently A. In some embodiments, each M 34 is independently absent. In some embodiments, each M 34 is independently an amino acid selected from the group consisting of T, A, V, I, P, F, Q, N, S, E, G, D, K, Y, C, L, and H. In some embodiments, each M 34 is independently T. In some embodiments, each M 35 is independently absent. In some embodiments, each M 35are independently selected from the group consisting of G, S, R, N, H, D, P, A, T, Q, E, C, Y, V, I, L, W, F, K, and M. In some embodiments, each M 35 is independently G. In some embodiments, each M 36 is independently absent. In some embodiments, each M 36 is independently an amino acid selected from the group consisting of T, Q, S, A, E, D, K, H, P, Y, V, W, I, F, L, N, G, and C. In some embodiments, each M 36 is independently T. In some embodiments, each M 37 is independently absent. In some embodiments, each M 37 is independently an amino acid selected from the group consisting of I, L, W, V, and M. In some embodiments, each M 37 is independently I. In some embodiments, each M 38 is independently absent. In some embodiments, each M 38 is independently an amino acid selected from the group consisting of A, G, S, Q, N, K, D, C, P, R, Y, E, V, W, T, H, M, and F. In some embodiments, each M 38 is independently A. In some embodiments, each M 39 is independently absent. In some embodiments, each M 39 is independently an amino acid selected from the group consisting of S, T, E, P, V, A, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M 39 is independently S. In some embodiments, each M 40 is independently absent. In some embodiments, each M 40 is independently an amino acid selected from the group consisting of T, S, A, D, P, M, Q, E, K, H, Y, V, W, I, F, L, N, G, and C. In some embodiments, each M 40 is independently T. In some embodiments, each M 41is independently absent. In some embodiments, each M 41 is independently an amino acid selected from the group consisting of L, F, I, V, Y, A, T, Q, S, D, M, N, K, P, E, R, H, G, and C. In some embodiments, each M 41 is independently L. In some embodiments, each M 42 is independently absent. In some embodiments, each M 42 is independently an amino acid selected from the group consisting of P, Y, A, T, Q, S, N, W, G, I, E, D, L, K, and H. In some embodiments, each M 43 is independently absent. In some embodiments, each M 43 is independently an amino acid selected from the group consisting of S, E, P, V, T, A, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M 43 is independently S. In some embodiments, each M 44 is independently absent. In some embodiments, each M 44 is independently an amino acid selected from the group consisting of N, Q, S, E, D, T, H, K, G, A, P, W, and F. In some embodiments, each M 45 is independently absent. In some embodiments, each M 45 is independently an amino acid selected from the group consisting of V, I, L, F, C, A, and T. In some embodiments, each M 45 is independently an amino acid selected from the group consisting of V, I, and L. In some embodiments, each M 46 is independently absent. In some embodiments, each M 46 is independently an amino acid selected from the group consisting of A, T, S, N, R, Y, K, D, H, M, L, F, G, Q, C, P, E, V, and W. In some embodiments, each M 46 is independently A. In some embodiments, each M 47 is independently absent. In some embodiments, each M 47is independently an amino acid selected from the group consisting of I, L, and V. In some embodiments, each M 47 is independently I. In some embodiments, each M 48 is independently absent. In some embodiments, each M 48 is independently an amino acid selected from the group consisting of S, P, V, E, T, A, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M 48 is independently S. In some embodiments, each M 49 is independently absent. In some embodiments, each M 49 is independently an amino acid selected from the group consisting of F, V, A, T, Q, N, S, E, G, D, and H. In some embodiments, each M 50 is independently absent. In some embodiments, each M 50 is independently an amino acid selected from the group consisting of L, F, I, V, Y, A, T, Q, S, D, M, N, K, P, E, R, H, G, and C. In some embodiments, each M 50 is independently L. In some embodiments, each M 51 is independently absent. In some embodiments, each M 51 is independently an amino acid selected from the group consisting of G, S, R, H, D, P, N, A, T, Q, E, C, Y, V, I, L, W, F, K, and M. In some embodiments, each M 51 is independently G. In some embodiments, each M 52 is independently absent. In some embodiments, each M 52 is independently an amino acid selected from the group consisting of T, N, S, G, C, R, H, A, D, P, M, Q, E, K, Y, V, W, I, F, and L. In some embodiments, each M 52 is independently T. In some embodiments, each M 53 is independently absent. In some embodiments, each M 53is independently an amino acid selected from the group consisting of I, L, W, V, and M. In some embodiments, each M 53 is independently I. In some embodiments, each M 54 is independently absent. In some embodiments, each M 54 is independently an amino acid selected from the group consisting of P, K, Y, A, T, Q, S, G, D, R, C, V, I, L, and H. In some embodiments, each M 54 is independently P. In some embodiments, each M 55 is independently absent. In some embodiments, each M 55 is independently an amino acid selected from the group consisting of D, E, Q, N, S, K, G, A, Y, P, F, T, R, and V. In some embodiments, each M 55 is independently D. In some embodiments, each M 56 is independently absent. In some embodiments, each M 56 is independently an amino acid selected from the group consisting of L, F, I, V, Y, P, A, T, Q, N, S, G, E, D, K, H, M, C, and R. In some embodiments, each M 56 is independently L. In some embodiments, each M 57 is independently absent. In some embodiments, each M 57 is independently an amino acid selected from the group consisting of S, P, V, E, T, A, F, L, N, R, G, Q, K, H, D, I, C, Y, M, and W. In some embodiments, each M 57 is independently S. In some embodiments, each M 58 is independently absent. In some embodiments, each M 58 is independently an amino acid selected from the group consisting of P, M, V, I, L, and F. In some embodiments, each M 59 is independently absent. In some embodiments, each M 59 is independently an amino acid selected from the group consisting of N, Q, S, E, D, T, R, K, G, A, and Y. In some embodiments, each M 60is independently absent. In some embodiments, each M 60 is independently an amino acid selected from the group consisting of G, S, H, P, R, D, N, A, T, Q, E, C, Y, V, I, L, W, F, K, and M. In some embodiments, each M 60 is independently G. In some embodiments, each M 61 is independently absent. In some embodiments, each M 61 is independently an amino acid selected from the group consisting of S, P, V, T, A, R, K, E, H, C, Y, I, F, L, N, Q, G, D, M, and W. In some embodiments, each M 61 is independently S. In some embodiments, each M 62 is independently absent. In some embodiments, each M 62 is independently an amino acid selected from the group consisting of P, K, A, Y, T, Q, S, G, D, R, C, V, I, L, and H. In some embodiments, each M 62 is independently P. In some embodiments, each M 63 is independently absent. In some embodiments, each M 63 is independently an amino acid selected from the group consisting of A, G, S, N, E, K, D, H, M, V, W, I, L, F, T, R, Y, Q, C, and P. In some embodiments, each M 63 is independently A. In some embodiments, each M 64 is independently absent. In some embodiments, each M 64 is independently an amino acid selected from the group consisting of D, E, Q, T, K, P, F, N, S, G, A, Y, R, and V. In some embodiments, each M 64 is independently D. In some embodiments, each M 65 is independently absent. In some embodiments, each M 65 is independently an amino acid selected from the group consisting of L, V, F, I, Y, P, A, T, Q, N, S, G, E, D, K, H, M, C, and R. In some embodiments, each M 65is independently L. In some embodiments, each M 66 is independently absent. In some embodiments, each M 66 is independently an amino acid selected from the group consisting of S, N, R, T, G, K, E, H, D, A, P, V, C, Y, I, F, L, Q, M, and W. In some embodiments, each M 66 is independently S. In some embodiments, each M 67 is independently an amino acid selected from the group consisting of K, R, H, S, G, N, Q, D, E, T, A, C, P, Y, M, V, W, I, L, and F. In some embodiments, each M 67 is independently an amino acid selected from the group consisting of K, R, H, and S. In some embodiments, each M 68 is independently an amino acid selected from the group consisting of R, K, H, S, G, N, Q, D, E, T, A, C, P, Y, M, V, W, I, L, and F. In some embodiments, each M 68 is independently an amino acid selected from the group consisting of R, K, H, and S. In some embodiments, each M 69 is independently an amino acid selected from the group consisting of S, A, N, Q, R, T, G, K, E, H, D, A, C, P, Y, M, V, W, I, F, and L. In some embodiments, each M 69 is independently an amino acid selected from the group consisting of S, A, N, Q, R, and T. In some embodiments, each M 70 is independently an amino acid selected from the group consisting of T, Q, N, S, A, E, G, D, C, R, K, H, P, Y, M, V, W, I, F, and L. In some embodiments, each M 70 is independently an amino acid selected from the group consisting of T, Q, N, S, A, and E.

[0142] In some embodiments, the proprotein signal peptide comprises the amino acid sequence of SEQ ID NO:74.

[0143] Variant of SEQ ID NO: 75 (Formula XV) In some embodiments, the proprotein signal peptide comprises an amino acid sequence represented by: (N 1 ) b -(N 2 ) b -(N 3 ) b -(N 4 ) b -(N 5 ) b -(N 6 ) b -(N 7 ) b -(N 8 ) b -(N 9 ) b -(N 10 ) b -(N 11 ) b -(N 12 ) b -(N 13 ) b -(N 14 ) b -(N 15 ) b -(N 16 ) b -(N 17 ) b -(N 18 ) b -(N 19 ) b -(N 20 ) b -(N 21 ) b -(N 22 ) b -(N 23 ) b -(N 24 ) b -(N 25 ) b -(N 26 ) b -(N 27 ) b -(N 28 ) b -(N 29 ) b -(N 30 ) b -(N 31 ) b -(N32 ) b -(N 33 ) b -(N 34 ) b -(N 35 ) b -(N 36 ) b -(N 37 ) b -(N 38 ) b -(N 39 ) b -(N 40 ) b -(N 41 ) b -(N 42 ) b -(N 43 ) b -(N 44 ) b -(N 45 ) b -(N 46 ) b -(N 47 ) b -(N 48 ) b -(N 49 ) b -(N 50 ) b -(N 51 ) b -(N 52 ) b -(N 53 ) b -(N 54 ) b -(N 55 ) b -(N 56 ) b -(N 57 ) b -(N 58 ) b -(N 59 ) b -(N 60 ) b -(N 61 ) b -(N 62 ) b -(N 63 ) b -(N 64 ) b -(N 65) b -(N 66 ) b -(N 67 ) c -(N 68 ) c -(N 69 ) c -(N 70 ) c -(N 71 ) c (Formula XV) wherein each b is independently 0, 1, 2, or 3, and each c is independently 1 or 2. Table 16 below lists the various amino acids that may be used at each position, with preferred amino acids underlined. [Table 16] TIFF2024511941000030.tif240160TIFF2024511941000031.tif223163TIFF20245119410 00032.tif237162TIFF2024511941000033.tif231163TIFF2024511941000034.tif147161

[0144] In some embodiments, amino acid position N 1 ~N 66 may be omitted or may be repeated up to two extra times (i.e., included 0-3 times), with each repeat being independently selected from the amino acids shown. 1 ~N 66 It should be understood that the omission or repetition of is independent of the omission or repetition of any amino acid at another position. 67 ~N 71 may be repeated up to one extra time (i.e., 1-2 times), with each repeat being independently selected from the amino acids shown. 67 ~N 71 It should be understood that the repetition of is independent of the repetition of any amino acid at another position.

[0145] In some embodiments, each N 1 is independently absent. In some embodiments, each N 1 is independently an amino acid selected from the group consisting of S, N, D, Q, R, T, G, E, H, A, P, M, V, K, Y, W, F, L, I, and C. In some embodiments, each N 1 is independently S. In some embodiments, each N 2 is independently absent. In some embodiments, each N 2 is independently an amino acid selected from the group consisting of P, A, S, Y, V, T, G, I, E, and C. In some embodiments, each N 2 is independently P. In some embodiments, each N 3 is independently absent. In some embodiments, each N 3 is independently an amino acid selected from the group consisting of T, S, G, D, C, A, L, N, R, P, Y, V, W, I, and F. In some embodiments, each N 3 is independently T. In some embodiments, each N 4 is independently absent. In some embodiments, each N 4 is independently an amino acid selected from the group consisting of S, R, E, A, Q, K, N, D, T, G, H, C, P, Y, I, F, L, M, V, and W. In some embodiments, each N 4 is independently S. In some embodiments, each N 5 is independently absent. In some embodiments, each N 5 is independently an amino acid selected from the group consisting of T, Q, N, G, C, M, S, A, E, D, Y, V, I, F, L, and W. In some embodiments, each N 5 is independently T. In some embodiments, each N 6 is independently absent. In some embodiments, each N 6 is independently an amino acid selected from the group consisting of I, V, L, F, W, Y, A, T, S, E, D, and H. In some embodiments, each N 6is independently an amino acid selected from the group consisting of I and V. In some embodiments, each N 7 is independently absent. In some embodiments, each N 7 is independently an amino acid selected from the group consisting of P, V, A, S, N, G, E, L, and K. In some embodiments, each N 8 is independently absent. In some embodiments, each N 8 is independently an amino acid selected from the group consisting of A, G, Q, T, S, N, P, R, D, V, K, C, Y, W, I, L, and F. In some embodiments, each N 8 is independently an amino acid selected from the group consisting of A, G, and Q. In some embodiments, each N 9 is independently absent. In some embodiments, each N 9 is independently an amino acid selected from the group consisting of F, Y, A, T, N, and R. In some embodiments, each N 9 is independently an amino acid selected from the group consisting of F and Y. In some embodiments, each N 10 is independently absent. In some embodiments, each N 10 is independently an amino acid selected from the group consisting of T, Q, N, R, K, M, S, E, D, H, P, V, W, I, F, and L. In some embodiments, each N 10 is independently T. In some embodiments, each N 11 is independently absent. In some embodiments, each N 11 is independently an amino acid selected from the group consisting of A, G, Q, T, S, N, P, R, D, V, K, C, Y, W, I, L, and F. In some embodiments, each N 11 is independently an amino acid selected from the group consisting of A, G, and Q. In some embodiments, each N 12 is independently absent. In some embodiments, each N 12is independently an amino acid selected from the group consisting of S, N, Q, R, T, G, K, E, H, D, A, P, L, M, V, Y, W, F, I, and C. In some embodiments, each N 12 is independently S. In some embodiments, each N 13 is independently absent. In some embodiments, each N 13 is independently an amino acid selected from the group consisting of L, F, I, W, V, M, Y, C, A, T, Q, N, S, G, E, D, and R. In some embodiments, each N 14 is independently absent. In some embodiments, each N 14 is independently an amino acid selected from the group consisting of V, I, L, A, T, S, G, R, P, Y, N, H, C, M, F, Q, E, K, and D. In some embodiments, each N 14 is independently V. In some embodiments, each N 15 is independently absent. In some embodiments, each N 15 is independently an amino acid selected from the group consisting of S, N, Q, T, G, K, E, H, D, A, C, P, Y, I, F, L, R, M, V, and W. In some embodiments, each N 15 is independently S. In some embodiments, each N 16 is independently absent. In some embodiments, each N 16 is independently an amino acid selected from the group consisting of T, N, S, A, D, R, P, Y, V, W, I, F, and L. In some embodiments, each N 16 is independently T. In some embodiments, each N 17 is independently absent. In some embodiments, each N 17 is independently an amino acid selected from the group consisting of S, N, Q, R, K, E, D, A, T, G, H, C, P, Y, I, F, L, M, V, and W. In some embodiments, each N 17 is independently S. In some embodiments, each N 18 is independently absent. In some embodiments, each N 18is independently an amino acid selected from the group consisting of V, A, T, S, G, R, W, I, C, L, F, E, D, K, P, Y, N, H, M, and Q. In some embodiments, each N 18 is independently V. In some embodiments, each N 19 is independently absent. In some embodiments, each N 19 is independently an amino acid selected from the group consisting of T, Q, N, S, A, E, G, D, Y, M, V, I, F, L, and W. In some embodiments, each N 19 is independently T. In some embodiments, each N 20 is independently absent. In some embodiments, each N 20 is independently an amino acid selected from the group consisting of S, Q, R, K, E, A, N, D, T, G, H, C, P, Y, I, F, L, M, V, and W. In some embodiments, each N 20 is independently S. In some embodiments, each N 21 is independently absent. In some embodiments, each N 21 is independently an amino acid selected from the group consisting of V, W, I, C, L, F, A, T, S, E, D, K, G, R, P, Y, N, H, M, and Q. In some embodiments, each N 21 is independently V. In some embodiments, each N 22 is independently absent. In some embodiments, each N 22 is independently an amino acid selected from the group consisting of T, Q, N, S, A, D, C, K, P, Y, M, V, W, I, F, G, E, H, R, and L. In some embodiments, each N 22 is independently T. In some embodiments, each N 23 is independently absent. In some embodiments, each N 23 is independently an amino acid selected from the group consisting of L, F, I, V, P, A, T, Q, S, G, R, K, H, M, Y, and D. In some embodiments, each N 23is independently an amino acid selected from the group consisting of L, F, I, V, P, A, T, Q, S, G, R, K, and H. In some embodiments, each N 24 is independently absent. In some embodiments, each N 24 is independently an amino acid selected from the group consisting of T, Q, S, A, G, P, Y, I, K, H, V, F, L, N, D, C, M, W, E, and R. In some embodiments, each N 24 is independently T. In some embodiments, each N 25 is independently absent. In some embodiments, each N 25 is independently an amino acid selected from the group consisting of S, R, E, A, Q, K, N, D, T, G, H, C, P, Y, I, F, L, M, V, and W. In some embodiments, each N 25 is independently S. In some embodiments, each N 26 is independently absent. In some embodiments, each N 26 is independently an amino acid selected from the group consisting of T, N, D, S, A, R, P, Y, V, W, I, F, and L. In some embodiments, each N 26 is independently T. In some embodiments, each N 27 is independently absent. In some embodiments, each N 27 is independently an amino acid selected from the group consisting of D, N, R, E, Q, S, H, T, K, G, W, I, P, and Y. In some embodiments, each N 27 is independently an amino acid selected from the group consisting of D and N. In some embodiments, each N 28 is independently absent. In some embodiments, each N 28 is independently an amino acid selected from the group consisting of V, A, T, S, G, R, W, I, C, L, F, E, D, K, P, Y, N, H, M, and Q. In some embodiments, each N 28 is independently V. In some embodiments, each N 29 is independently absent. In some embodiments, each N 29is independently an amino acid selected from the group consisting of T, S, A, D, C, L, N, R, P, Y, V, W, I, and F. In some embodiments, each N 29 is independently T. In some embodiments, each N 30 is independently absent. In some embodiments, each N 30 is independently an amino acid selected from the group consisting of P, Y, V, A, T, S, G, I, E, and C. In some embodiments, each N 30 is independently P. In some embodiments, each N 31 is independently absent. In some embodiments, each N 31 is independently an amino acid selected from the group consisting of T, Q, S, A, G, K, H, P, Y, V, I, F, L, N, D, C, M, W, E, and R. In some embodiments, each N 31 is independently T. In some embodiments, each N 32 is independently absent. In some embodiments, each N 32 is independently an amino acid selected from the group consisting of S, R, E, A, Q, K, N, D, T, G, H, C, P, Y, I, F, L, M, V, and W. In some embodiments, each N 32 is independently S. In some embodiments, each N 33 is independently absent. In some embodiments, each N 33 is independently an amino acid selected from the group consisting of E, D, Q, N, S, T, H, R, G, A, P, F, and L. In some embodiments, each N 34 is independently absent. In some embodiments, each N 34 is independently an amino acid selected from the group consisting of D, N, R, E, Q, S, H, T, K, G, W, I, P, and Y. In some embodiments, each N 34 are independently D and N In some embodiments, each N 35 is independently absent. In some embodiments, each N 35is independently an amino acid selected from the group consisting of T, Q, S, A, G, P, Y, I, K, H, V, F, L, N, D, C, M, W, E, and R. In some embodiments, each N 35 is independently T. In some embodiments, each N 36 is independently absent. In some embodiments, each N 36 is independently an amino acid selected from the group consisting of G, S, K, A, T, Q, D, C, P, Y, V, W, I, L, and F. In some embodiments, each N 37 is independently absent. In some embodiments, each N 37 is independently an amino acid selected from the group consisting of F, Y, A, T, N, and R. In some embodiments, each N 37 is independently an amino acid selected from the group consisting of F and Y. In some embodiments, each N 38 is independently absent. In some embodiments, each N 38 is independently an amino acid selected from the group consisting of V, A, T, S, G, R, W, I, C, L, F, E, D, K, P, Y, N, H, M, and Q. In some embodiments, each N 38 is independently V. In some embodiments, each N 39 is independently absent. In some embodiments, each N 39 is independently an amino acid selected from the group consisting of L, F, I, W, V, M, C, A, T, Q, N, S, G, D, R, K, and H. In some embodiments, each N 40 is independently absent. In some embodiments, each N 40 is independently an amino acid selected from the group consisting of P, A, S, Y, V, T, G, I, E, and C. In some embodiments, each N 40 is independently P. In some embodiments, each N 41 is independently absent. In some embodiments, each N 41 is independently an amino acid selected from the group consisting of D, N, R, G, Y, E, Q, S, H, T, K, W, and I. In some embodiments, each N 41is independently an amino acid selected from the group consisting of D and N. In some embodiments, each N 42 is independently absent. In some embodiments, each N 42 is independently an amino acid selected from the group consisting of S, R, E, A, N, T, G, P, V, Q, K, H, D, Y, M, I, F, L, C, and W. In some embodiments, each N 42 is independently S. In some embodiments, each N 43 is independently absent. In some embodiments, each N 43 is independently an amino acid selected from the group consisting of G, S, R, K, A, N, Q, H, E, D, P, W, L, and F. In some embodiments, each N 44 is independently absent. In some embodiments, each N 44 is independently an amino acid selected from the group consisting of T, Q, S, A, G, P, Y, I, N, E, D, C, K, H, R, V, L, M, F, and W. In some embodiments, each N 44 is independently T. In some embodiments, each N 45 is independently absent. In some embodiments, each N 45 is independently an amino acid selected from the group consisting of S, T, G, A, V, I, R, E, N, P, Q, K, H, D, Y, M, F, L, C, and W. In some embodiments, each N 45 is independently S. In some embodiments, each N 46 is independently absent. In some embodiments, each N 46 is independently C. In some embodiments, each N 47 is independently absent. In some embodiments, each N 47 is independently an amino acid selected from the group consisting of S, N, R, T, G, K, E, H, D, A, P, Y, V, W, I, L, Q, M, F, and C. In some embodiments, each N 47 is independently S. In some embodiments, each N 48 is independently absent. In some embodiments, each N 48is independently an amino acid selected from the group consisting of G, S, R, K, N, T, Q, H, E, D, P, I, and L. In some embodiments, each N 49 is independently absent. In some embodiments, each N 49 is independently an amino acid selected from the group consisting of T, S, G, D, C, A, L, N, R, P, Y, V, W, I, and F. In some embodiments, each N 49 is independently T. In some embodiments, each N 50 is independently absent. In some embodiments, each N 50 is independently an amino acid selected from the group consisting of V, A, T, S, G, I, R, P, Y, L, N, H, C, M, F, Q, E, and K. In some embodiments, each N 50 is independently V. In some embodiments, each N 51 is independently absent. In some embodiments, each N 51 is independently an amino acid selected from the group consisting of A, T, G, S, Q, N, R, Y, E, H, M, V, W, I, L, and F. In some embodiments, each N 52 is independently absent. In some embodiments, each N 52 is independently an amino acid selected from the group consisting of D, E, Q, N, S, T, K, A, Y, P, M, W, I, F, and L. In some embodiments, each N 53 is independently absent. In some embodiments, each N 53 is independently an amino acid selected from the group consisting of A, T, C, G, S, N, P, R, K, D, H, M, and F. In some embodiments, each N 54 is independently absent. In some embodiments, each N 54 is independently an amino acid selected from the group consisting of L, F, I, V, P, A, T, Q, S, G, R, K, H, M, Y, and D. In some embodiments, each N 54 is independently an amino acid selected from the group consisting of L, F, I, V, P, A, T, Q, S, G, R, K, and H. In some embodiments, each N 55is independently absent. In some embodiments, each N 55 is independently an amino acid selected from the group consisting of E, D, N, T, R, K, G, A, and V. In some embodiments, each N 56 is independently absent. In some embodiments, each N 56 is independently an amino acid selected from the group consisting of A, G, Q, T, S, N, P, R, D, V, W, K, C, Y, I, L, and F. In some embodiments, each N 56 is independently an amino acid selected from the group consisting of A, G, and Q. In some embodiments, each N 57 is independently absent. In some embodiments, each N 57 is independently an amino acid selected from the group consisting of Y, C, N, I, F, and L. In some embodiments, each N 58 is independently absent. In some embodiments, each N 58 is independently an amino acid selected from the group consisting of S, T, G, H, A, P, Y, V, F, L, N, R, K, E, D, W, I, Q, M, and C. In some embodiments, each N 58 is independently S. In some embodiments, each N 59 is independently absent. In some embodiments, each N 59 is independently an amino acid selected from the group consisting of I, V, and L. In some embodiments, each N 59 is independently an amino acid selected from the group consisting of I and V. In some embodiments, each N 60 is independently absent. In some embodiments, each N 60 is independently S. In some embodiments, each N 61 is independently absent. In some embodiments, each N 61 is independently an amino acid selected from the group consisting of G, S, R, K, A, N, T, Q, E, D, P, and Y. In some embodiments, each N 62 is independently absent. In some embodiments, each N 62is independently an amino acid selected from the group consisting of I, V, L, F, W, Y, A, T, S, E, D, and H. In some embodiments, each N 62 is independently an amino acid selected from the group consisting of I and V. In some embodiments, each N 63 is independently absent. In some embodiments, each N 63 is independently an amino acid selected from the group consisting of T, Q, N, G, C, M, S, A, E, D, Y, V, I, F, L, and W. In some embodiments, each N 63 is independently T. In some embodiments, each N 64 is independently absent. In some embodiments, each N 64 is independently an amino acid selected from the group consisting of S, N, Q, R, G, K, E, D, P, Y, W, F, T, H, A, V, L, I, M, and C. In some embodiments, each N 64 is independently S. In some embodiments, each N 65 is independently absent. In some embodiments, each N 65 is independently an amino acid selected from the group consisting of A, C, G, S, Q, N, R, Y, E, K, D, H, M, V, I, and L. In some embodiments, each N 66 is independently absent. In some embodiments, each N 66 is independently an amino acid selected from the group consisting of V, I, A, T, S, G, R, P, Y, L, N, H, C, M, F, Q, E, K, and D. In some embodiments, each N 66 is independently V. In some embodiments, each N 67 is independently an amino acid selected from the group consisting of S, N, Q, R, T, G, K, E, H, D, A, C, P, Y, M, V, W, I, F, and L. In some embodiments, each N 67 is independently an amino acid selected from the group consisting of S, N, Q, R, and T. In some embodiments, each N 68is independently an amino acid selected from the group consisting of K, R, H, S, G, N, Q, D, E, T, A, C, P, Y, M, V, W, I, L, and F. In some embodiments, each N 68 is independently an amino acid selected from the group consisting of K, R, H, and S. In some embodiments, each N 69 is independently an amino acid selected from the group consisting of K, R, H, S, G, N, Q, D, E, T, A, C, P, Y, M, V, W, I, L, and F. In some embodiments, each N 69 is independently an amino acid selected from the group consisting of K, R, H, and S. In some embodiments, each N 70 is independently an amino acid selected from the group consisting of D, E, Q, N, S, H, T, R, K, G, A, C, Y, P, M, V, W, I, F, and L. In some embodiments, each N 70 is independently an amino acid selected from the group consisting of D, E, Q, and N. In some embodiments, each N 71 is independently an amino acid selected from the group consisting of A, T, C, G, S, Q, N, P, R, Y, E, K, D, H, M, V, W, I, L, and F. In some embodiments, each N 71 is independently an amino acid selected from the group consisting of A, T, C, and G.

[0146] In some embodiments, the proprotein signal peptide comprises the amino acid sequence of SEQ ID NO:75.

[0147] In some embodiments, a synthetic pre-protein signal peptide is provided. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula IX, and Formula XIII. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula I. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula II. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula III. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula IV. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula V. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula IX. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula XIII.

[0148] In some embodiments, the synthetic preprotein signal peptide comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. In some embodiments, the synthetic preprotein signal peptide comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO:1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. In some embodiments, the pre-protein signal peptide further comprises the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 69, or Formula XII.

[0149] In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:1. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:2. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:3. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:4. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:5. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:6. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:7. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:8. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:9. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:10. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:11. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:13. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO:14. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 15. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 16. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 28. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 31. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 32. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 33. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 55.In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 70. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 71. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 72. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 73.

[0150] In some embodiments, a synthetic proprotein signal peptide is provided. In some embodiments, the synthetic proprotein signal peptide comprises an amino acid sequence selected from the group consisting of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, and Formula XV. In some embodiments, the synthetic proprotein signal peptide comprises an amino acid sequence of Formula VI. In some embodiments, the synthetic proprotein signal peptide comprises an amino acid sequence of Formula VII. In some embodiments, the synthetic proprotein signal peptide comprises an amino acid sequence of Formula VIII. In some embodiments, the synthetic proprotein signal peptide comprises an amino acid sequence of Formula X. In some embodiments, the synthetic proprotein signal peptide comprises an amino acid sequence of Formula XI. In some embodiments, the synthetic proprotein signal peptide comprises an amino acid sequence of Formula XIV. In some embodiments, the synthetic proprotein signal peptide comprises an amino acid sequence of Formula XV.

[0151] In some embodiments, the synthetic proprotein signal peptide comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the synthetic proprotein signal peptide comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. In some embodiments, the synthetic proprotein signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 34, 35, 36, 37, 38, 56, 57, 58, 74, and 75. In some embodiments, the proprotein signal peptide further comprises the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 69, or Formula XII.

[0152] In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 17. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 19. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 20. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 21. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 23. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 24. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 25. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 27. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 29. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 34. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 35. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 36. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 37. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 56. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 57. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 58. In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO: 74.In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of SEQ ID NO:75.

[0153] In some embodiments, a preprotein plus proprotein signal peptide is provided, in some embodiments, the preprotein plus proprotein signal peptide comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the amino acid sequence of SEQ ID NO:30.

[0154] In some embodiments, a recombinant polypeptide is provided, the recombinant polypeptide comprising (X 1 ) n -(Y 1 ) m -Z 1 wherein X 1 is a synthetic preprotein signal peptide, and Y 1 is a synthetic proprotein signal peptide, and Z 1 is a payload protein, n is 0 or 1, m is 0 or 1, and n and m cannot simultaneously be 0. In some embodiments, n is 0 and m is 1, and the recombinant polypeptide is 1 )-Z 1 In some embodiments, n is 1, m is 0, and the recombinant polypeptide comprises the formula: 1 )-Z 1 In some embodiments, n is 1, m is 1, and the recombinant polypeptide comprises the formula: 1 )-(Y 1 )-Z 1 Contains the formula:

[0155] In some embodiments, the recombinant polypeptide comprises payload protein Z. 1 In some embodiments, the amino acid sequence of SEQ ID NO: 68, SEQ ID NO: 69, or formula XII is further comprised at the N-terminus of the 1 ) n -(Y 1 ) m -Z 1The formula is (X 1 ) n -(Y 1 ) m -(K 1 ) p -Z 1 wherein X 1 is a synthetic preprotein signal peptide, and Y 1 is a synthetic proprotein signal peptide, and K 1 is a sequence selected from the group consisting of SEQ ID NO: 68, SEQ ID NO: 69, and formula XII; Z 1 is a payload protein, where n is 0 or 1, m is 0 or 1, and p is 0 or 1, and n and m cannot simultaneously be 0. In some embodiments, n is 0, m is 1, and p is 0, and the recombinant polypeptide is 1 )-Z 1 In some embodiments, n is 0, m is 1, p is 1, and the recombinant polypeptide comprises the formula: 1 )-(K 1 )-Z 1 In some embodiments, n is 1, m is 0, p is 0, and the recombinant polypeptide comprises the formula: 1 )-Z 1 In some embodiments, n is 1, m is 0, p is 1, and the recombinant polypeptide comprises the formula: 1 )-(K 1 )-Z 1 In some embodiments, n is 1, m is 1, p is 0, and the recombinant polypeptide comprises the formula: 1 )-(Y 1 )-Z 1 In some embodiments, n is 1, m is 1, p is 1, and the recombinant polypeptide comprises the formula: 1 )-(Y 1 )-(K 1 )-Z 1 Contains the formula:

[0156] In some embodiments, n is 1 and X 1comprises an amino acid sequence selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula IX, and Formula XIII. 1 comprises the amino acid sequence of Formula I. In some embodiments, X 1 comprises the amino acid sequence of formula II. 1 comprises the amino acid sequence of formula III. 1 In some embodiments, X comprises the amino acid sequence of formula IV. 1 comprises the amino acid sequence of formula V. In some embodiments, X 1 In some embodiments, X comprises the amino acid sequence of formula IX. 1 In some embodiments, X comprises the amino acid sequence of formula XIII. 1 comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. 1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. 1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73.

[0157] In some embodiments, m is 1 and Y 1comprises an amino acid sequence selected from the group consisting of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, and Formula XV. 1 comprises the amino acid sequence of formula VI. In some embodiments, Y 1 comprises the amino acid sequence of formula VII. 1 In some embodiments, Y comprises the amino acid sequence of formula VIII. 1 comprises an amino acid sequence of formula X. In some embodiments, Y 1 In some embodiments, Y comprises the amino acid sequence of formula XI. 1 In some embodiments, Y comprises the amino acid sequence of formula XIV. 1 In some embodiments, Y comprises the amino acid sequence of formula XV. 1 comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. 1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. 1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75.

[0158] In some embodiments, X 1 and Y 1is combined and represented by the preprotein plus proprotein signal peptide and comprises an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity to the amino acid sequence of SEQ ID NO:30.

[0159] In some embodiments, Z 1 is any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an antiviral, insulin, an incretin, an enzyme, an enzyme inhibitor, a hormone, a cytokine, an antibody, an antimicrobial peptide, a mucosal protein, an insecticide, a bactericide, a herbicide, a fungicide, a nematicide, an acaricide, a plant growth regulator, a plant growth stimulant, or a fertilizer), a vaccine, a diagnostic protein, a feed converting enzyme, a flavoring agent, or a nutritional protein.

[0160] In some embodiments, Z 1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO:59; or APVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLDKREEGEPKSMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTNWEDQPIAIA PKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSLDGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKS YWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEVSASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK (SEQ ID NO:59) or is substantially similar to SEQ ID NO:59, or is an active fragment of SEQ ID NO:59. 1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 59. In some embodiments, Z 1 comprises the amino acid sequence of SEQ ID NO:59.

[0161] In some embodiments, Z 1comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 60; or SMTNETSDRPLVHFTPNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTNWEDQPIAIAPKRNDSGAFSGSMVVDYNNTSGFFNDTIDPRQRCVAIWTYNTPESEEQYISYSL DGGYTFTEYQKNPVLAANSTQFRDPKVFWYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQDPSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDN QSRVVDFGKDYYALQTFFNTDPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPILNISNAGPWSRFATNTTLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVFADLSLWFKGLEDPEEYLRMGFEVSASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSENDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQVREVK (SEQ ID NO: 60) or is substantially similar to SEQ ID NO:60, or is an active fragment of SEQ ID NO:60. 1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 60. 1 comprises the amino acid sequence of SEQ ID NO:60.

[0162] In some embodiments, Z 1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 61; or KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACQLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV (SEQ ID NO:61) or is substantially similar to SEQ ID NO:61, or is an active fragment of SEQ ID NO:61. 1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:61. 1 comprises the amino acid sequence of SEQ ID NO:61.

[0163] In some embodiments, Z 1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 62; or IKHRLNGFTILEHPDPAKRDLLQDIVTWDDKSLFINGERIMLFSGEVHPFRLPVPSLWLDIFHKIRALGFNCVSFYIDWALLEGKPGDYRAEGIFALEPFFDAAKEAGIYLIARPGSYINAEV SGGGFPGWLQRVNGTLRSSDEPFLKATDNYIANAAAAVAKAQITNGGPVILYQPENEYSGGCCGVKYPDADYMQYVMDQARKADIVVPFISNDASPSGHNAPGSGTSAVDIYGHDSYPLGFDC ANPSVWPEGKLPDNFRTLHLEQSPSTPYSLLEFQAGAFDPWGGPGFEKCYALVNHEFSRVFYRNDLSFFGVSTFNLYMTFGGTNWGNLGHPGGYTSYDYGSPITETRNVTREKYSDIKLLANFV KASPSYLTATPRNLTTGVYTDTSDLAVTPLIGDSPGSFFVVRHTDYSSQESTSYKLKLPTSAGNLTIPQLEGTLSLNGRDSKIHVVDYNVSGTNIIYSTAEVFTWKKFDGNKVLVLYGGPKEH HELAIASKSNVTIIEGSDSGIVSTRKGSSVIIGWDVSSTRRIVQVGDLRVFLLDRNSAYNYWVPELPTEGTSPGFSTSKTTASSIIVKAGYLLRGAHLDGADLHLTADFNATTPIEVIGAPTG AKNLFVNGEKASHTVDKNGIWSSEVKYAAPEIKLPGLKDLDWKYLDTLPEIKSSYDDSAWVSADLPKTKNTHRPLDTPTSLYSSDYGFHTGYLIYRGHFVANGKESEFFIRTQGGSAFGSSVW LNETYLGSWTGADYAMDGNSTYKLSQLESGKNYVITVVIDNLGLDENWTVGEETMKNPRGILSYKLSGQDASAITWKLTGNLGGEDYQDKVRGPLNEGGLYAERQGFHQPQPPSESWESGSPLEGLSKPGIGFYTAQFDLDLPKGWDVPLYFNFGNNTQAARAQLYVNGYQYGKFTGNVGPQTSFPVPEGILNYRGTNYVALSLWALESDGAKLGSFELSYTTPVLTGYGNVESPEQPKYEQRKGAY (SEQ ID NO: 62) or is substantially similar to SEQ ID NO:62, or is an active fragment of SEQ ID NO:62. 1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:62. 1 comprises the amino acid sequence of SEQ ID NO:62.

[0164] In some embodiments, Z 1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 63; or EVQLVESGGGLVQPGGSLRLSCAASGFTFSDYWMYWVRQAPGKGLEWVSEINTNGLITKYPDSVGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCARSPSGFNRGQGTLVTVSS (SEQ ID NO: 63) or is substantially similar to SEQ ID NO:63, or is an active fragment of SEQ ID NO:63. 1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 63. 1 comprises the amino acid sequence of SEQ ID NO:63.

[0165] In some embodiments, Z 1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 64; or IEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLAEITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFTWPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNIDTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELAKDPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTRITK (SEQ ID NO: 64) or is substantially similar to SEQ ID NO:64, or is an active fragment of SEQ ID NO:64. 1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:64. 1 comprises the amino acid sequence of SEQ ID NO:64.

[0166] In some embodiments, Z 1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 65; or AQSEPELKLESVVIVSRGVRAPTKATQLMQDVTPDAWPTWPVKLGELTPRGGELLAYLGHYWRQRLVADGLLPKCGCPQSGQVAILADVDERTRKTGEAFAAGLAPDCAITVHTQADTSSPDPLFNPLKTGVCQLDNANVTDAILERAGGSLADFTGHYQTAFRELERVLNFPQSNLCLKREKQDESCSLTQALPSELKVSADCVSLTGAVSLASMLTEIFLLQQAQGMPEPGWGRITDSHQWNTLLSLHNAQFDLLQRTPEVARSRATPLLDLIKTALTPHPPQKQAYGVTLPTSVLFLAGHDTNLANLGGALELNWTLPGQPDNTPPGGELVFERWRRLSDNSQWIQVSLVFQTLQQMRDKTPLSLNTPPGEVKLTLAGCEERNAQGMCSLAGFTQIVNEARIPACSL (SEQ ID NO: 65) or is substantially similar to SEQ ID NO:65, or is an active fragment of SEQ ID NO:65. 1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 65. 1 comprises the amino acid sequence of SEQ ID NO:65.

[0167] In some embodiments, Z 1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 66; or FVNQHLCGSHLVEALYLVCGERGFFYTPKEWKGIVEQCCTSICSLYQLENYCN (SEQ ID NO:66) or is substantially similar to SEQ ID NO:66, or is an active fragment of SEQ ID NO:66. 1comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 66. 1 comprises the amino acid sequence of SEQ ID NO:66.

[0168] In some embodiments, Z 1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO:67; or GPETLCGAELVDALQFVCGPRGFYFNKPTGYGSSIRRAPQTGIVDECCFRSCDLRRLEMYCAPLKPTKAARSIRAQRHTDMPKTQKEVHLKNTSRGSAGNKTYRM (SEQ ID NO:67) or is substantially similar to SEQ ID NO:67, or is an active fragment of SEQ ID NO:67. 1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO:67. 1 comprises the amino acid sequence of SEQ ID NO:67.

[0169] In some embodiments, Z 1 comprises an amino acid sequence having at least 70% identity to SEQ ID NO: 85; or KVFERCELARTLKRLGMDGYRGISLANWMCLAKWESGYNTRATNYNAGDRSTDYGIFQINSRYWCNDGKTPGAVNACQLSCSALLQDNIADAVACAKRVVRDPQGIRAWVAWRNRCQNRDVRQYVQGCGV (SEQ ID NO: 85) or is substantially similar to SEQ ID NO: 85, or is an active fragment of SEQ ID NO: 85. 1 comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 85. 1 comprises the amino acid sequence of SEQ ID NO:85.

[0170] In any of the embodiments herein, Z 1 may further comprise an affinity tag. The affinity tag may be utilized, for example, for purification or detection of the protein. The affinity tag may be utilized in any method known in the art in which affinity tags are utilized. Affinity tags are known in the art, and any such affinity tag may be utilized. Non-limiting examples of affinity tags that may be utilized include 6XHIS, FLAG, GST, MBP, streptavidin peptide, GFP, and the like. In some embodiments, any peptide sequence that may be utilized for purification or detection may be utilized.

[0171] In some embodiments, the recombinant polypeptide comprises a polypeptide selected from the group consisting of: (X 1 ) n -(Y 1 ) m -Z 1 wherein n is 0 or 1, m is 0 or 1, and wherein n and m cannot be 0 at the same time; X 1comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73; 1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75; and Z 1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 60, 61, 62, 63, 64, 65, 66, 67, and 85. In some embodiments, component X 1 , Y 1 , and Z 1 In some embodiments, component X is directly fused to 1 , Y 1 , and Z 1 is indirectly fused, for example, via a peptide linker as provided herein.

[0172] In some embodiments, the recombinant polypeptide comprises payload protein Z. 1 In some embodiments, the amino acid sequence of SEQ ID NO: 68 is further included at the N-terminus of (X 1 ) n -(Y 1 ) m -Z 1 The formula is (X 1 ) n -(Y 1 ) m -(K 1 ) p -Z 1 wherein X 1 is a synthetic preprotein signal peptide, and Y 1 is a synthetic proprotein signal peptide, 1 is a sequence selected from the group consisting of SEQ ID NO: 68, SEQ ID NO: 69, and formula XII; Z 1is a payload protein, n is 0 or 1, m is 0 or 1, and p is 0 or 1, and n and m cannot simultaneously be 0. In some embodiments, n is 0, m is 1, and p is 0, and the recombinant polypeptide is 1 )-Z 1 In some embodiments, n is 0, m is 1, p is 1, and the recombinant polypeptide comprises the formula: 1 )-(K 1 )-Z 1 In some embodiments, n is 1, m is 0, p is 0, and the recombinant polypeptide comprises the formula: 1 )-Z 1 In some embodiments, n is 1, m is 0, p is 1, and the recombinant polypeptide comprises the formula: 1 )-(K 1 )-Z 1 In some embodiments, n is 1, m is 1, p is 0, and the recombinant polypeptide comprises the formula: 1 )-(Y 1 )-Z 1 In some embodiments, n is 1, m is 1, p is 1, and the recombinant polypeptide comprises the formula: 1 )-(Y 1 )-(K 1 )-Z 1 Contains the formula:

[0173] In some embodiments, a nucleic acid is provided. In some embodiments, the nucleic acid encodes a recombinant polypeptide provided herein. In some embodiments, the recombinant polypeptide comprises a synthetic signal peptide and a payload protein. In some embodiments, the synthetic signal peptide is as provided herein. In some embodiments, the payload protein is as provided herein.

[0174] In some embodiments, engineered yeast is provided. In some embodiments, the engineered yeast comprises (X 1 ) n -(Y 1) m -Z 1 and a nucleic acid encoding a recombinant polypeptide having the formula: 1 is a synthetic preprotein signal peptide, and Y 1 is a synthetic proprotein signal peptide, and Z 1 is a payload protein, n is 0 or 1, m is 0 or 1, and n and m cannot be 0 simultaneously.

[0175] In some embodiments, the recombinant polypeptide comprises payload protein Z. 1 In some embodiments, the amino acid sequence of SEQ ID NO: 68 is further included at the N-terminus of (X 1 ) n -(Y 1 ) m -Z 1 The formula is (X 1 ) n -(Y 1 ) m -(K 1 ) p -Z 1 wherein X 1 is a synthetic preprotein signal peptide, and Y 1 is a synthetic proprotein signal peptide, 1 is a sequence selected from the group consisting of SEQ ID NO: 68, SEQ ID NO: 69, and formula XII; Z 1 is a payload protein, where n is 0 or 1, m is 0 or 1, and p is 0 or 1, and n and m cannot simultaneously be 0. In some embodiments, n is 0, m is 1, and p is 0, and the recombinant polypeptide is 1 )-Z 1 In some embodiments, n is 0, m is 1, p is 1, and the recombinant polypeptide comprises the formula: 1 )-(K 1 )-Z 1 In some embodiments, n is 1, m is 0, p is 0, and the recombinant polypeptide comprises the formula: 1 )-Z 1In some embodiments, n is 1, m is 0, p is 1, and the recombinant polypeptide comprises the formula: 1 )-(K 1 )-Z 1 In some embodiments, n is 1, m is 1, p is 0, and the recombinant polypeptide comprises the formula: 1 )-(Y 1 )-Z 1 In some embodiments, n is 1, m is 1, p is 1, and the recombinant polypeptide comprises the formula: 1 )-(Y 1 )-(K 1 )-Z 1 Contains the formula:

[0176] In some embodiments, n is 1 and X 1 comprises an amino acid sequence selected from the group consisting of Formula I, Formula II, Formula III, Formula IV, Formula V, Formula IX, and Formula XIII. 1 comprises the amino acid sequence of Formula I. In some embodiments, X 1 comprises the amino acid sequence of formula II. 1 comprises the amino acid sequence of formula III. 1 In some embodiments, X comprises the amino acid sequence of formula IV. 1 comprises the amino acid sequence of formula V. In some embodiments, X 1 In some embodiments, X comprises the amino acid sequence of formula IX. 1 In some embodiments, X comprises the amino acid sequence of formula XIII. 1 comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. 1comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. 1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73.

[0177] In some embodiments, m is 1 and Y 1 comprises an amino acid sequence selected from the group consisting of Formula VI, Formula VII, Formula VIII, Formula X, Formula XI, Formula XIV, and Formula XV. 1 comprises the amino acid sequence of formula VI. In some embodiments, Y 1 comprises the amino acid sequence of formula VII. 1 In some embodiments, Y comprises the amino acid sequence of formula VIII. 1 comprises an amino acid sequence of formula X. In some embodiments, Y 1 In some embodiments, Y comprises the amino acid sequence of formula XI. 1 In some embodiments, Y comprises the amino acid sequence of formula XIV. 1 In some embodiments, Y comprises the amino acid sequence of formula XV. 1 comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. 1comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. 1 comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75.

[0178] In some embodiments, Z 1 is any peptide or protein. In some embodiments, the payload protein is selected from the group comprising an antiviral, insulin, an incretin, an enzyme, an enzyme inhibitor, a hormone, a cytokine, an antibody, an antimicrobial peptide, a mucosal protein, an insecticide, a bactericide, a herbicide, a fungicide, a nematicide, an acaricide, a plant growth regulator, a plant growth stimulant, or a fertilizer), a vaccine, a diagnostic protein, a feed converting enzyme, a flavoring agent, or a nutritional protein.

[0179] In some embodiments, Z 1 comprises an amino acid sequence having at least 70% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 60, 61, 62, 63, 64, 65, 66, and 67. 1comprises an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 60, 61, 62, 63, 64, 65, 66, 67, and 85. 1 comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 59, 60, 61, 62, 63, 64, 65, 66, 67, and 85.

[0180] In some embodiments, component X 1 , Y 1 , and Z 1 In some embodiments, component X is directly fused to 1 , Y 1 , and Z 1 is indirectly fused, for example, via a peptide linker as provided herein.

[0181] In some embodiments, X 1 , Y 1 , and Z 1 The identity of is influenced by the strain of yeast utilized. In some embodiments, the yeast strain is any yeast provided herein. In some embodiments, the yeast is selected from the group consisting of Kluyveromyces, Pichia, Saccharomyces, Trichoderma, and Aspergillus. Specific yeasts, such as X. 1 , Y 1 , and Z 1Combinations of are described and provided below. It should be understood that the embodiments provided below are merely exemplary and are not intended to limit the scope of the invention in any way. Thus, although a particular embodiment may not refer to the use of a particular preprotein or proprotein sequence number, this should not be interpreted as excluding a particular sequence number from use in a particular yeast. Furthermore, although a particular embodiment may not refer to the inclusion of any synthetic preprotein or proprotein signal peptide, this should not be interpreted as excluding a preprotein or proprotein signal peptide from use in a particular yeast. For example, if a recombinant polypeptide is described for use in a particular yeast and the recombinant polypeptide is said to include a synthetic preprotein signal peptide domain and a payload protein domain, this should not be interpreted as excluding a synthetic proprotein signal domain from being included in the particular yeast. Similarly, if a recombinant polypeptide is described for use in a particular yeast and the recombinant polypeptide is said to include a synthetic proprotein signal peptide domain and a payload protein domain, this should not be interpreted as excluding a synthetic preprotein signal domain from being included in the particular yeast.

[0182] Synthetic preprotein signal peptides in Kluyveromyces yeasts and their uses In some embodiments, synthetic pre-protein signal peptides are provided that can be fused to a payload protein to facilitate secretion of the payload protein from Kluyveromyces yeast (e.g., K. lactis). In some embodiments, Kluyveromyces yeast (e.g., K. lactis) can be genetically modified with a nucleic acid molecule that codes for expression of a recombinant polypeptide that includes a synthetic pre-protein signal peptide fused either directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide includes the amino acid sequence of Formula I or SEQ ID NO:1. In some embodiments, the nucleic acid molecule is any nucleic acid molecule that codes for a peptide that includes the amino acid sequence of Formula I or SEQ ID NO:1. For example, SEQ ID NO:39 can be used to code for a synthetic pre-protein signal peptide that includes the amino acid sequence of SEQ ID NO:1. It should be understood that the foregoing examples are not intended to be limiting in any way. One of skill in the art would know how to develop a suitable nucleotide sequence that would direct expression of a synthetic signal peptide that includes the amino acid sequence of Formula I or SEQ ID NO:1. In some embodiments, the signal peptide that includes the amino acid sequence of Formula I or SEQ ID NO:1 can be fused directly or indirectly to a natural constitutive pro-protein signal peptide or to a synthetic signal peptide disclosed herein.

[0183] In some embodiments, a recombinant polypeptide is provided that includes a synthetic pre-protein signal peptide that includes the amino acid sequence of Formula I or SEQ ID NO:1 and a payload protein. In some embodiments, the inclusion of a pre-protein signal peptide that includes the amino acid sequence of Formula I or SEQ ID NO:1 results in the payload protein being more easily secreted by the yeast in which it is produced. Thus, in another embodiment, a method is provided for producing a payload protein using a Kluyveromyces yeast (e.g., K. lactis), the method comprising: providing a nucleic acid molecule encoding a recombinant peptide that includes a payload protein and a synthetic pre-protein signal peptide that includes the amino acid sequence of Formula I SEQ ID NO:1; genetically modifying a Kluyveromyces yeast (e.g., K. lactis) with the nucleic acid molecule, thereby producing an engineered yeast; and culturing the engineered yeast under conditions effective to express the recombinant polypeptide. In some embodiments, the nucleic acid molecule encoding the synthetic signal peptide of SEQ ID NO:1 is SEQ ID NO:39. In some embodiments, the nucleic acid molecule encoding the synthetic signal peptide amino acid of Formula I or SEQ ID NO:1 is any nucleic acid molecule that encodes the amino acid sequence.

[0184] In some embodiments, a method for increasing extracellular secretion of a payload protein from a Kluyveromyces yeast (e.g., K. lactis) is provided, the method comprising providing a nucleic acid encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide, genetically modifying a Kluyveromyces yeast (e.g., K. lactis) with the nucleic acid, thereby producing an engineered yeast, and culturing the engineered yeast under conditions effective to produce and secrete an increased amount of the payload protein, as compared to the amount of payload protein secreted by a Kluyveromyces yeast (e.g., K. lactis) using a recombinant polypeptide comprising the payload protein and the signal peptide α-MF, or any other commonly utilized signal peptide, such as SUC2, PHO5, or HSA. In some embodiments, the synthetic pre-protein signal peptide comprises the amino acid sequence of Formula I or SEQ ID NO:1. In some embodiments, the synthetic pre-protein signal peptide further comprises a naturally occurring pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is connected to the payload protein via a peptide linker provided herein.

[0185] In some embodiments, an engineered Kluyveromyces yeast (e.g., K. lactis) is provided, where the yeast is genetically modified with a nucleic acid molecule encoding expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide directly or indirectly fused to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises the amino acid sequence of Formula I or SEQ ID NO:1. In some embodiments, the synthetic pre-protein signal peptide further comprises a naturally occurring pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide provided herein. In some embodiments, the synthetic pre-protein signal peptide is directly fused to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is indirectly fused to the payload protein via a connecting linker peptide sequence provided herein. In some embodiments, the nucleic acid molecule used to encode the synthetic pre-protein signal peptide comprising the amino acid sequence of SEQ ID NO:1 is given by SEQ ID NO:39. In some embodiments, the nucleic acid molecule used to encode the synthetic pre-protein signal peptide comprising the amino acid sequence of Formula I or SEQ ID NO:1 is any nucleic acid molecule encoding the amino acid sequence.

[0186] In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group including enzymes (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), growth factors (e.g., IGF-1), insulin, incretins (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), cytokines, antibodies, antimicrobial peptides), mucosal proteins (e.g., trefoil factors, Reg3 proteins, superoxide dismutase), agricultural products (e.g., insecticides, bactericides, herbicides, fungicides, nematicides, acaricides, plant growth regulators, plant growth stimulants, or fertilizers), vaccines, diagnostic proteins, feed converting enzymes, flavorings, or nutritional proteins. The listed examples are provided for clarity only and are not intended to be limiting in any way. Thus, for example, the present disclosure is not intended to be limited to IGF-1 with respect to "growth factors," but rather encompasses and includes all growth factors known in the art.

[0187] Synthetic preprotein signal peptides and their uses in Pichia yeasts In some embodiments, synthetic pre-protein signal peptides are provided for use in the yeast species Pichia (e.g., P. pastoris). In some embodiments, Pichia yeast can be genetically engineered with a nucleic acid molecule that encodes the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal comprises an amino acid sequence represented by Formula II or SEQ ID NO: 2, 3, 4, 5, 6, or 7. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein and connected via a peptide linker provided herein. In some embodiments, any nucleic acid encoding Formula II or SEQ ID NO: 2, 3, 4, 5, 6, or 7 can be utilized to direct expression of the synthetic signal peptide. -Those of skill in the art will know how to develop suitable nucleotide sequences that will direct expression of a synthetic pre-protein signal represented by Formula II or SEQ ID NO: 2, 3, 4, 5, 6, or 7. In some embodiments, the synthetic preprotein signal peptide of Formula II or SEQ ID NO: 2, 3, 4, 5, 6, or 7 may be further fused directly or indirectly to a native constitutive proprotein signal peptide or a synthetic signal peptide disclosed herein. In some embodiments, the synthetic preprotein signal peptide is further fused to a native constitutive proprotein signal peptide. In some embodiments, the synthetic preprotein signal peptide is further fused to a synthetic signal peptide disclosed herein. In some embodiments, the synthetic preprotein signal peptide of Formula II or SEQ ID NO: 2, 3, 4, 5, 6, or 7 is further fused to a synthetic proprotein signal peptide selected from the group consisting of SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, 24, 25, 34, 35, 36, 37, 38, 56, 57, and 58.In some embodiments, the synthetic preprotein signal peptide of Formula II or SEQ ID NO:2, 3, 4, 5, 6, or 7 is further fused to a synthetic proprotein signal peptide represented by SEQ ID NO:17.

[0188] In some embodiments, a recombinant polypeptide is provided that includes a synthetic pre-protein signal peptide comprising the amino acid sequence of Formula II or SEQ ID NO: 2, 3, 4, 5, 6, or 7, and a payload protein. In some embodiments, the inclusion of a pre-protein signal peptide comprising the amino acid sequence of Formula II or SEQ ID NO: 2, 3, 4, 5, 6, or 7 results in the payload protein being more easily secreted by the yeast in which it is produced. Thus, in some embodiments, a method of producing a payload protein using a Pichia yeast (e.g., P. pastoris) is provided, the method comprising providing a nucleic acid molecule encoding a recombinant peptide comprising a payload protein and a synthetic pre-protein signal peptide comprising the amino acid sequence of Formula II or SEQ ID NO: 2, 3, 4, 5, 6, or 7, genetically modifying a Pichia yeast (e.g., P. pastoris) with the nucleic acid molecule, thereby producing an engineered yeast, and culturing the engineered yeast under conditions effective to express the recombinant polypeptide. In some embodiments, the nucleic acid molecule encoding the amino acid sequence of Formula II or SEQ ID NO: 2, 3, 4, 5, 6, or 7 is any nucleic acid molecule encoding the amino acid sequence.

[0189] In some embodiments, a method of increasing extracellular secretion of a payload protein from a Pichia yeast is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic preprotein signal peptide, genetically modifying a Pichia yeast (e.g., P. pastoris) with the nucleic acid, thereby generating an engineered yeast, and culturing the engineered yeast under conditions effective to produce and secrete an increased amount of the payload protein when compared to the amount of payload protein secreted by a Pichia yeast genetically modified to express a recombinant polypeptide comprising a payload protein and a preprotein signal peptide α-MF (α-MF consisting of the amino acid sequence represented by SEQ ID NO:27). In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of Formula II or SEQ ID NO:2, 3, 4, 5, 6, or 7. In some embodiments, the synthetic preprotein signal peptide further comprises a naturally occurring proprotein signal peptide. In some embodiments, the synthetic preprotein signal peptide further comprises a synthetic proprotein signal peptide provided herein. In some embodiments, the synthetic preprotein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is indirectly fused to the payload protein, for example, via a peptide linker provided herein.

[0190] In some embodiments, an engineered Pichia yeast (e.g., P. pastoris) is provided, where the yeast is genetically modified with a nucleic acid encoding expression of a recombinant polypeptide comprising a synthetic preprotein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of Formula II or SEQ ID NO: 2, 3, 4, 5, 6, or 7. In some embodiments, the synthetic preprotein signal peptide further comprises a naturally occurring proprotein signal peptide. In some embodiments, the synthetic preprotein signal peptide further comprises a synthetic proprotein signal peptide provided herein. In some embodiments, the synthetic preprotein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic preprotein signal peptide is fused indirectly to the payload protein, for example, via a peptide linker provided herein. In some embodiments, the payload protein can be any peptide or protein. In some embodiments, the payload protein is selected from the group including enzymes (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), growth factors (e.g., IGF-1), insulin, incretins (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), cytokines, antibodies, antimicrobial peptides), mucosal proteins (e.g., trefoil factors, Reg3 proteins, superoxide dismutase), agricultural products (e.g., insecticides, bactericides, herbicides, fungicides, nematicides, acaricides, plant growth regulators, plant growth stimulants, or fertilizers), vaccines, diagnostic proteins, feed converting enzymes, flavorings, or nutritional proteins. The listed examples are provided for clarity only and are not intended to be limiting in any way. Thus, for example, the present disclosure is not limited to IGF-1 for "growth factors," but rather encompasses and includes all growth factors known in the art.

[0191] Synthetic preprotein signal peptides in Saccharomyces yeasts and their usesIn another embodiment, a synthetic pre-protein signal peptide is provided for use in the yeast species Saccharomyces (e.g., S. boulardii or S. cerevisiae). In some embodiments, S. cerevisiae yeast can be genetically engineered with a nucleic acid molecule encoding expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises the amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein, for example, via a peptide linker provided herein. In some embodiments, any nucleic acid encoding Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16 can be utilized to direct expression of the synthetic pre-protein signal peptide. One of skill in the art would know how to develop suitable nucleic acids that would direct expression of a synthetic signal peptide comprising the amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, a pre-protein signal peptide comprising the amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16 may be fused directly or indirectly to a naturally occurring constitutive pro-protein signal peptide. In some embodiments, a pre-protein signal peptide comprising the amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16 may be fused directly or indirectly to a synthetic signal peptide disclosed herein, such as Formula VI, Formula VII, Formula VIII, or SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, the synthetic pre-protein signal peptide is fused directly to a natural or synthetic pro-protein signal peptide.In some embodiments, the synthetic preprotein signal peptide is indirectly fused to a natural or synthetic proprotein signal peptide, for example, via a peptide linker provided herein.

[0192] In some embodiments, a recombinant polypeptide is provided that includes a synthetic preprotein signal peptide comprising the amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16, and a payload protein. In some embodiments, the inclusion of a synthetic preprotein signal peptide comprising the amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16 results in the payload protein being more readily secreted by the yeast in which it is produced. Thus, in another embodiment, a method of producing a payload protein using Saccharomyces yeast is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic preprotein signal peptide comprising the amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16, genetically modifying a Saccharomyces yeast with the nucleic acid, thereby producing an engineered yeast, and culturing the engineered yeast under conditions effective to express the recombinant polypeptide. In some embodiments, a nucleic acid molecule encoding the amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16 is any nucleic acid molecule encoding the amino acid sequence.

[0193] In some embodiments, a method of increasing extracellular secretion of a payload protein from a Saccharomyces yeast is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide, genetically modifying a Saccharomyces yeast with the nucleic acid, thereby generating an engineered yeast, and culturing the engineered yeast under conditions effective to produce and secrete an increased amount of the payload protein when compared to the amount of payload protein secreted by a Saccharomyces yeast genetically modified to express a recombinant polypeptide comprising the payload protein and the pre-protein signal peptide α-MF or yeast aspartic protease 3 (YAP). In some embodiments, the synthetic pre-protein signal peptide comprises an amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the synthetic pre-protein signal peptide further comprises a naturally occurring pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide provided herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein, hi some embodiments, the synthetic pre-protein signal peptide is fused indirectly to the payload protein, for example, via a peptide linker provided herein.

[0194] In some embodiments, an engineered Saccharomyces yeast (e.g., S. boulardii or S. cerevisiae) is provided, where the yeast is genetically modified with a nucleic acid molecule encoding expression of a recombinant polypeptide comprising a synthetic preprotein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16. In some embodiments, the synthetic preprotein signal peptide further comprises a naturally occurring proprotein signal peptide. In some embodiments, the synthetic preprotein signal peptide further comprises a synthetic proprotein signal peptide provided herein. In some embodiments, the synthetic preprotein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic preprotein signal peptide is fused indirectly to the payload protein, for example, via a peptide linker provided herein. In some embodiments, the payload protein can be any peptide or protein. In some embodiments, the payload protein is selected from the group including enzymes (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), growth factors (e.g., IGF-1), insulin, incretins (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), cytokines, antibodies, antimicrobial peptides), mucosal proteins (e.g., trefoil factors, Reg3 proteins, superoxide dismutase), agricultural products (e.g., insecticides, bactericides, herbicides, fungicides, nematicides, acaricides, plant growth regulators, plant growth stimulants, or fertilizers), vaccines, diagnostic proteins, feed converting enzymes, flavorings, or nutritional proteins. The listed examples are provided for clarity only and are not intended to be limiting in any way. Thus, for example, the present disclosure does not limit "growth factors" to IGF-1, but rather encompasses and includes all growth factors known in the art.

[0195] Synthetic preprotein signal peptides in Trichoderma yeasts and their uses In some embodiments, synthetic pre-protein signal peptides are provided for use in the yeast species Trichoderma (e.g., T. reesei or T. viride). In some embodiments, Trichoderma yeast can be genetically engineered with a nucleic acid molecule that encodes the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises the amino acid sequence of Formula IX or SEQ ID NO: 31, 32, or 33. In some embodiments, the synthetic pre-protein signal peptide is fused directly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to a payload protein, for example, via a peptide linker provided herein. In some embodiments, any nucleic acid molecule encoding Formula IX or SEQ ID NO: 31, 32, or 33 can be utilized to direct expression of the synthetic signal peptide. One of skill in the art will know how to develop a suitable nucleotide sequence that will direct expression of a synthetic pre-protein signal peptide comprising the amino acid sequence of Formula IX or SEQ ID NO: 31, 32, or 33. In some embodiments, a synthetic preprotein signal peptide comprising the amino acid sequence of Formula IX or SEQ ID NO:31, 32, or 33 may be further fused directly or indirectly to a native constitutive proprotein signal peptide or a synthetic signal peptide disclosed herein. In some embodiments, the synthetic preprotein signal peptide is further fused to a native constitutive proprotein signal peptide. In some embodiments, the synthetic preprotein signal peptide is further fused to a synthetic signal peptide disclosed herein.

[0196] In some embodiments, a recombinant polypeptide is provided that includes a synthetic pre-protein signal peptide comprising the amino acid sequence of Formula IX or SEQ ID NO: 31, 32, or 33, and a payload protein. In some embodiments, the inclusion of a pre-protein signal peptide comprising the amino acid sequence of Formula IX or SEQ ID NO: 31, 32, or 33 results in the payload protein being more easily secreted by the yeast in which it is produced. Thus, in another embodiment, a method is provided for producing a payload protein using a Trichoderma yeast (e.g., T. reesei or T. viride), the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide comprising the amino acid sequence of Formula IX or SEQ ID NO: 31, 32, or 33, genetically modifying a T. reesei yeast with the nucleic acid molecule, thereby producing an engineered yeast, and culturing the engineered yeast under conditions effective to express the recombinant polypeptide. In some embodiments, the nucleic acid molecule encoding the amino acid sequence of Formula IX or SEQ ID NO: 31, 32, or 33 is any nucleic acid molecule encoding the amino acid sequence.

[0197] In some embodiments, a method is provided for increasing extracellular secretion of a payload protein from a Trichoderma yeast (e.g., T. reesei or T. viride), the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide, genetically modifying a Trichoderma yeast with the nucleic acid, thereby producing an engineered yeast, and culturing the engineered yeast under conditions effective to secrete an increased amount of the payload protein when compared to the amount of payload protein secreted by a Trichoderma yeast genetically modified to express a recombinant polypeptide comprising a payload protein and a pre-protein signal peptide comprising a native pre-protein signal peptide sequence provided herein or a control pre-protein signal peptide sequence provided herein. In some embodiments, the synthetic pre-protein signal peptide comprises the amino acid sequence of Formula IX or SEQ ID NO: 31, 32, or 33. In some embodiments, the synthetic pre-protein signal peptide further comprises a native pro-protein signal peptide. In some embodiments, the synthetic pre-protein signal peptide further comprises a synthetic pro-protein signal peptide provided herein. In some embodiments, the synthetic pre-protein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic pre-protein signal peptide is indirectly fused to the payload protein, for example, via a peptide linker provided herein.

[0198] In some embodiments, an engineered Trichoderma yeast (e.g., T. reesei or T. viride) is provided, where the yeast is genetically modified with a nucleic acid molecule encoding expression of a recombinant polypeptide comprising a synthetic preprotein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of Formula IX or SEQ ID NO: 31, 32, or 33. In some embodiments, the synthetic preprotein signal peptide further comprises a naturally occurring proprotein signal peptide. In some embodiments, the synthetic preprotein signal peptide further comprises a synthetic proprotein signal peptide provided herein. In some embodiments, the synthetic preprotein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic preprotein signal peptide is fused indirectly to the payload protein, for example, via a peptide linker provided herein.

[0199] In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group including enzymes (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), growth factors (e.g., IGF-1), insulin, incretins (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), cytokines, antibodies, antimicrobial peptides), mucosal proteins (e.g., trefoil factors, Reg3 proteins, superoxide dismutase), agricultural products (e.g., insecticides, bactericides, herbicides, fungicides, nematicides, acaricides, plant growth regulators, plant growth stimulants, or fertilizers), vaccines, diagnostic proteins, feed converting enzymes, flavorings, or nutritional proteins. The listed examples are provided for clarity only and are not intended to be limiting in any way. Thus, for example, the present disclosure is not limited to "growth factors" only IGF-1, but rather encompasses and includes all growth factors known in the art.

[0200] Synthetic preprotein signal peptides and their use in Aspergillus yeast strains. In some embodiments, synthetic pre-protein signal peptides are provided for use in the yeast species Aspergillus (e.g., A. niger). In some embodiments, Aspergillus yeast can be genetically engineered with a nucleic acid molecule that encodes the expression of a recombinant polypeptide comprising a synthetic pre-protein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide comprises the amino acid sequence of Formula XIII or SEQ ID NO: 70, 71, 72, or 73. In some embodiments, the synthetic pre-protein signal peptide is fused directly to a payload protein. In some embodiments, the synthetic pre-protein signal peptide is fused indirectly to a payload protein, for example, via a peptide linker provided herein. In some embodiments, any nucleic acid molecule encoding Formula XIII or SEQ ID NO: 70, 71, 72, or 73 can be utilized to direct expression of the synthetic signal peptide. One of skill in the art will know how to develop a suitable nucleotide sequence that will direct expression of a synthetic pre-protein signal peptide comprising the amino acid sequence of Formula XIII or SEQ ID NO: 70, 71, 72, or 73. In some embodiments, a synthetic preprotein signal peptide comprising the amino acid sequence of Formula XIII or SEQ ID NO:70, 71, 72, or 73 may be further fused directly or indirectly to a native constitutive proprotein signal peptide or a synthetic signal peptide disclosed herein. In some embodiments, the synthetic preprotein signal peptide is further fused to a native constitutive proprotein signal peptide. In some embodiments, the synthetic preprotein signal peptide is further fused to a synthetic signal peptide disclosed herein.

[0201] In some embodiments, a recombinant polypeptide is provided that includes a synthetic pre-protein signal peptide comprising the amino acid sequence of Formula XIII or SEQ ID NO: 70, 71, 72, or 73, and a payload protein. In some embodiments, the inclusion of a pre-protein signal peptide comprising the amino acid sequence of Formula XIII or SEQ ID NO: 70, 71, 72, or 73 results in the payload protein being more easily secreted by the yeast in which it is produced. Thus, in another embodiment, a method is provided for producing a payload protein using an Aspergillus yeast (e.g., A. niger), the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide comprising the amino acid sequence of Formula XIII or SEQ ID NO: 70, 71, 72, or 73, genetically modifying an Aspergillus yeast with the nucleic acid molecule, thereby producing an engineered yeast, and culturing the engineered yeast under conditions effective to express the recombinant polypeptide. In some embodiments, the nucleic acid molecule encoding the amino acid sequence of Formula XIII or SEQ ID NO: 70, 71, 72, or 73 is any nucleic acid molecule encoding the amino acid sequence.

[0202] In some embodiments, a method is provided for increasing extracellular secretion of a payload protein from an Aspergillus yeast (e.g., A. niger), the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic pre-protein signal peptide, genetically modifying an Aspergillus yeast with the nucleic acid, thereby producing an engineered yeast, and culturing the engineered yeast under conditions effective to secrete an increased amount of the payload protein when compared to the amount of payload protein secreted by an Aspergillus yeast genetically modified to express a recombinant polypeptide comprising a payload protein and a pre-protein signal peptide comprising a native pre-protein signal peptide sequence provided herein or a control pre-protein signal peptide sequence provided herein. In some embodiments, the control pre-protein signal peptide is: MSFRSLLALSGLVCTGLA (SEQ ID NO:76)

[0203] In some embodiments, the control preprotein signal peptide is a glucoamylase protein, represented by SEQ ID NO:77 below. MSFRSLLALSGLVCTGLANVISKRATLDSWLSNEATVARTAILNNIGADGAWVSGADSGIVVASPSTDNPDYFYTWTRDSGLVLKTLVDLFRNGDTSLLSTIENYISAQAIVQGISNPSGDLSSGAGLGEPKFNVDETAYTGSWGRPQRDGPALRATAMI GFGQWLLDNGYTSTATDIVWPLVRNDLSYVAQYWNQTGYDLWEEVNGSSFFTIAVQHRALVEGSAFATAVGSSCSWCDSQAPEILCYLQSFWTGSFILANFDSSRSGKDANTLLGSIHTFDPEAACDDSTFQPCSPRALANHKEVVDSFRSIYTLNDGLS DSEAVAVGRYPEDTYYNGNPWFLCTLAAAEQLYDALYQWDKQGSLEVTDVSLDFFKALYSDAATGTYSSSSSTYSSIVDAVKTFADGFVSIVETHAASNGSMSEQYDKSDGEQLSARDLTWSYAALLTANNRRNSVVPASWGETSASSVPGTCAATSAIGTYSSVTVTSWPSIVATGGTTTTATPTGSGSVTSTSSKTTATASKTSTSTSSTSCTTPTAVAVTFDLTATTTYGENIYLVGSISQLGDWETSDGIALSADKYTSSDPLWYVTVTLPAGESFEYKFIRIESDDSVEWESDPNREYTVPQACGTSTATVTDTWR (SEQ ID NO: 77)

[0204] In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of Formula XIII or SEQ ID NO: 70, 71, 72, or 73. In some embodiments, the synthetic preprotein signal peptide further comprises a naturally occurring proprotein signal peptide. In some embodiments, the synthetic preprotein signal peptide further comprises a synthetic proprotein signal peptide provided herein. In some embodiments, the synthetic preprotein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic preprotein signal peptide is fused indirectly to the payload protein, for example, via a peptide linker provided herein.

[0205] In some embodiments, an engineered Aspergillus yeast (e.g., A. niger) is provided, where the yeast is genetically modified with a nucleic acid molecule encoding expression of a recombinant polypeptide comprising a synthetic preprotein signal peptide fused directly or indirectly to a payload protein. In some embodiments, the synthetic preprotein signal peptide comprises the amino acid sequence of Formula XIII or SEQ ID NO: 70, 71, 72, or 73. In some embodiments, the synthetic preprotein signal peptide further comprises a naturally occurring proprotein signal peptide. In some embodiments, the synthetic preprotein signal peptide further comprises a synthetic proprotein signal peptide provided herein. In some embodiments, the synthetic preprotein signal peptide is fused directly to the payload protein. In some embodiments, the synthetic preprotein signal peptide is fused indirectly to the payload protein, for example, via a peptide linker provided herein.

[0206] In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group including enzymes (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), growth factors (e.g., IGF-1), insulin, incretins (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), cytokines, antibodies, antimicrobial peptides), mucosal proteins (e.g., trefoil factors, Reg3 proteins, superoxide dismutase), agricultural products (e.g., insecticides, bactericides, herbicides, fungicides, nematicides, acaricides, plant growth regulators, plant growth stimulants, or fertilizers), vaccines, diagnostic proteins, feed converting enzymes, flavorings, or nutritional proteins. The listed examples are provided for clarity only and are not intended to be limiting in any way. Thus, for example, the present disclosure is not limited to "growth factors" only IGF-1, but rather encompasses and includes all growth factors known in the art.

[0207] Synthetic proprotein signal peptides in Saccharomyces, Pichia, and Kluyveromyces yeast strains In some embodiments, various synthetic proprotein signal peptides are provided that, in addition to being suitable for use in combination with the preprotein signal peptides described above, may also be used without the synthetic preprotein signal peptide. In some embodiments, the proprotein signal peptide may comprise the amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24, any of which may be used in any yeast strain provided herein, such as Saccharomyces (e.g., S. cerevisiae, S. boulardii), Pichia (e.g., P. pastoris), and / or Kluyveromyces (e.g., K. lactis). In some embodiments, the synthetic signal peptide may comprise only the proprotein signal peptide comprising the amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, the synthetic signal peptide may further comprise any naturally occurring constitutive preprotein signal peptide. In some embodiments, the synthetic signal peptide may further comprise any synthetic preprotein signal peptide described herein. In some embodiments, when used in combination with a preprotein signal peptide (natural or synthetic), the N-terminus of the proprotein signal peptide may be fused directly or indirectly to the C-terminus of the preprotein signal peptide. The proprotein signal peptide may then be fused directly or indirectly to the N-terminus of the payload protein, optionally via a KR site, a Ste13 cleavage site, and / or a spacer. In some embodiments, indirect fusion may be achieved, for example, through the inclusion of a linker peptide as provided herein.

[0208] Thus, in some embodiments, a synthetic signal peptide is provided, which comprises a proprotein signal peptide comprising the amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24 fused directly or indirectly to a payload protein. In some embodiments, the synthetic signal peptide further comprises a preprotein signal peptide. In some embodiments, the preprotein signal peptide is a naturally occurring signal peptide. In some embodiments, the preprotein signal peptide is a synthetic signal peptide. In some embodiments, the preprotein signal peptide comprises the amino acid sequence of Formula III, Formula IV, Formula V, or SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16.

[0209] In some embodiments, a recombinant polypeptide is provided that includes a synthetic proprotein signal peptide comprising the amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24, and a payload protein. In some embodiments, the inclusion of a proprotein signal peptide comprising the amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24 results in the payload protein being more easily secreted by the yeast in which it is produced. Thus, in another embodiment, a method of producing a payload protein using a yeast strain is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide that includes a payload protein and a synthetic signal peptide comprising the amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24, genetically modifying the yeast with the nucleic acid, thereby producing an engineered yeast, and culturing the engineered yeast under conditions effective to express the recombinant polypeptide. In some embodiments, the yeast strain is selected from the group including Saccharomyces (e.g., S. cerevisiae, S. boulardii), Pichia (e.g., P. pastoris), and / or Kluyveromyces (e.g., K. lactis). In some embodiments, the nucleic acid molecule encoding the amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24 is any nucleic acid molecule encoding the amino acid sequence.

[0210] In some embodiments, a method of increasing extracellular secretion of a payload protein from a yeast strain is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic proprotein signal peptide, genetically modifying a yeast with the nucleic acid molecule, thereby producing an engineered yeast, and culturing the engineered yeast under conditions effective to secrete an increased amount of the payload protein when compared to the amount of payload protein secreted by a yeast genetically modified to express the recombinant polypeptide comprising the payload protein and a native proprotein signal peptide. In some embodiments, the yeast strain is selected from the group comprising Saccharomyces (e.g., S. cerevisiae, S. boulardii), Pichia (e.g., P. pastoris), and / or Kluyveromyces (e.g., K. lactis). In some embodiments, the synthetic proprotein signal peptide comprises the amino acid sequence of Formula VI, Formula VII, Formula VIII, or SEQ ID NO: 17, 18, 19, 20, 21, 22, 23, or 24. In some embodiments, the synthetic proprotein further comprises a native preprotein signal peptide. In some embodiments, the synthetic proprotein further comprises a synthetic preprotein signal peptide as provided herein. In some embodiments, the synthetic proprotein signal peptide is directly fused to the payload protein. In some embodiments, the synthetic proprotein signal peptide is indirectly fused to the payload protein, for example, via a peptide linker as provided herein.

[0211] In some embodiments, the payload protein may be any peptide or protein. In some embodiments, the payload protein is selected from the group including enzymes (e.g., invertase, isomaltase, lactase, lysozyme, An-PEP), growth factors (e.g., IGF-1), insulin, incretins (e.g., GLP-1, GLP-2, leptin, apelin, ghrelin, PYY, nesfatin), cytokines, antibodies, antimicrobial peptides), mucosal proteins (e.g., trefoil factors, Reg3 proteins, superoxide dismutase), agricultural products (e.g., insecticides, bactericides, herbicides, fungicides, nematicides, acaricides, plant growth regulators, plant growth stimulants, or fertilizers), vaccines, diagnostic proteins, feed converting enzymes, flavorings, or nutritional proteins. The listed examples are provided for clarity only and are not intended to be limiting in any way. Thus, for example, the present disclosure is not limited to "growth factors" only IGF-1, but rather encompasses and includes all growth factors known in the art.

[0212] Synthetic proprotein signal peptides in Trichoderma yeast strains. In some embodiments, various synthetic proprotein signal peptides are provided that, in addition to being suitable for use in combination with the preprotein signal peptides described above, may also be used without the synthetic preprotein signal peptide. In some embodiments, the proprotein signal peptide may comprise the amino acid sequence of Formula X, Formula XI, or SEQ ID NO: 34, 35, 36, 37, or 38, any of which may be used in any yeast species within the Trichoderma strain (e.g., T. reesei, T. viride). In some embodiments, the synthetic signal peptide may comprise only the amino acid sequence of Formula X, Formula XI, or SEQ ID NO: 34, 35, 36, 37, or 38. In some embodiments, the synthetic signal peptide may further comprise any naturally occurring constitutive preprotein signal peptide. In some embodiments, the synthetic signal peptide may further comprise any of the synthetic preprotein signal peptides provided herein. In some embodiments, when used in combination with a preprotein signal peptide (natural or synthetic), the N-terminus of the proprotein signal peptide may be fused directly or indirectly to the C-terminus of the preprotein signal peptide. The proprotein signal peptide can then be fused directly or indirectly to the N-terminus of the payload protein, optionally via a KR site, a Ste13 cleavage site, and / or a spacer, In some embodiments, indirect fusion can be achieved, for example, through the inclusion of a linker peptide as provided herein.

[0213] Thus, in some embodiments, a synthetic signal peptide is provided, which comprises a proprotein signal peptide comprising the amino acid sequence of Formula X, Formula XI, or SEQ ID NO: 34, 35, 36, 37, or 38 fused directly or indirectly to a payload protein. In some embodiments, the synthetic signal peptide further comprises a preprotein signal peptide. In some embodiments, the preprotein signal peptide is a naturally occurring preprotein signal peptide. In some embodiments, the preprotein signal peptide is a synthetic preprotein signal peptide provided herein. In some embodiments, the preprotein signal peptide comprises the amino acid sequence of Formula IX or SEQ ID NO: 31, 32, or 33.

[0214] In some embodiments, a recombinant polypeptide is provided that includes a synthetic proprotein signal peptide comprising the amino acid sequence of Formula X, Formula XI, or SEQ ID NO: 34, 35, 36, 37, or 38, and a payload protein. In some embodiments, the inclusion of a proprotein signal peptide comprising the amino acid sequence of Formula X, Formula XI, or SEQ ID NO: 34, 35, 36, 37, or 38 results in the payload protein being more easily secreted by the yeast in which it is produced. Thus, in another embodiment, a method of producing a payload protein using a yeast strain is provided, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic signal peptide comprising the amino acid sequence of Formula X, Formula XI, or SEQ ID NO: 34, 35, 36, 37, or 38, genetically modifying the yeast with the nucleic acid, thereby producing an engineered yeast, and culturing the engineered yeast under conditions effective to express the recombinant polypeptide. In some embodiments, the yeast strain is a Trichoderma yeast strain (e.g., T. reesei, T. viride). In some embodiments, a nucleic acid molecule encoding the amino acid sequence of Formula X, Formula XI, or SEQ ID NO: 34, 35, 36, 37, or 38 is any nucleic acid molecule encoding the amino acid sequence.

[0215] In some embodiments, a method is provided for increasing extracellular secretion of a payload protein from a yeast strain, the method comprising providing a nucleic acid molecule encoding a recombinant polypeptide comprising a payload protein and a synthetic proprotein signal peptide, genetically modifying yeast with the nucleic acid molecule, thereby producing an engineered yeast, and culturing the engineered yeast under conditions effective to secrete an increased amount of the payload protein when compared to the amount of payload protein secreted by a yeast genetically modified to express the recombinant polypeptide comprising the payload protein and a native proprotein signal peptide. In some embodiments, the yeast strain is a Trichoderma yeast strain (e.g., T. reesei, T. viride). In som...

Claims

**Claim 1**: A pre-protein signal peptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 31, 32, 33, 70, 71, 72, and 73. **Claim 2**: The pre-protein signal peptide according to claim 1, wherein the pre-protein signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15, 16, 28, 31, 32, 33, 55, 70, 71, 72, or 73. **Claim 3**: A pro-protein signal peptide comprising an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 17, 18, 19, 20, 21, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. **Claim 4**: The pro-protein signal peptide according to claim 3, wherein the pro-protein signal peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22, 17, 18, 19, 20, 21, 23, 24, 25, 27, 29, 34, 35, 36, 37, 38, 56, 57, 58, 74, or 75. **Claim 5**: A polypeptide comprising the formula (X1)n - (Y1)m - Z1, wherein X1 is the pre-protein signal peptide according to claim 1, Y1 is the pro-protein signal peptide according to claim 3, Z1 is a payload protein, n is 0 to 1, m is 0 to 1, n and m cannot be 0 simultaneously. **Claim 6**: The polypeptide according to claim 5, wherein Z1 is selected from the group consisting of antiviral agents, insulin, incretin, enzymes, enzyme inhibitors, hormones, cytokines, antibodies, antibacterial peptides, mucosal proteins, insecticides, fungicides, herbicides, fungicides, nematicides, acaricides, plant growth regulators, plant growth stimulants, or fertilizers, vaccines, diagnostic proteins, feed conversion enzymes, flavoring agents, and nutritional proteins. **Claim 7**: A yeast comprising a heterologous nucleic acid molecule encoding a polypeptide having the formula (X1)n-(Y1)m-Z1, wherein X1 is the preprotein signal peptide according to claim 1, Y1 is the proprotein signal peptide according to claim 3, Z1 is a payload protein, n is 0 to 1, m is 0 to 1, provided that both n and m are not 0. **Claim 8**: The yeast according to claim 7, wherein the yeast is selected from the group consisting of Kluyveromyces, Pichia, Saccharomyces, Trichoderma, and Aspergillus, and optionally, i) the yeast is Kluyveromyces yeast, X1 comprises the amino acid sequence of SEQ ID NO: 1, and Y1 comprises an amino acid sequence selected from SEQ ID NO: 20 or SEQ ID NO: 21; ii) the yeast is Pichia yeast (e.g., P. pastoris), X1 comprises an amino acid sequence selected from SEQ ID NO: 2, 3, 4, 5, 6, or 7, and Y1 comprises an amino acid sequence selected from SEQ ID NO: 20 or SEQ ID NO: 21; iii) the yeast is Saccharomyces yeast, X1 comprises an amino acid sequence selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16, and Y1 comprises an amino acid sequence selected from SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, or 25; iv) the yeast is Trichoderma yeast, X1 comprises an amino acid sequence selected from SEQ ID NO: 31, 32, or 33, and Y1 comprises an amino acid sequence selected from SEQ ID NO: 34, 35, 36, 37, or 38, or v) the yeast is an Aspergillus yeast (e.g., A. niger), X1 comprises an amino acid sequence selected from SEQ ID NO: 70, 71, 72, or 73, and Y1 comprises an amino acid sequence selected from SEQ ID NO: 74 or SEQ ID NO: 75, yeast. **Claim 9** The yeast according to claim 7 or 8, wherein Z1 is selected from the group consisting of antiviral agents, insulin, incretin, enzymes, enzyme inhibitors, hormones, insecticides, cytokines, antibodies, antibacterial peptides, mucosal proteins, fungicides herbicides, fungicides, nematicides, acaricides, plant growth regulators, plant growth stimulants, or fertilizers, vaccines, diagnostic proteins, feed conversion enzymes, flavoring agents, and nutritional proteins. **Claim 10** A method for producing a payload protein, i) transfecting a yeast with a nucleic acid encoding the polypeptide according to claim 5 or 6 to produce a engineered yeast; ii) culturing the engineered yeast in an environment effective for growing the engineered yeast; iii) inducing secretion of the payload protein by the engineered yeast, wherein the step of inducing secretion of the payload protein comprises culturing the yeast under conditions sufficient to express the polypeptide according to claim 5 or 6, and the presence of the signal peptide induces secretion of the payload protein, the step of inducing secretion of the payload protein. A method comprising the above steps. **Claim 11** In the method according to claim 10, the yeast is selected from the group consisting of Kluyveromyces, Pichia, Saccharomyces, Trichoderma, and Aspergillus, and optionally, i) the yeast is a Kluyveromyces yeast, X1 comprises the amino acid sequence of SEQ ID NO: 1, and Y1 comprises an amino acid sequence selected from SEQ ID NO: 20 or SEQ ID NO: 21; ii) the yeast is a Pichia yeast (e.g., P. pastoris), X1 comprises an amino acid sequence selected from SEQ ID NO: 2, 3, 4, 5, 6, or 7, and Y1 comprises an amino acid sequence selected from SEQ ID NO: 20 or SEQ ID NO:

21. iii) the yeast is Saccharomyces yeast, X1 comprises an amino acid sequence selected from SEQ ID NO: 8, 9, 10, 11, 12, 13, 14, 15, or 16, and Y1 comprises an amino acid sequence selected from SEQ ID NO: 18, 19, 20, 21, 22, 23, 24, or 25; iv) the yeast is Trichoderma yeast, X1 comprises an amino acid sequence selected from SEQ ID NO: 31, 32, or 33, and Y1 comprises an amino acid sequence selected from SEQ ID NO: 34, 35, 36, 37, or 38; or v) the yeast is Aspergillus yeast (e.g., A. niger), X1 comprises an amino acid sequence selected from SEQ ID NO: 70, 71, 72, or 73, and Y1 comprises an amino acid sequence selected from SEQ ID NO: 74 or SEQ ID NO: 75; Method. **Claim 12**: The method according to claim 10 or 11, wherein Z1 is selected from the group consisting of an antiviral agent, insulin, incretin, cytokine, antibody, antibacterial peptide, mucosal protein, enzyme, enzyme inhibitor, hormone, insecticide, bactericide, herbicide, fungicide, nematicide, acaricide, plant growth regulator, plant growth stimulant, fertilizer, vaccine, diagnostic protein, feed conversion enzyme, flavoring agent, or nutritional protein. **Claim 13**: The yeast according to any one of claims 7 to 9, for use in the treatment of a disease or condition in a subject in need thereof. **Claim 14**: The yeast for use according to claim 13, wherein the disease or condition is selected from an infectious disease, an autoimmune disease, a primary (congenital) enzyme deficiency disorder, an enzyme deficiency disorder secondary to a functional bowel disorder, diabetes, obesity, a metabolic disorder, an abnormal overgrowth of intestinal bacteria, an intestinal infectious disease, bacterial vaginitis, an inflammatory bowel disease, irritable bowel syndrome, small intestine syndrome, celiac disease, gluten intolerance, colitis, peptic ulcer, or another gastrointestinal condition or disorder. **Claim 15**: The yeast for use according to claim 13 or 14, wherein i) the disease or condition is an enzyme deficiency disorder and the payload protein is an enzyme; or ii) the disease or condition is congenital sucrase-isomaltase deficiency and the payload protein is one or both of invertase and isomaltase; or iii) the disease or condition is one or both of sucrose and isomaltase intolerance secondary to functional bowel disorder, and the payload protein is one or both of invertase and isomaltase, or, iv) the disease or condition is one or more of gluten intolerance, refractory sprue, or celiac disease, and the payload protein is one or more of An-PEP, Mx-PEP, Aspergillus tubigensis prolyl endopeptidase, subtilisin, sedolisin, and larototide, or, v) the disease or condition is pancreatitis or exocrine pancreatic insufficiency, and the payload protein is selected from one or more of triacylglycerol lipase, colipase, alpha-amylase, trypsin, and chymotrypsin, or, vi) the disease or condition is enteropeptidase deficiency or enterokinase deficiency, and the payload protein is one or all of enteropeptidase, proenteropeptidase, and enterokinase, or, vii) the disease or condition is small intestinal bacterial overgrowth, inflammatory bowel disease, irritable bowel syndrome, C. difficile infection, cystic fibrosis, necrotizing enterocolitis, and diabetes, and the payload protein is intestinal alkaline phosphatase, or, viii) the disease or condition is short bowel syndrome, and the payload protein is IGF-1, GLP-2, or a synthetic derivative of GLP-2, or, ix) the disease or condition is lactose hypersensitivity or lactose intolerance, and the payload protein is lactase, or, x) the disease or condition is trehalose hypersensitivity or lactose intolerance, and the payload protein is trehalase, or, xi) the disease or condition is maltose hypersensitivity or lactose intolerance, and the payload protein is maltase, or, xii) the disease or condition is pernicious anemia, and the payload protein is intrinsic factor, or, xiii) the disease or condition is a bacterial overgrowth disorder, and the payload protein is lysozyme, nisin, defensin, magainin, catestatin, or any combination thereof, or, xiv) the disease or condition is type 1 diabetes or type 2 diabetes, and the payload protein is insulin or an incretin, or, xv) the disease or condition has an inflammatory component, and the payload protein is IL-10, IL-22, TGFβ, an anti-TNFα antibody or fragment thereof, or any combination thereof, or, xvi) the condition is a bacterial infection caused by one or more of E. coli, C. difficile, vibrio cholera, Shigella, Salmonella, Cryptosporidium, or any combination thereof, or, xvii) the condition is a viral infection, Yeast.

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