Fusion polypeptides

Abstract

Provided herein, inter alia, are fusion DNA constructs comprising improved protease recognition sequences for expression and purification of one or more polypeptides of interest, and methods for producing one or more polypeptides of interest in recombinant host cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Patent Application No. 62 / 815,912, filed March 8, 2019, the disclosure of which is incorporated herein by reference in its entirety. Technical Field

[0003] Provided herein, inter alia, are compositions comprising improved cleavable fusion polypeptides and methods of using the same to produce one or more polypeptides of interest in host cells. Background Art

[0004] It has been reported that fusion polypeptides are produced in a variety of organisms including Escherichia coli (E. coli), yeast and filamentous fungi. In some of these fusion proteins, a protease recognition site has been inserted between the target polypeptide and the carrier protein (e.g., Contreras et al., 1991, Biotechnology [biotechnology] (New York), 9 (4): 378-81 and Ward et al., 1995, Biotechnology [biotechnology] (New York), 13 (5): 498-503). However, incomplete cleavage has been observed in many cases, which results in poor yields of one or more target polypeptides. Therefore, there is a need for compositions and methods for improved fusion protein production and efficient and complete protease-mediated fusion protein cleavage. The subject matter disclosed herein meets these needs and also provides additional benefits. Summary of the Invention

[0005] Provided herein are, inter alia, non-naturally occurring engineered fusion polypeptides having altered amino acid sequences that result in improved or complete protease-mediated (e.g., KEX2-mediated) cleavage when producing and purifying one or more polypeptides of interest in a host cell, and methods of utilizing the fusion polypeptides. The disclosed methods, engineered fusion polypeptides, and recombinant host cells provide for enhanced secretion and / or purification of the fusion polypeptides of one or more polypeptides of interest compared to fusion polypeptides that do not contain the disclosed altered amino acid sequences and / or that are not used according to the methods disclosed herein.

[0006] Thus, in some aspects, provided herein is a fusion polypeptide comprising the amino acid sequence T1-S2-V-A3-V4-E5-X1-X2-Q6-V7- (SEQ ID NO: 1), wherein: 1) X1 and X2 are basic amino acids; and 2) the amino acid sequence has one or more substitutions selected from the group consisting of: T1 is substituted with an amino acid selected from the group consisting of hydrophobic amino acids, aromatic amino acids, acidic amino acids, and basic amino acids; S2 is substituted with an amino acid selected from the group consisting of hydrophobic amino acids, aromatic amino acids, basic amino acids, and amino acids that influence chain orientation; A3 is substituted with an amino acid selected from the group consisting of basic amino acids and M; V4 is substituted with L; E5 is substituted with an aromatic amino acid; Q6 is substituted with an amino acid selected from the group consisting of acidic amino acids and amino acids that influence chain orientation; and / or V7 is substituted with an amino acid selected from the group consisting of L, I, and aromatic amino acids. In some embodiments, T1 is substituted with an amino acid selected from the group consisting of A, F, M, Q, R, N, E, and Y; S2 is substituted with an amino acid selected from the group consisting of F, H, K, L, M, P, Q, R, N, and V; A3 is substituted with an amino acid selected from the group consisting of H, K, M, and R; E5 is substituted with an amino acid selected from the group consisting of F and W; Q6 is substituted with an amino acid selected from the group consisting of D and G; and / or V7 is substituted with an amino acid selected from the group consisting of L, I, and F.

[0007] In some embodiments of any of the embodiments disclosed herein, the amino acid sequence is selected from the group consisting of ASVAVEKRQV (SEQ ID NO:3), FSVAVEKRQV (SEQ ID NO:2), MSVAVEKRQV (SEQ ID NO:4), QSVAVEKRQV (SEQ ID NO:5), RSVAVEKRQV (SEQ ID NO:6), and YSVAVEKRQV (SEQ ID NO:7). In some embodiments of any of the embodiments disclosed herein, the amino acid sequence is selected from the group consisting of: TFVAVEKRQV (SEQ ID NO: 8), THVAVEKRQV (SEQ ID NO: 9), TKVAVEKRQV (SEQ ID NO: 10), TLVAVEKRQV (SEQ ID NO: 11), TMVAVEKRQV (SEQ ID NO: 12), TPVAVEKRQV (SEQ ID NO: 13), TQVAVEKRQV (SEQ ID NO: 14), TRVAVEKRQV (SEQ ID NO: 15), and TVVAVEKRQV (SEQ ID NO: 16). In some embodiments of any of the embodiments disclosed herein, the amino acid sequence is selected from the group consisting of: TSVHVEKRQV (SEQ ID NO: 17), TSVKVEKRQV (SEQ ID NO: 18), and TSVRVEKRQV (SEQ ID NO: 19). In some embodiments of any embodiment disclosed herein, the amino acid sequence is TSVALEKRQV (SEQ ID NO: 20). In some embodiments of any embodiment disclosed herein, the amino acid sequence is selected from the group consisting of TSVAVFKRQV (SEQ ID NO: 21) and TSVAVWKRQV (SEQ ID NO: 22). In some embodiments of any embodiment disclosed herein, the amino acid sequence is selected from the group consisting of TSVAVEKRDV (SEQ ID NO: 23) and TSVAVEKRGV (SEQ ID NO: 24). In some embodiments of any embodiment disclosed herein, the amino acid sequence is selected from the group consisting of TSVAVEKRQF (SEQ ID NO: 25) and TSVAVEKRQL (SEQ ID NO: 26). In some embodiments of any embodiment disclosed herein, the amino acid sequence further comprises two additional amino acids T8-L9 (SEQ ID NO: 64) at the C-terminus, wherein T8 is substituted with an amino acid selected from the group consisting of an acidic amino acid and a hydrophobic amino acid; and / or L9 is substituted with an amino acid selected from the group consisting of I and V. In some embodiments, T8 is substituted with E or F.In some embodiments of any of the embodiments disclosed herein, the amino acid sequence is TSVAVEKRQVEL (SEQ ID NO: 27). In some embodiments of any of the embodiments disclosed herein, the amino acid sequence is selected from the group consisting of TSVAVEKRQVTI (SEQ ID NO: 28) and TSVAVEKRQVTV (SEQ ID NO: 29). In some embodiments of any of the embodiments disclosed herein, the fusion polypeptide comprises two or more substitutions. In some embodiments, at least one of the two or more substitutions is at S2 or V4. In some embodiments, the substitution at S2 is H or N. In some embodiments, the amino acid sequence is selected from the group consisting of THVAVEKRQVTI (SEQ ID NO: 30), THVAVEKRDVTL (SEQ ID NO: 31), THVAVEKRQVAL (SEQ ID NO: 32), EHVAVEKRQVTL (SEQ ID NO: 33), TNVAVEKRDVTL (SEQ ID NO: 34), and TNVAVEKRQVAL (SEQ ID NO: 35). In some embodiments, the substitution at V4 is L. In some embodiments, the amino acid sequence is selected from the group consisting of: TSVALWKRQVTL (SEQ ID NO: 36), TSVMLEKRQVTL (SEQ ID NO: 37), TSVALEKRQITL (SEQ ID NO: 38), and TSVALEKRQVAL (SEQ ID NO: 39). In some embodiments, the fusion polypeptide comprises substitutions at S2 and V4. In some embodiments, the amino acid sequence is THVALEKRQVTL (SEQ ID NO: 40). In some embodiments, the amino acid sequence is TSVAVEKRDVAL (SEQ ID NO: 41). In some embodiments of any of the embodiments disclosed herein, the fusion polypeptide comprises three or more substitutions.In some embodiments, the amino acid sequence is selected from the group consisting of THVMLEKRQVTL (SEQ ID NO:42), TKVMLEKRQVTL (SEQ ID NO:43), THVAVEKRDVAL (SEQ ID NO:44), THVALWKRQVTL (SEQ ID NO:45), TKVAVEKRDLTL (SEQ ID NO:46), TNVAVEKRDLTL (SEQ ID NO:47), EHVAVWKRQVTL (SEQ ID NO:48), EHVALEKRQVTL (SEQ ID NO:49), ESVALWKRQVTL (SEQ ID NO:50), RSVRVEKRDVTL (SEQ ID NO:51), and TSVALEKRDVAL (SEQ ID NO:52). In some embodiments of any of the embodiments disclosed herein, the fusion polypeptide comprises four or more substitutions. In some embodiments, the amino acid sequence is selected from the group consisting of THVALEKRDVAL (SEQ ID NO: 53), TKVRVEKRDLTL (SEQ ID NO: 54), TNVALEKRDVAL (SEQ ID NO: 55), EHVALWKRQVTL (SEQ ID NO: 56), EPVALWKRQVTL (SEQ ID NO: 57), NHVALWKRQVTL (SEQ ID NO: 58), RSVRVEKRDLTL (SEQ ID NO: 59), RKVRVEKRDVTL (SEQ ID NO: 60), RKVRVEKRQLTL (SEQ ID NO: 61), and RKVAVEKRDLTL (SEQ ID NO: 62). In some embodiments of any of the embodiments disclosed herein, the fusion polypeptide comprises five or more substitutions. In some embodiments, the amino acid sequence is RKVRVEKRDLTL (SEQ ID NO: 63). In some embodiments of any of the embodiments disclosed herein, X1 and X2 are selected from the group consisting of KK, RR, KR, and RK. In some embodiments of any of the embodiments disclosed herein, the amino acid sequence is completely cleaved by one or more proteases. In some embodiments, the protease is Kex2 serine peptidase (EC 3.4.21.61). In some embodiments of any of the embodiments disclosed herein, the fusion polypeptide further comprises a polypeptide encoding a signal sequence. In some embodiments, the polypeptide encoding the signal sequence is located at the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 1. In some embodiments of any of the embodiments disclosed herein, the fusion polypeptide further comprises a polypeptide encoding a carrier protein.In some embodiments, the polypeptide encoding the carrier protein is located at the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 1. In some embodiments of any of the embodiments disclosed herein, the polypeptide encoding the carrier protein is adjacent to the polypeptide encoding the signal sequence. In some embodiments of any of the embodiments disclosed herein, the carrier protein comprises CBH1 or a fragment thereof. In some embodiments of any of the embodiments disclosed herein, the fusion polypeptide further comprises a polypeptide of interest. In some embodiments, the polypeptide encoding the polypeptide of interest is located at the N-terminus or C-terminus of the amino acid sequence of SEQ ID NO: 1. In some embodiments of any of the embodiments disclosed herein, the polypeptide of interest is an enzyme. In some embodiments, the enzyme is selected from the group consisting of an active or inactive carbohydrate-degrading enzyme, a protease, a lipase, and a lytic enzyme. In some embodiments of any of the embodiments disclosed herein, the polypeptide of interest is a therapeutic protein. In some embodiments, the therapeutic protein is an antibody or a functional fragment thereof. In some embodiments, the antibody is a light chain or heavy chain monoclonal antibody. In some embodiments of any of the embodiments disclosed herein, the signal sequence is a CBH1 signal sequence, the carrier protein is a carrier protein containing CBH1, and the polypeptide of interest is an antibody light chain or a functional fragment thereof. In some embodiments of any of the embodiments disclosed herein, the signal sequence is a CBH1 signal sequence, the carrier protein is a carrier protein containing CBH1, and the polypeptide of interest is an antibody heavy chain or a functional fragment thereof. In some embodiments of any of the embodiments disclosed herein, the signal sequence is a CBH1 signal sequence, the carrier protein is a carrier protein containing CBH1, and the polypeptide of interest is an antibody heavy chain or a fragment thereof and an antibody light chain or a functional fragment thereof. In some embodiments, the antibody or its functional fragment is a single domain antibody (sdAb). In some embodiments, the antibody or its functional fragment is selected from the group consisting of: Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules (e.g., scFv), and multispecific antibodies formed by antibody fragments. In some embodiments of any of the embodiments disclosed herein, the antibody or functional fragment thereof is an anti-respiratory syncytial virus (RSV) antibody, an anti-Ebola virus antibody, an anti-aggregated β-amyloid protein (Aβ) antibody, an anti-human immunodeficiency virus (HIV) antibody, an anti-herpes simplex virus (HSV) antibody, an anti-sperm antibody (such as an anti-human contraceptive antigen (HCA) antibody), and an anti-HER2 / neu antibody.

[0008] In other aspects, provided herein are nucleic acids encoding any of the fusion polypeptides disclosed herein.

[0009] In a further aspect, provided herein is a vector encoding any of the nucleic acids disclosed herein. In some embodiments, the vector further comprises a nucleic acid sequence encoding a promoter.

[0010] In yet other aspects, provided herein is a host cell comprising any fusion polypeptide disclosed herein, any nucleic acid disclosed herein, and / or any vector disclosed herein. In certain embodiments, the host cell is selected from the group consisting of a mammalian host cell, a bacterial host cell, and a fungal host cell. In certain embodiments, the mammalian cell is a Chinese hamster ovary (CHO) cell. In certain embodiments, the bacterial cell is an Escherichia coli cell. In certain embodiments, the fungal cell is a yeast cell or a filamentous fungal cell. In certain embodiments, the yeast cell is a Saccharomyces species. In some embodiments of any of the embodiments disclosed herein, the fungal cell is selected from the group consisting of Trichoderma species, Penicillium species, Humicola species, Chrysosporium species, Gliocladium species, Aspergillus species, Fusarium species, Mucor species, Neurospora species, Hypocrea species, Myceliophthora species, and Emericella species. In some embodiments, the fungal cell is selected from the group consisting of Trichoderma reesei, Trichoderma viride, Trichoderma koningii, Trichoderma harzianum, Humicola insolens, Humicola grisea, Chrysosporium lucknowense, Aspergillus oryzae, Aspergillus niger, Aspergillus nidulans, Aspergillus skawachi, Aspergillus aculeatus, Aspergillus japonicus, Aspergillus sojae, Myceliophthora thermophila, and Aspergillus awamori.

[0011] In other aspects, provided herein are methods for producing any of the fusion polypeptides disclosed herein, comprising: culturing any of the host cells disclosed herein under conditions suitable for producing the fusion polypeptide. In some embodiments, the method further comprises isolating the fusion polypeptide. In some embodiments of any of the embodiments disclosed herein, the method further comprises cleaving the fusion polypeptide with a protease. In some embodiments, the protease is a Kex2 serine peptidase (EC 3.4.21.61). In some embodiments of any of the embodiments disclosed herein, the cleavage of the fusion polypeptide is enhanced compared to the cleavage of an equivalent fusion polypeptide lacking the amino acid sequence of SEQ ID NO: 1. In some embodiments, the method further comprises isolating one or both products of the cleaved fusion polypeptide. In some embodiments of any of the embodiments disclosed herein, the secretion of the fusion polypeptide is enhanced compared to the secretion of an equivalent fusion polypeptide lacking the amino acid sequence of SEQ ID NO: 1.

[0012] In additional aspects, provided herein are methods of cleaving a fusion polypeptide, the methods comprising: contacting any of the fusion polypeptides disclosed herein with a protease. In some embodiments, the protease is Kex2 serine peptidase (EC 3.4.21.61).

[0013] In other aspects, provided herein are kits comprising: a) written instructions for producing any fusion polypeptide disclosed herein; and b) one or more nucleic acids disclosed herein; 2) any vector disclosed herein; and / or 3) any host cell disclosed herein. In some embodiments, the kit further comprises one or more of the following: 4) a composition comprising a Kex2 serine peptidase (EC 3.4.21.61); and / or 5) a nucleic acid encoding a Kex2 serine peptidase. In some embodiments of any embodiment disclosed herein, the kit further comprises a host cell expressing the Kex2 serine peptidase. In some embodiments of any embodiment disclosed herein, the kit further comprises one or more of the following: 6) a composition comprising an additional protease; and / or 7) a nucleic acid encoding an additional protease.

[0014] Each of the aspects and embodiments described herein can be used together unless expressly or clearly excluded from the context of that embodiment or aspect.

[0015] Throughout this specification, various patents, patent applications, and other types of publications (e.g., journal articles, electronic database entries, etc.) are cited. The disclosures of all patents, patent applications, and other publications cited herein are hereby incorporated by reference in their entirety for all purposes. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 is a schematic diagram depicting a fusion polypeptide including the pre-sequence and post-sequence of the KEX2 site.

[0017] Figure 2 Depicted are representative SDS-PAGE gels showing the cleavage efficiency of selected engineered KEX2 recognition sequences. Asterisks (*) indicate unchanged sequences.

[0018] Figure 3 Depicted is a schematic diagram of the entry clone used for Synagis HC heavy chain SEL library construction.

[0019] Figure 4 A schematic diagram of the expression vector pTTTpyr2-ISceI-Synagis HC_Geneart_SEL heavy chain is depicted.

[0020] Figure 5A The nucleotide sequence of the Synagis antibody heavy chain is depicted. Figure 5B The amino acid sequence of the Synagis antibody heavy chain is depicted.

[0021] Figure 6 A schematic diagram of the pAS25 expression vector is depicted.

[0022] Figure 7A A schematic diagram of the trastuzumab heavy chain vector with the TKVAVEKR kex sequence is depicted. Figure 7B A schematic diagram of the trastuzumab heavy chain vector with the TSVAVEKR kex sequence is depicted.

[0023] Figure 8A A schematic diagram of the trastuzumab light chain vector with the TKVAVEKR kex sequence is depicted. Figure 8B A schematic diagram of the trastuzumab light chain vector with the TSVAVEKR kex sequence is depicted.

[0024] Figure 9 Depicted are the results of a Western blot showing cleavage of trastuzumab from CBH1.

[0025] Figure 10A The amino acid sequence of trastuzumab HC is depicted. Figure 10B The nucleotide sequence of the trastuzumab HC in pJC159 is depicted. Figure 10C The nucleotide sequence of the trastuzumab HC in pJC158 is depicted.

[0026] Figure 11A The amino acid sequence of trastuzumab LC is depicted. Figure 11B The nucleotide sequence of trastuzumab LC is depicted.

[0027] Figure 12A The amino acid sequence of CBH1 is depicted. Figure 12B The nucleotide sequence of CBH1 is depicted. DETAILED DESCRIPTION

[0028] The invention disclosed herein is based in part on the inventors' observation that protein secretion and / or proteolysis in fusion polypeptides is enhanced when a protease recognition site is engineered to include one or more alternative substituted amino acids in the pre- and / or post-sequence of the protease recognition site.

[0029] Therefore, this paper provides fusion DNA construct, vector, fusion polypeptide, host cell expressing fusion DNA construct and / or fusion polypeptide and the method for secretion and / or cracking of the fusion polypeptide produced in the host cell for enhancing.More specifically, and in some non-limiting aspects, the KEX2 site presequence and / or postsequence of through engineering approaches have been included in the fusion polypeptide to enhance or improve the cracking target polypeptide from the fusion polypeptide.Compared with the fusion polypeptide that does not comprise the protease recognition site presequence and / or postsequence of through engineering approaches disclosed herein, the fusion polypeptide disclosed herein shows secretion and / or purification of better target polypeptide.Therefore, the disclosure provides the alternative and improved method for protein production (particularly therapeutic protein production, such as antibody production), which causes relatively fast amplification time and high-level purified protein, and the risk of limited uncleaved product pollution.

[0030] I. Definition

[0031] The term "polypeptide" or "protein" means a polymer containing any of the 20 naturally occurring amino acids, regardless of size. Although the term "protein" is generally used to refer to relatively large proteins, and "peptide" is generally used to refer to small polypeptides, the use of these terms in the art generally overlaps. Therefore, unless otherwise indicated, the term "polypeptide" generally refers to proteins, polypeptides, and peptides. Conventional one-letter or three-letter codes for amino acid residues are used herein.

[0032] As used herein, "fusion polypeptide" or "fusion protein" refers to a polypeptide comprising two or more different polypeptides or active fragments thereof that are not naturally present in the same polypeptide. In some embodiments, the two or more different polypeptides are effectively linked together in a covalent manner, for example, by chemical linkage or in-frame fusion via a peptide bond.

[0033] The term "protease recognition site" refers to a cleavage motif in a polypeptide amino acid sequence that is cleaved by a protease.

[0034] The term "protease recognition site presequence" refers to the 2 to 6 consecutive amino acids [(X) n , where n is 2 to 6].

[0035] The term "post-protease recognition site sequence" refers to the 2 to 6 consecutive amino acids [(X) n , where n is 2 to 6].

[0036] The term "KEX2" refers to a calcium-dependent endopeptidase active according to the IUBMB enzyme nomenclature defined as EC 3.4.21.61. During protein secretion, KEX2 cleaves the peptide bond immediately C-terminal to a pair of basic amino acids (KEX2 cleavage site).

[0037] The term "KEX2 region" refers to a continuous region of 4 to 12 amino acid residues (such as any one of 4, 5, 6, 7, 8, 9, 10, 11, or 12 amino acids) located in a polypeptide (e.g., a fusion polypeptide). The KEX2 region consists of a KEX2 site, a KEX2 site presequence, and a KEX2 site postsequence.

[0038] The term "KEX2 site" refers to a two-amino acid KEX2 protease recognition site cleavage motif in a polypeptide. A KEX2 site contains two consecutive basic amino acids (e.g., lysine, histidine, and / or arginine) arranged in any order (e.g., KK, RR, KR, or RK).

[0039] The term "KEX2 site presequence" refers to the 2 to 8 consecutive amino acids [(X) n , wherein n is 2 to 8, such as any one of 2, 3, 4, 5, 6, 7, or 8]. For example, if the KEX2 region is defined as TSVAVEKRQV (SEQ ID NO: 80), then the "KR" motif is the KEX2 site of the region, n is 6 and the "TSVAVE" motif (SEQ ID NO: 81) corresponds to the KEX2 site presequence of the region.

[0040] The term "post-KEX2 site sequence" refers to one or two (or in other embodiments, one to four) consecutive amino acids [(X) n, wherein n is 1 to 4, such as any one of 1, 2, 3, or 4]. For example, if the KEX2 region is defined as TSVAVEKRQV (SEQ ID NO: 80), the "KR" motif is the KEX2 site of the region, n is 2 and the "QV" motif corresponds to the sequence after the KEX2 site of the region.

[0041] The term "nucleic acid" or "polynucleotide" encompasses single-stranded or double-stranded DNA, RNA, and chemical modifications thereof. Herein, the terms "nucleic acid" and "polynucleotide" are used interchangeably. Because the genetic code is degenerate, more than one codon can be used to encode a specific amino acid, and the present subject matter encompasses nucleotide sequences that encode a specific amino acid sequence.

[0042] With respect to polypeptides, the terms "wild-type," "wildtype," "parental," or "reference" refer to naturally occurring polypeptides that do not include artificial substitutions, insertions, or deletions at one or more amino acid positions. Similarly, with respect to polynucleotides, the terms "wild-type," "wild type," "parental," or "reference" refer to naturally occurring polynucleotides that do not include artificial nucleoside changes. However, polynucleotides encoding wild-type, parental, or reference polypeptides are not limited to naturally occurring polynucleotides, but encompass any polynucleotides encoding wild-type, parental, or reference polypeptides.

[0043] As used herein, the term "non-naturally occurring" refers to any substance not found in nature (e.g., recombinant nucleic acid and protein sequences produced in the laboratory), such as modifications of wild-type nucleic acid and / or amino acid sequences. In some embodiments, a non-naturally occurring polypeptide comprises an amino acid substitution (i.e., a mutation) that is not found in the corresponding wild-type or naturally occurring amino acid sequence.

[0044] As used herein, a "derivative" or "variant" of a polypeptide means a polypeptide derived from a precursor polypeptide (e.g., a native polypeptide) by the addition of one or more amino acids to one or both of the C-terminus and the N-terminus, the substitution of one or more amino acids at one or more of the different sites in the amino acid sequence, the deletion of one or more amino acids at one or both termini of the polypeptide or at one or more sites in the amino acid sequence, or the insertion of one or more amino acids at one or more sites in the amino acid sequence.

[0045] As used herein, "variant polynucleotide" encodes a variant polypeptide, has a certain degree of homology / identity with the parent polynucleotide, or hybridizes under stringent conditions with the parent polynucleotide or its complement. Suitably, the variant polynucleotide has at least 80%, at least 85%, 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 even at least 99% nucleotide sequence identity with the parent polynucleotide or the complement of the parent polynucleotide. Methods for determining percent identity are known in the art.

[0046] The term "derived from" encompasses the terms "originate from," "obtained from," "obtainable from," "isolated from," and "produced from," and generally indicates that one specified material finds its source in, or has characteristics that can be described with reference to, another specified material.

[0047] "Control sequences" are defined herein as including all components that are necessary or advantageous for the expression of a polynucleotide or polypeptide of interest. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader sequence, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence, and a transcription terminator. At a minimum, the control sequences include a promoter, and transcriptional and translational termination signals. These control sequences may be provided with a plurality of linkers for the purpose of introducing specific restriction sites that facilitate ligation of these control sequences to the coding region of the nucleic acid sequence encoding the polypeptide.

[0048] "Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed in a functional relationship (i.e., at a position relative to) a polynucleotide or polypeptide of interest (such as a coding sequence in a DNA sequence) such that the control sequence directs or regulates the expression of the polynucleotide and / or polypeptide.

[0049] As used herein, "DNA construct" means a DNA sequence that is operably linked to suitable control sequences that enable protein expression in a suitable host. Such control sequences may include a promoter that enables transcription, an optional operator sequence that controls transcription, a sequence encoding a suitable ribosome binding site on the mRNA, an enhancer, and sequences that control transcription and translation termination.

[0050] The term "fusion DNA construct" or "fusion nucleic acid" refers to a nucleic acid comprising multiple polynucleotide sequences operatively linked from 5' to 3' (for example, but not limited to, a DNA molecule encoding a signal sequence, a DNA molecule encoding a carrier protein, a DNA molecule encoding a KEX2 site, and a DNA molecule encoding a polypeptide of interest) and encoding a fusion polypeptide.

[0051] "Vector" refers to a polynucleotide sequence designed to introduce a nucleic acid into one or more cell types. Vectors include cloning vectors, expression vectors, shuttle vectors, plasmids, phage particles, cassettes, and the like.

[0052] An "expression vector" is a vector that has the ability to incorporate and express a DNA fragment in a cell. Many prokaryotic and eukaryotic expression vectors are commercially available.

[0053] A "promoter" or "promoter sequence" is a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest (such as a coding region). Typically, the promoter sequence contains transcriptional control sequences that mediate expression of the polynucleotide of interest. The promoter can be any nucleic acid sequence that shows transcriptional activity in the selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0054] The term "signal sequence" refers to an amino acid sequence at the amino terminus of a protein that directs the protein to a secretion system for secretion from a cell. The signal sequence is cleaved from the protein prior to secretion. In some cases, a signal sequence may be referred to as a "signal peptide" or "leader peptide." The definition of a signal sequence is a functional definition. The mature form of an extracellular protein lacks a signal sequence that is cleaved during secretion.

[0055] As used herein, the term "carrier protein" refers to a protein that acts on or promotes the secretion of a polypeptide (such as a fusion polypeptide) from a host cell. Exemplary carrier proteins are discussed in more detail below.

[0056] The term "recombinant" when used in reference to a subject cell, nucleic acid, polypeptide / enzyme or vector indicates that the subject cell, nucleic acid, polypeptide / enzyme or vector has been modified from its native state. Thus, for example, a recombinant cell expresses genes not found in the native (non-recombinant) form of the cell, or expresses native genes at levels or under conditions that are different from those found in nature. The recombinant nucleic acid can differ from the native sequence by one or more nucleotides and / or be operably linked to a heterologous sequence, e.g., a heterologous promoter, a signal sequence that allows secretion in an expression vector, etc. The recombinant polypeptide / enzyme can differ from the native sequence by one or more amino acids and / or be fused to a heterologous sequence. A vector comprising a nucleic acid encoding an antibody heavy chain is, for example, a recombinant vector.

[0057] As used herein, "microorganism" refers to bacteria, fungi, viruses, protozoa, and other microorganisms or microscopic organisms.

[0058] "Host strain" or "host cell" refers to a suitable host for expressing a polynucleotide vector or DNA construct comprising a polypeptide encoding the present disclosure (particularly a recombinant fusion polypeptide). In specific embodiments, the host strain can be a filamentous fungal cell or a mammalian cell. The term "host cell" includes cells and protoplasts.

[0059] The term "filamentous fungi" refers to all filamentous forms of the subdivision Eumycotina (see, Alexopoulos, CJ (1962), INTRODUCTORY MYCOLOGY, Wiley, New York). These fungi are characterized by a vegetative mycelium with a cell wall composed of chitin, cellulose, and other complex polysaccharides. The filamentous fungi disclosed herein differ from yeast morphology, physiology, and genetics. Vegetative growth of filamentous fungi is by hyphal extension, and carbon catabolism is obligately aerobic.

[0060] The term "culturing" refers to growing a population of microbial cells in a liquid or solid medium under suitable conditions.

[0061] The term "heterologous" with respect to a polynucleotide or polypeptide refers to a polynucleotide or polypeptide that does not naturally occur in the host cell. In some embodiments, the protein is a commercially important industrial protein, and in some embodiments, the heterologous protein is a therapeutic protein. The term is intended to encompass proteins encoded by naturally occurring genes, mutant genes, and / or synthetic genes.

[0062] The term "endogenous" with reference to a polynucleotide or protein refers to a polynucleotide or protein that naturally occurs in the host cell.

[0063] As used herein, the terms "recovered," "isolated," and "separated" refer to a protein (eg, a polypeptide of interest), cell, nucleic acid, or amino acid that is removed from at least one associated component.

[0064] As used herein, the terms "transformed," "stably transformed," and "transgenic," with respect to cells, mean that the cells have a non-native (e.g., heterologous) nucleic acid sequence or an additional copy of a native (e.g., homologous) nucleic acid sequence integrated into the genome, or have an episomal plasmid that is maintained through multiple generations.

[0065] As used herein, the term "expression" refers to the process of producing a polypeptide based on the nucleic acid sequence of a gene, including both transcription and translation.

[0066] The term "secreted protein" refers to a region of a polypeptide that is released from a cell during protein secretion. In some embodiments, a secreted protein is a protein that is released or cleaved from a recombinant fusion polypeptide.

[0067] The term "secretion" refers to the selective movement of a protein across a host cell membrane into the extracellular space and surrounding medium.

[0068] Certain ranges are presented herein where a numerical value is preceded by the term "about". The term "about" is used herein to provide literal support for the exact number that follows it, as well as for numbers that are close to or approximately the number that follows the term. In determining whether a number is close to or approximately a specifically recited number, the unrecited number that is close or approximately can be a number that provides a substantial equivalent to the specifically recited number in the context in which it is presented. For example, with respect to a numerical value, the term "about" refers to a range of -10% to +10% of the numerical value, unless the term is otherwise specifically defined in the context.

[0069] As used herein, the singular terms "a," "an," and "the" include plural references unless the context dictates otherwise.

[0070] It is also noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or utilization of a "negative" limitation.

[0071] It is also noted that the term "consisting essentially of" as used herein refers to a composition wherein the component or components following the term, in the presence of other known component or components, constitutes a total amount of less than 30% by weight of the overall composition and does not contribute to or interfere with the action or activity of the component or components.

[0072] It is further noted that the term "comprising" as used herein is intended to include, but is not limited to, the one or more components following the term "comprising." The one or more components following the term "comprising" are required or mandatory, but the composition comprising the one or more components may further include other non-mandatory or optional components.

[0073] It is also noted that the term "consisting of, as used herein, is meant to include and be limited to the component or components following the term "consisting of. Thus, the component or components following the term "consisting of" are required or mandatory, and one or more other components are not present in the composition.

[0074] Every maximum numerical limitation given throughout this specification is intended to include every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0075] Unless otherwise defined herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0076] Additional definitions of terms may appear throughout this specification.

[0077] II. Composition

[0078] A. Fusion polypeptide

[0079] Provided herein are non-naturally occurring fusion polypeptides, fragments thereof, or variants thereof with improved expression and / or cleavage properties. The subject fusion polypeptide may include, but is not limited to, one or more of the following: a) signal sequence, b) carrier protein, c) protease recognition region comprising: i) protease cleavage site; ii) a protease cleavage site presequence immediately adjacent to the N-terminus of the protease cleavage site; and / or iii) a protease cleavage site postsequence immediately adjacent to the C-terminus of the protease cleavage site; and d) one or more polypeptides of interest (such as, but not limited to, antibody heavy or light chains). In certain embodiments, the protease cleavage site is a KEX2 protease cleavage site, including a KEX2 protease cleavage pre-cleavage site and / or a post-cleavage site.

[0080] Figure 1 Representative fusion polypeptides are shown. The various portions of the subject polypeptide (i.e., "signal sequence," carrier protein, "linker" (containing the protease recognition sequence), and "desired protein" (i.e., "polypeptide of interest") are labeled as such for clarity and convenience only. It is recognized that the fusion polypeptide may also be referred to as a "preprotein" or "precursor protein" because it typically contains an N-terminal region that is cleaved off during secretion and a C-terminal region that is secreted.

[0081] 1. Signal sequence

[0082] The signal sequence of theme fusion polypeptide can be any signal sequence that promotes protein secretion from host cell (for example, filamentous fungal host cell).In a specific embodiment, described fusion polypeptide can comprise the signal sequence of known protein highly secreted from the host cell that will produce described fusion protein.The signal sequence adopted can be endogenous or non-endogenous for the host cell that will produce described fusion polypeptide.

[0083] Suitable signal sequences are known in the art (see, e.g., Ward et al., Bio / Technology 1990 8:435-440; and Paloheimo et al., Applied and Environmental Microbiology 2003 69:7073-7082). Non-limiting examples of suitable signal sequences include those for cellobiohydrolase I, cellobiohydrolase II, endoglucanases I, II, and III, α-amylase, aspartyl protease, glucoamylase, phytase, mannanase, α and β glucosidase, bovine chymosin, human interferon, and human tissue plasminogen activator, and synthetic consensus eukaryotic signal sequences such as those described by Gwynne et al., (1987) Bio / Technology 5:713-719.

[0084] In some embodiments, if Trichoderma (e.g., Trichoderma reesei) is used as the host cell, the signal sequence or vector of Trichoderma reesei mannanase I (Man5A, or MANI), Trichoderma reesei cellobiohydrolase II (Cel6A or CBHII), endoglucanase I (Cel7b or EGI), endoglucanase II (Cel5a or EGII), endoglucanase III (Cel12A or EGIII), xylanase I or II (XynIIa or XynIIb), or Trichoderma reesei cellobiohydrolase I (Cel7a or CBHI) can be used to fusion polypeptides.

[0085] In other embodiments, if Aspergillus (e.g., Aspergillus niger) is used as a host cell, the signal sequence or carrier of Aspergillus niger glucoamylase (GlaA) or alpha amylase can be used in the fusion polypeptide. Aspergillus niger and Aspergillus awamori glucoamylase have the same amino acid sequence. Two forms of enzymes are generally identified in the culture supernatant. GAI is a full-length form (amino acid residues 1-616), while GAII is a natural proteolytic fragment comprising amino acid residues 1-512. It is known that GAI folds into two separate domains connected by an extended joint region. The two domains are a catalytic domain (amino acid 1-471) of 471 residues and a starch binding domain (amino acid 509-616) of 108 residues, and the joint region between the two domains is 36 residues (amino acid 472-508). GAII lacks the starch binding domain. With reference to Libby et al., (1994) Protein Engineering [protein engineering] 7: 1109-1114. In some embodiments, the glucoamylase used as a carrier protein and including a signal sequence will have greater than 95%, 96%, 97%, 98%, and 99% sequence identity to the catalytic domain of an Aspergillus or Trichoderma glucoamylase. The term "catalytic domain" refers to a structural portion or region of the amino acid sequence of a protein that has the catalytic activity of the protein.

[0086] 2. Carrier

[0087] In certain embodiments, the fusion polypeptide may comprise a "carrier protein" that functions to secrete or facilitates the secretion of the polypeptide from the host cell.

[0088] The carrier protein can include all or part of the mature sequence of a secreted polypeptide. In some embodiments, a full-length secreted carrier protein polypeptide is used. However, a functional portion of a secreted carrier protein polypeptide can be used. As used herein, a "portion" or grammatical equivalent of a secreted carrier protein polypeptide refers to a truncated secreted carrier protein polypeptide that retains the ability to fold into a normal but truncated configuration.

[0089] Typically, if the carrier protein is a truncated protein, it is C-terminally truncated (i.e., comprising a complete N-terminus). Alternatively, the carrier protein can be N-terminally truncated, or optionally truncated at both ends to leave a functional portion. Typically, such portions of secreted proteins comprising the carrier protein comprise greater than 50%, greater than 70%, greater than 80%, and greater than 90% of the secreted protein, and in some embodiments, comprise the N-terminal portion of the secreted protein. In certain embodiments, the carrier protein will also include a linker region in addition to the catalytic domain. In certain embodiments, a portion of the linker region of the CBHI protein can be used for a carrier protein.

[0090] In some embodiments, the first amino acid sequence comprising a signal sequence that functions as a secretory sequence is encoded by a first DNA molecule. The second amino acid sequence comprising the carrier protein is encoded by a second DNA sequence. However, as described above, the signal sequence and the carrier protein can be obtained from the same gene.

[0091] 3.KEX2 area

[0092] During protein secretion in fungal cells, certain proteins are cleaved by KEX2, a member of the KEX2 or "kexin" family of serine peptidases (EC 3.4.21.61). KEX2 is a highly specific, calcium-dependent endopeptidase that cleaves the peptide bond immediately C-terminal to a pair of basic amino acids (i.e., the "KEX2 site") in protein substrates during protein secretion. KEX2 proteins typically contain a cysteine ​​residue near the histidine residue in their active site and are inhibited by para-mercuric benzoic acid. The KEX2 peptidase of Saccharomyces cerevisiae, a founding member of this group (Fuller et al., 1989, Proc. Natl. Acad. Sci. USA 86:1434-1438), cleaves α-factor pheromones and killer toxin precursors during their secretion.

[0093] In some embodiments, the protease recognition site is a KEX2 region. The KEX2 region comprises a KEX2 site (X1-X2), a KEX2 site presequence (X1-X2) immediately adjacent to the N-terminus of the KEX2 site, and a KEX2 site presequence (X2). n =2-6, such as any one of 2, 3, 4, 5, or 6), and a KEX2 site presequence (X n =2-4, such as any one of 2, 3, or 4). In some embodiments, the KEX2 region provides a means for cleavage (i.e., separation) of the fusion polypeptide at the amino terminus of the polypeptide of interest in vivo. The KEX2 region of the fusion polypeptide as disclosed herein is not a naturally occurring region between the carrier protein and the polypeptide of interest.

[0094] The KEX2 cleavage site can be cleaved by a natural filamentous fungal protease (e.g., a natural Aspergillus KEXB-like protease or a natural Trichoderma KEX2 protease) or can be cleaved by one or more other proteases present in eukaryotic (such as yeast or mammalian) cells. The polypeptide of interest is cleaved from the fusion polypeptide immediately downstream (i.e., C-terminal) of the KEX2 cleavage site.

[0095] The KEX2 site comprises an amino acid sequence "X1-X2," wherein X1 and X2 are independently basic amino acids. The KEX2 site may comprise any one of KK, KR, RK, or RR. In some embodiments, the KEX2 site is KR.

[0096] The KEX2 site presequence may include the amino acid sequence X n = 2-8, wherein X is any amino acid and n is 2-8, such as any one of 2, 3, 4, 5, 6, 7, or 8. At the C-terminus of the carrier protein in the fusion polypeptide disclosed herein, the KEX2 region as defined herein is not naturally found in the carrier protein. In some embodiments, the KEX2 site presequence is different from the contiguous X naturally occurring at the C-terminus of the carrier protein. n = amino acid sequence of 2-8 amino acid residues. However, the continuous X n = 2-6 amino acid residues can be found in other parts of the carrier protein and can be linked to the KEX2 site (X1-X2), but the KEX2 region is not attached to the N-terminal polypeptide of interest of the carrier protein.

[0097] Amino acid substitutions in the KEX2 site pre- and / or post-sequences can be the result of replacing one amino acid with another having similar structural and / or chemical properties, such as replacing leucine with serine, i.e., conservative substitutions; or the result of replacing one amino acid with another having different structural or chemical properties, such as replacing asparagine with aspartic acid, i.e., non-conservative substitutions. Naturally occurring residues are divided into several groups based on common side chain properties: (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acidic: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues affecting chain orientation: gly, pro; and (6) aromatic: trp, tyr, phe. Non-conservative substitutions require exchanging a member of one of these categories for another. Such substituted residues can also be introduced into the conservative substitution site or the non-conservative site.

[0098] In some embodiments, when the KEX2 site presequence is defined as T1-S2-V-A3-V4-E5-X1-X2, T1 is substituted by a hydrophobic amino acid, an aromatic amino acid, an acidic amino acid, or a basic amino acid; S2 is substituted by a hydrophobic amino acid, an aromatic amino acid, a basic amino acid, or an amino acid that affects chain orientation; A3 is substituted by a basic amino acid or M; V4 is substituted by L; and / or E5 is substituted by an aromatic amino acid.

[0099] In other embodiments, when the sequence after the KEX2 site is defined as X1-X2-Q6-V7, Q6 is substituted by an acidic amino acid or an amino acid that affects chain orientation; and / or V7 is substituted by an L, I, or aromatic amino acid.

[0100] In additional embodiments, the KEX2 region is selected from the group consisting of ASVAVEKRQV (SEQ ID NO:3), FSVAVEKRQV (SEQ ID NO:2), MSVAVEKRQV (SEQ ID NO:4), QSVAVEKRQV (SEQ ID NO:5), RSVAVEKRQV (SEQ ID NO:6), YSVAVEKRQV (SEQ ID NO:7), TFVAVEKRQV (SEQ ID NO:7), NO:8),THVAVEKRQV(SEQ ID NO:9),TKVAVEKRQV(SEQ ID NO:10),TLVAVEKRQV(SEQ ID NO:11),TMVAVEKRQV(SEQ ID NO:12),TPVAVEKRQV(SEQ ID NO:13),TQVAVEKRQV(SEQ ID NO:14),TRVAVEKRQV(SEQ ID NO:15), TVVAVEKRQV (SEQ ID NO:16), TSVHVEKRQV (SEQ ID NO:17), TSVKVEKRQV (SEQ ID NO:18), TSVRVEKRQV (SEQ ID NO:19), TSVALEKRQV (SEQ ID NO:20), TSVAVFKRQV (SEQ ID NO:21), TSVAVWKRQV (SEQ ID NO:22), TSVAVEKRDV (SEQ ID NO:23), TSVAVEKRGV (SEQ ID NO:24), TSVAVEKRQF (SEQ ID NO:25), or TSVAVEKRQL (SEQ ID NO:26).

[0101] When a variant with multiple substitutions (such as any of 2, 3, 4, or 5 substitutions) is generated in the KEX2 region, in additional embodiments, the sequence after the KEX2 site can be defined as X1-X2-Q6-V7-T8-L9 (SEQ ID NO: 64), wherein Q6 is substituted with an acidic amino acid or an amino acid that affects chain orientation; V7 is substituted with an L, I, or aromatic amino acid; T8 is substituted with an acidic amino acid or a hydrophobic amino acid; and / or L9 is substituted with I or V. In some embodiments, the KEX2 region is selected from TSVAVEKRQVEL (SEQ ID NO: 27), TSVAVEKRQVTI (SEQ ID NO: 28), or TSVAVEKRQVTV (SEQ ID NO: 29).

[0102] In further embodiments, when variants are generated with multiple substitutions (such as any of 2, 3, 4, or 5 substitutions) in the KEX2 region, in additional embodiments, the KEX2 site presequence may be defined as G -2 -P -1 -T1-S2-V-A3-V4-E5-X1-X2 (SEQ ID NO:65), where G -2 replaced by tyrosine and / or P -1 Substituted with threonine or leucine.

[0103] In additional embodiments, the KEX2 region has two or more substitutions (such as any of 2, 3, 4, or 5 substitutions) compared to an unaltered KEX2 region amino acid sequence selected from the group consisting of THVAVEKRQVTI (SEQ ID NO:30), THVAVEKRDVTL (SEQ ID NO:31), THVAVEKRQVAL (SEQ ID NO:32), EHVAVEKRQVTL (SEQ ID NO:33), TNVAVEKRDVTL (SEQ ID NO:34), TNVAVEKRQVAL (SEQ ID NO:35), TSVALWKRQVTL (SEQ ID NO:36), TSVMLEKRQVTL (SEQ ID NO:37), TSVALEKRQITL (SEQ ID NO:38), TSVALEKRQVAL (SEQ ID NO:39), THVAVEKRQVTL (SEQ ID NO:40), TSVAVEKRDVAL (SEQ ID NO:41), NO:41), THVMLEKRQVTL (SEQ ID NO:42), TKVMLEKRQVTL (SEQ ID NO:43), THVAVEKRDVAL (SEQ ID NO:44), THVALWKRQVTL (SEQ ID NO:45), TKVAVEKRDLTL (SEQ ID NO:46), TNVAVEKRDLTL (SEQID NO:47), EHVAVWKRQVTL (SEQ ID NO:48), EHVALEKRQVTL (SEQ ID NO:49), ESVALWKRQVTL (SEQ ID NO:50), RSVRVEKRDVTL (SEQ ID NO:51), TSVALEKRDVAL (SEQ ID NO:52), THVALEKRDVAL (SEQ ID NO:53), TKVRVEKRDLTL (SEQ ID NO:54), TNVALEKRDVAL (SEQ ID NO:55), EHVALWKRQVTL (SEQ ID NO:56), EPVALWKRQVTL (SEQ ID NO:57), NHVALWKRQVTL (SEQ ID NO:58), RSVRVEKRDLTL (SEQ ID NO:59), RKVRVEKRDVTL (SEQ ID NO:60), RKVRVEKRQLTL (SEQ ID NO:61), RKVAVEKRDLTL (SEQ ID NO:62), RKVRVEKRDLTL (SEQ ID NO:63), YLTSVMLEKRQV (SEQID NO:83), YLTHVMLEKRQV (SEQ ID NO:84), YPTHVMLEKRQV (SEQ ID NO:85), YPTHVALEKRQV (SEQ ID NO:86), GLTSVMVEKRQV (SEQ ID NO:87), or GLTHVMLEKRQV (SEQ ID NO:88).

[0104] In yet other embodiments, the KEX2 site presequence is not KSRS (SEQ ID NO: 66), SRIS (SEQ ID NO: 67), GGGS (SEQ ID NO: 68), TSTY (SEQ ID NO: 69), ASIS (SEQ ID NO: 70), ATAS (SEQ ID NO: 71), TASQ (SEQ ID NO: 72), TASL (SEQ ID NO: 73), SVIS (SEQ ID NO: 74), NVIS (SEQ ID NO: 75), GGG, TSRD (SEQ ID NO: 76), SPMD (SEQ ID NO: 77), DLGE (SEQ ID NO: 78), or TPTA (SEQ ID NO: 79). In another embodiment, the KEX2 site presequence is not any of the KEX2 site presequences disclosed in U.S. Patent No. 8,198,046, the disclosure of which is hereby incorporated by reference in its entirety.

[0105] The engineered KEX2 site pre- and / or post-sequences provided herein result in enhanced cleavage and / or secretion of a polypeptide of interest from a host cell, compared to cleavage and / or secretion of the polypeptide of interest from an equivalent fusion polypeptide lacking the KEX2 site pre- and / or post-sequences provided herein.

[0106] The KEX2 site pre-sequence and / or post-sequence provided herein can be an optimized KEX2 site pre-sequence and / or post-sequence. The optimized KEX2 pre-sequence and / or post-sequence are KEX2 pre-sequences and / or post-sequences described in the present disclosure, but compared with other variant KEX2 site pre-sequences and / or post-sequences, they provide greater or more efficient cracking or secretion from host cells (i.e., relative to unmodified sequence, there is greater than about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% cracking or secretion). Arbitrary fusion polypeptide disclosed herein can include the optimized KEX2 pre-sequence and / or post-sequence as the KEX2 pre-sequence and / or post-sequence. The optimized KEX2 pre-sequence and / or post-sequence can adopt any signal sequence, any carrier region from secreted protein, any KEX2 site or any target polypeptide. The KEX2 region containing the optimized KEX2 site pre-sequence and / or post-sequence can be non-naturally occurring. In certain embodiments, the KEX2 region containing the optimized KEX2 site pre- and / or post-sequences is not found in any protein secreted from a host cell, such as, but not limited to, a filamentous fungal host cell.

[0107] 4. Target peptide

[0108] The polypeptide of interest in the fusion polypeptide can be any portion of a protein that can be secreted from a host cell (such as a eukaryotic host cell, for example, a mammalian or filamentous fungal host cell), including so-called industrial enzymes, therapeutic proteins, hormones, structural proteins, plasma proteins, food additives, and foods. The polypeptide of interest can be a heterologous protein or a homologous protein, and can include a hybrid polypeptide comprising a combination of partial or complete polypeptides, each of which can be homologous or heterologous to the expression host. The secreted polypeptide of interest can be derived from bacteria (such as Bacillus species and Pseudomonas species), fungi (such as Aspergillus, Trichoderma, Humicola, or Mucor species), viruses (such as hepatitis A or B or adenovirus), mammals (such as humans, rats, or mice), or plant sources. The polypeptide of interest can additionally include naturally occurring protein allele variations and engineered variations. In additional embodiments, the polypeptide of interest can be a heterotetramer, such as an antibody. In further embodiments, the polypeptide of the heterotetramer can be produced by one or more fusion polypeptide cleavage events.

[0109] In one embodiment, the polypeptide of interest can be an enzyme, such as a carbohydrase, such as a starch hydrolyzing α-amylase, alkaline α-amylase, β-amylase, cellulase; a glucanase, α-glucosidase, α-galactosidase, glucoamylase, hemicellulase, pentosanases, xylanases, invertases, lactases, naringinase, pectinase or pullulanase; a protease, such as an acid protease, an alkaline protease, bromelain, ficin, a neutral protease, papain, pepsin, peptidase, rennet, rennin, chymosin, subtilisin, thermolysin, aspartate a protease, or trypsin; a granular starch hydrolase, such as a glucoamylase or an alpha-amylase; a lipase or esterase, such as a triglyceride esterase, a phospholipase, a forestomach esterase, a phosphatase, a phytase, an amidase, an iminoacylase, a glutaminase, a lysozyme, or a penicillin acylase; an isomerase, such as a glucose isomerase; a phenol oxidase, for example, a laccase; an oxidoreductase, for example, an amino acid oxidase, a catalase, a chloroperoxidase, a glucose oxidase, a hydroxysteroid dehydrogenase, or a peroxidase; a lyase, such as acetolactate decarboxylase, aspartate β-decarboxylase, a fumarate, or a histidase; a transferase, such as a cyclodextrin glycosyltransferase or an acyltransferase; or a ligase, for example. In particular embodiments, the protein can be an aminopeptidase, a carboxypeptidase, a chitinase, a glucoamylase, an alpha-amylase, a cutinase, a phytase, a deoxyribonuclease, an alpha-galactosidase, a beta-galactosidase, a beta-glucosidase, a laccase, a mannosidase, a deformase, a pectinolytic enzyme, a polyphenol oxidase, a ribonuclease, or a transglutaminase.

[0110] In other embodiments, the polypeptide of interest can be a therapeutic protein (i.e., a protein with therapeutic biological activity). Examples of suitable therapeutic proteins include: erythropoietin, cytokines such as interferon-α, interferon-β, interferon-γ, interferon-o, and granulocyte-CSF (GM-CSF), coagulation factors such as factor VIII, factor IX, and human protein C, antithrombin III, thrombin, soluble IgE receptor α-chain, immunoglobulins such as immunoglobulin G (IgG), IgG fragments, IgG fusions, IgM or IgA; interleukins, urokinase, chymotrypsin, and urea trypsin inhibitor, IGF-binding protein, epidermal growth factor, growth hormone-releasing factor, annexin V fusion protein, angiostatin, vascular endothelial growth factor-2, myeloid progenitor cell inhibitory factor-1, osteoprotegerin, α-1-antitrypsin, α-fetoprotein, DNase II, kringle of human plasminogen activator. 3. Glucocerebrosidase, TNF-binding protein 1, follicle-stimulating hormone, cytotoxic T-lymphocyte-associated antigen 4-Ig, transmembrane activator and calcium regulator and cyclophilin ligand, soluble TNF receptor Fc fusion, glucagon-like protein 1, and IL-2 receptor agonist.

[0111] In some embodiments, the polypeptide of interest is an immunoglobulin (i.e., an antibody) from any class (G, A, M, E, or D) (see U.S. Pat. No. 4,816,567, incorporated herein by reference, and references therein for discussing immunoglobulin structure). In other embodiments, the antibody protein is a monoclonal antibody comprising a heavy chain or a light chain and functional fragments thereof. In further embodiments, a humanized antibody is a polypeptide of interest (e.g., trastuzumab). In some embodiments, the antibody or functional fragment thereof is an anti-respiratory syncytial virus (RSV) antibody, an anti-Ebola virus antibody, an anti-aggregated β-amyloid protein (Aβ) antibody, an anti-human immunodeficiency virus (HIV) antibody, an anti-herpes simplex virus (HSV) antibody, an anti-sperm antibody (such as an anti-human contraceptive antigen (HCA) antibody), and an anti-HER2 / neu antibody. Some specific examples of monoclonal antibody fragments are truncated forms of the heavy chain to remove part of the constant region, such as Fab fragments, wherein the heavy chain (Fd) lacks the hinge region and the CH2 and CH3 domains; Fab' fragments, wherein the heavy chain includes the hinge region but lacks the CH2 and CH3 domains; and F(ab')2 fragments, which include the Fab portion connected by the hinge region. (Verma et al. (1998) J. Immunological Methods 216:165-181 and Pennell and Eldin (1998) Res. Immunol. 149:599-603, incorporated herein by reference.) Also of interest are single-chain antibodies (ScFv) and single-domain antibodies (e.g., camelid antibodies), as well as fusion proteins in which a protein is stably fused to a portion of an antibody (e.g., Fc fusion proteins). In some embodiments, the antibodies are engineered to improve one or more properties (e.g., stability, manufacturability, and / or binding to the antigen).

[0112] In some embodiments, the fusion polypeptide will comprise a signal sequence, a carrier protein, a KEX2 region, and a polypeptide of interest in operable linkage.

[0113] B. polynucleotides

[0114] Another aspect of the compositions and methods disclosed herein is a polynucleotide or nucleic acid sequence encoding a fusion polypeptide, such as any of the engineered KEX2 regions containing a fusion polypeptide disclosed herein.

[0115] Provided herein is a fusion DNA construct encoding a fusion polypeptide as disclosed above, comprising an operably linked promoter; a first DNA molecule encoding a signal sequence; a second DNA molecule encoding a carrier protein; a third DNA molecule encoding a KEX2 region, the KEX2 region comprising a KEX2 site and a KEX2 site presequence immediately 5′ to the KEX2 site and a KEX2 site postsequence immediately 3′ to the KEX2 site; and a fourth DNA molecule encoding a polypeptide of interest. The components of the fusion DNA construct can appear in any order. Since the genetic code is known, given the description of the fusion polypeptides disclosed herein, the design and production of these nucleic acids are well within the skill of a person of ordinary skill. In certain embodiments, the nucleic acids can be codon-optimized to express the fusion polypeptide in a specific host cell. Since codon usage tables are available for many species, for example, mammalian cells and filamentous fungi, the design and production of codon-optimized nucleic acids encoding the subject fusion polypeptides will be well within the skill of a person of ordinary skill.

[0116] C. promoter

[0117] Examples of suitable promoters for directing transcription of nucleic acids in a host cell (e.g., a filamentous fungal host cell) are promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase (Korman et al. (1990) Curr. Genet. 17:203-212; Gines et al. (1989) Gene 79:107-117), Aspergillus niger or Aspergillus awamori glucoamylase (glaA) (Nunberg et al. (1984) Mol. Cell Biol. 4:2306-2315; Boel E. et al. (1984) EMBO J. [European Molecular Biology Society] 3:1581-1585), Rhizomucor miehei lipase, Aspergillus oryzae alkaline proteinase, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase (Hyner et al., (1983) Mol. Cell. Biol. [Molecular Cell Biology] 3:1430-1439), Fusarium venenatum amyloglucosidase, Fusarium oxysporum trypsin-like protease (WO 96 / 00787), Trichoderma reesei cellobiohydrolase I (Shoemaker et al. (1984) EPA EPO 0137280), Trichoderma reesei cellobiohydrolase II, T. reesei endoglucanase I, Trichoderma reesei endoglucanase II, Trichoderma reesei endoglucanase III, Trichoderma reesei endoglucanase IV, Trichoderma reesei endoglucanase V, Trichoderma reesei xylanase I, Trichoderma reesei xylanase II, Trichoderma reesei β-xylosidase, and the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral α-amylase and Aspergillus oryzae triose phosphate isomerase); and mutants, truncations, and hybrid promoters thereof. Reference is also made to Yelton et al., (1984) Proc. Natl. Acad. Sci. USA 81: 1470-1474; Mullaney et al., (1985) Mol. Gen. Genet. 199: 37-45; Lockington et al., (1986) Gene 33: 137-149; Macknight et al., (1986) Cell 46: 143-147; Hynes et al., (1983) Mol. Cell Biol. 3: 1430-1439.Higher eukaryotic promoters such as the SV40 early promoter (Barclay et al. (1983) Molecular and Cellular Biology 3:2117-2130) may also be useful. The promoter may be constitutive or inducible. Exemplary promoters include Trichoderma reesei cellobiohydrolase I or II, Trichoderma reesei endoglucanase I, II or III, and Trichoderma reesei xylanase II.

[0118] D. carrier

[0119] The polynucleotide encoding any fusion polypeptide disclosed herein can be present in a vector, for example, a phage, plasmid, virus, or retroviral vector. In certain embodiments, the vector can be an expression vector for expressing the subject fusion polypeptide in a filamentous fungal cell.

[0120] Vectors for expressing recombinant proteins are well known in the art (Ausubel et al., Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995; Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., (1989) Cold Spring Harbor, New York).

[0121] Fusion DNA constructs can be constructed using well-known techniques, as generally described in, for example, European Patent Application Publication No. 0 215 594, the disclosure of which is incorporated herein by reference.

[0122] The synthetic polynucleotide fragment encoding the polypeptide of interest (eg, immunoglobulin) can be incorporated into a heterologous nucleic acid construct or vector capable of introduction into and replication in a host cell (eg, a filamentous fungal cell).

[0123] In case DNA construct is made or more specifically fused DNA construct, it can be incorporated into any number of carriers known in the art. Although in some embodiments, described DNA construct will include promoter sequence, in other embodiments, described carrier will include other regulatory sequences that work in host to be transformed, such as ribosome bind site, transcription initiation and termination sequence, terminator sequence, polyadenylation signal, enhancer and or activator. In certain embodiments, the polynucleotides encoding the described polypeptide of interest and KEX2 zone will be inserted into the carrier comprising promoter, signal sequence and carrier protein at suitable restriction endonuclease site by standard procedure. This type of program and relevant subcloning procedure are considered to be within the knowledge of those skilled in the art.

[0124] The terminator sequence that is recognized by the expression host to terminate transcription can be effectively connected to the 3 ' end of the fusion DNA construct encoding the fusion protein to be expressed. Those of ordinary skill in the art are familiar with the various terminator sequences that can be used for host cells such as filamentous fungi. Non-limiting examples include the terminator from Aspergillus nidulans trpC gene (Yelton M. et al., (1984) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 81: 1470-1474) or from the terminator of Aspergillus niger glucoamylase gene (Nunberg et al. (1984) Mol. Cell. Biol. [Molecular Cell Biology] 4: 2306-2353) or from the terminator of Trichoderma reesei cellobiohydrolase I gene.

[0125] Polyadenylation sequence is a DNA sequence that is recognized by the expression host when transcribed to add polyadenosine residues to transcribed mRNA. Examples include the polyadenylation sequence from the Aspergillus nidulans trpC gene (Yelton et al. (1984) Proc. Natl. Acad. Sci. USA [Proceedings of the National Academy of Sciences of the United States] 81; 1470-1474), the polyadenylation sequence from the Aspergillus niger glucoamylase gene (Nunberg et al. (1984) Mol. Cell. Biol. [Molecular Cell Biology] 4: 2306-2315), the polyadenylation sequence from Aspergillus oryzae or Aspergillus niger alpha amylase genes and the polyadenylation sequence from the Rhizomucor miehei carboxyproteinase gene.

[0126] In a further embodiment, the fusion DNA construct or the vector comprising the fusion DNA construct will contain a selectable marker gene to allow selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used. Examples of selectable markers include, but are not limited to, selectable markers that confer antimicrobial resistance (e.g., hygromycin, bleomycin, chloramphenicol, and phleomycin). Genes that confer metabolic advantages, such as nutritional selective markers, can also be used. Some of these markers include amdS. In addition, sequences encoding genes that complement auxotrophic defects can be used as selectable markers (e.g., pyr4 complements of pyr4-deficient Aspergillus nidulans, Aspergillus awamori, or Trichoderma reesei and argB complements of argB-deficient strains). See Kelley et al., (1985) EMBO J. 4:475-479; Penttila et al., (1987) Gene 61:155-164 and Kinghorn et al. (1992) Applied Molecular Genetics of Filamentous Fungi, Blackie Academic and Professional, Chapman and Hall, London, the disclosures of each of which are incorporated herein by reference.

[0127] E. host cells

[0128] The expression cassette or vector is introduced into a suitable expression host cell, which then expresses the corresponding nucleotide sequence encoding the fusion polypeptide.

[0129] Suitable host cells include cells of any microorganism (e.g., bacteria, protozoa, algae, fungi (e.g., yeast or filamentous fungi), or cells of other microorganisms), and can be cells of bacteria, yeast, or filamentous fungi. Fungal expression hosts can be, for example, yeast, which can also be used as ethanologenic microorganisms. Also suitable are mammalian expression hosts, such as mouse (e.g., NSO), Chinese hamster ovary (CHO), or baby hamster kidney (BHK) cell lines. Other eukaryotic hosts, such as insect cells or viral expression systems (e.g., bacteriophages such as M13, T7 phage, or lambda, or viruses such as baculovirus), are also suitable for producing the polypeptide.

[0130] Suitable host cells of bacterial genera include, but are not limited to, cells of the following genera: Escherichia, Proteus, Bacillus, Ralstonia, Lactobacillus, Lactococcus, Pseudomonas, Staphylococcus, and Streptomyces. Suitable bacterial species cells include, but are not limited to, cells of Escherichia coli, Bacillus subtilis, Bacillus licheniformis, Bacillus megaterium, Lactobacillus brevis, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas stutzerei, Staphylococcus carnosus, Lactococcus lactis, Ralstonia eutropha, Proteus mirabilis, and Streptomyces lividans.

[0131] Suitable host cells of the genus Saccharomyces include, but are not limited to, those of the genus Saccharomyces, Schizosaccharomyces, Candida, Hansenula, Pichia, Kluyveromyces, Yarrowia, and Phaffia. Suitable cells of yeast species include, but are not limited to, those of the genus Saccharomyces, Schizosaccharomyces pombe, Candida albicans, Hansenula polymorpha, Yarrowia lipolytica, Pichia pastoris, P. canadensis, Kluyveromyces marxianus, and Phaffia rhodozyma.

[0132] Suitable host cells of filamentous fungi include all filamentous forms of the subphylum Fungi. Suitable cells of filamentous fungi include but are not limited to following cells: Acremonium, Aspergillus, Aureobasidium, Bjerkandera, Ceriporiopsis, Chrysosporium, Coprinus, Coriolus, Corynascus, Chaertomium, Cryptococcus, Filobasidium, Fusarium, Gibberella, Humicola, Magnaporthe, Mucor, Myceliophthora, Mucor, Neoca and / or Trichoderma.

[0133] Suitable cells of filamentous fungal species include, but are not limited to, cells of Aspergillus awamori, Aspergillus fumigatus, Aspergillus foetidus, Aspergillus japonicus, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Chrysosporium lucknowense, Fusarium bactridioides, Fusarium cerealis, Fusarium crookwellense, Fusarium culmorum, Fusarium graminearum, Fusarium graminum, Fusarium heterosporum, Fusarium negundi, Fusarium oxysporum, Fusarium reticulatum, Fusarium roseum, Fusarium niger, Fusarium gramin ... sambucinum), Fusarium sarcochroum, Fusarium sporotrichioides, Fusarium sulphureum, Fusarium torulosum, Fusarium trichothecioides, Fusarium veneer, Bjerkandera adusta, Ceriporiopsis aneirina, Ceriporiopsis, Ceriporiopsis caregiea, Ceriporiopsis gilvescens, Ceriporiopsis pannocinta, Ceriporiopsis rivulosa, Ceriporiopsis subrufa, Ceriporiopsis subvermispora, Coprinus cinereus), Coriolushirsutus, Humicola insolens, Humicola lanuginosa, Mucor miehei, Myceliophthora thermophila, Neurospora crassa, Neurospora intermediaintermedia), Penicillium purpurogenum, Penicillium canescens, Penicillium solitum, Penicillium funiculosum, Phanerochaete chrysosporium, Phlebia radiate, Pleurotus eryngii, Talaromyces flavus, Thielavia terrestris, Trametes villosa, Trametes versicolor, Trichoderma harzianum, Trichoderma koningii, Trichoderma longibrachiatum, Trichoderma reesei, and Trichoderma viride.

[0134] Promoters and / or signal sequences associated with secreted proteins in a particular host of interest are candidates for heterologous production and secretion of fusion polypeptides in that or other hosts. As non-limiting examples, in filamentous fungal systems, promoters driving the genes for cellobiohydrolase 1 (cbhl), glucoamylase A (glaA), TAKA-amylase (amyA), xylanase (exlA), gpd-promoter cbhl, cbhll, endoglucanase genes egl-eg5, Cel61B, Cel74A, gpd promoter, Pgkl, pkil, EF-1alpha, tefl, cDNAl, and hexl are suitable and can be derived from many different organisms (e.g., Aspergillus niger, Trichoderma reesei, Aspergillus oryzae, Aspergillus awamori, Aspergillus nidulans).

[0135] In certain embodiments, the polynucleotide encoding the fusion polypeptide is recombined with the polynucleotide recombinant homology or heterologous signal sequence to which the encoding is applicable, resulting in secretion of the recombinant polypeptide into the extracellular (or periplasmic) space, thereby allowing direct detection of the enzymatic activity in the cell supernatant (or periplasmic space or lysate). The signal sequence applicable to Escherichia coli, other Gram-negative bacteria, and other organisms known in the art include those that drive the expression of HlyA, DsbA, Pbp, PhoA, PelB, OmpA, OmpT, or M13 phage Gill genes. For Bacillus subtilis, Gram-positive organisms, and other organisms known in the art, the signal sequence applicable further includes those that drive the expression of AprE, NprB, Mpr, AmyA, AmyE, Blac, SacB, and for Saccharomyces cerevisiae or other yeasts, includes killer toxin, Bar1, Suc2, mating factor α, Inu1A, or Ggplp signal sequence. The signal sequence can be cracked by many signal peptidases, thereby removing it from the remaining expressed protein.

[0136] In certain embodiments, the fusion polypeptide is expressed alone or as a fusion with an additional peptide, tag or protein (e.g., 6XHis, HA or FLAG tag) positioned at the N-terminus or C-terminus. Suitable fusions include tags, peptides or proteins (e.g., 6XHis, HA, chitin binding protein, thioredoxin or FLAG tags) that promote affinity purification or detection and those that promote target beta-glucosidase expression, secretion or processing. In addition to KEX2, further suitable processing sites include enterokinase, STE13 or other protease cleavage sites known in the art for in vivo or in vitro cracking.

[0137] A polynucleotide encoding a fusion polypeptide can be introduced into an expression host cell by a variety of transformation methods, including, but not limited to, electroporation, lipid-assisted transformation or transfection ("lipofection"), chemically mediated transfection (e.g., CaCl and / or CaP), lithium acetate-mediated transformation (e.g., of host cell protoplasts), biolistic "gene gun" transformation, PEG-mediated transformation (e.g., of host cell protoplasts), protoplast fusion (e.g., using bacterial or eukaryotic protoplasts), liposome-mediated transformation, Agrobacterium tumefaciens, adenovirus or other viral or phage transformation or transduction.

[0138] III. Methods

[0139] A. Fusion Peptide Production

[0140] This paper additionally provides the method for producing one or more fusion polypeptides disclosed herein in host cells (such as, but not limited to mammals or filamentous fungal host cells).In certain embodiments, these methods include obtaining a host cell comprising a fusion DNA construct disclosed herein or a carrier and cultivating the host cell under the suitable conditions of allowing the expression of the target polypeptide and secretion.Because the culture of the host cell (that is, the composition containing the host cell and growth medium) can contain the secretory protein of fusion polypeptide as herein described, in certain embodiments, the target polypeptide is reclaimed from the culture medium.In other embodiments, the target polypeptide is purified.Can be recovered and / or purified protein from growth medium by any convenient method known in the art.

[0141] In certain embodiments, subject host cells (such as, fungal host cells) can be cultivated under batch or continuous fermentation conditions. Classical batch fermentation is a closed system, wherein the composition of the culture medium is set at the beginning of the fermentation and is not subject to artificial changes during the fermentation process. Therefore, at the beginning of the fermentation, the culture medium is inoculated with the required organism. In this method, fermentation is allowed to occur without adding any components to the system. Typically, batch fermentation meets the qualification of "batch" with respect to adding a carbon source, and control factors (such as pH and oxygen concentration) are often attempted. The metabolites and biomass composition of the batch system are constantly changing until the fermentation stops. In batch culture, cells progress to the high growth logarithmic phase through the static lag phase and finally enter the stable phase where the growth rate decreases or stops. If left untreated, the cells in the stable phase eventually die. In general, cells in the logarithmic phase are responsible for the large-scale production of end products.

[0142] The variant of standard batch system is " fed-batch fermentation " system, and this system can also be used. In this variant of typical batch system, along with the progress of fermentation, substrate is added with increment. When catabolite inhibition easily suppresses the metabolism of described cell and in described substratum, wish to have under the situation of limited amount of substrate, fed-batch system is useful. The measurement of actual substrate concentration in fed-batch system is difficult and therefore it is estimated based on the variation of measurable factor (such as pH, dissolved oxygen and waste gas (such as CO ) partial pressure). Batch and fed-batch fermentation are commonly used and are known in the art.

[0143] Continuous fermentation is an open system in which a defined fermentation medium is continuously added to a bioreactor while an equal amount of conditioned medium is removed for processing. Continuous fermentation typically maintains the culture at a constant high density, with cells primarily in logarithmic phase growth.

[0144] Continuous fermentation allows a factor or any number of factors affecting cell growth and / or end product concentration to be regulated. For example, in one embodiment, limiting nutrients (such as carbon source or nitrogen source) are maintained at a fixed rate, and all other parameters are allowed to be regulated. In other systems, many factors affecting growth can constantly change, while the cell concentration measured by culture medium turbidity remains unchanged. Continuous systems strive to maintain stable growth conditions. Therefore, the cell loss caused by transferring culture medium must be balanced with the cell growth rate in the fermentation. The technology of regulating the nutrient and growth factor method for continuous fermentation process and maximizing product formation rate is known.

[0145] B. Expression and secretion

[0146] Production of a polypeptide of interest in a host cell (e.g., a filamentous fungal host cell) comprising a fusion DNA construct encoding the fusion polypeptide results in secretion of the polypeptide of interest of the fusion polypeptide. During the secretion process of, for example, a fungus, sugar chains can be attached to the protein to be secreted to produce a glycosylated protein. Thus, in some embodiments, the production of a polypeptide of interest (e.g., an antibody) can include glycosylated or non-glycosylated proteins.

[0147] In some embodiments, the secreted protein of the subject fusion polypeptide is typically present in the culture medium of the host cell in a higher amount than the secreted polypeptide of interest produced by an equivalent host cell (i.e., the same cell type, grown under the same conditions) lacking the engineered KEX2 site pre- and / or post-sequences disclosed herein. A culture of the subject cells producing a polypeptide of interest from a fusion polypeptide according to the methods disclosed herein can contain greater than about 5%, greater than about 10%, greater than about 20%, greater than about 40%, greater than about 60%, greater than about 80%, greater than about 100%, greater than about 150%, greater than about 200%, greater than about 300%, greater than about 500%, or greater than 1000% of the polypeptide of interest in the growth medium compared to a culture of equivalent cells expressing other equivalent proteins that do not have the engineered KEX2 site pre- and / or post-sequences as contemplated by the present disclosure.

[0148] In certain embodiments, the level of expression and secretion of the polypeptide of interest (e.g., full-length antibody) will be greater than 0.5 g / L. Typically, the polypeptide of interest can be recovered from culture medium at a level greater than 1.0 g / L. Reproducible levels greater than about 1.5, 2.0, 3.0, 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10, 1, 112, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 g / L can be obtained. In certain embodiments, the expression and secretion levels of the polypeptide of interest will be greater than about 30 g / L and even greater than about 40 g / L.

[0149] In other embodiments, the ratio of secreted cleaved polypeptide of interest to uncleaved secreted polypeptide of interest is greater than about 5:1, 10:1, 15:1, 20:1, 25:1, 30:1, 35:1, 40:1, 50:1, 55:1, 60:1, 65:1, 70:1, 75:1, 80:1, 85:1, 90:1, 95:1, 100:1, 150:1, 200:1, 250:1, 300:1, 350:1, 400:1, 450:1, 500:1, 1000:1, 5000:1, 7500:1, 10000:1, or 100000:1, including all values ​​falling between these ratios.

[0150] In some embodiments, the cleavage of the polypeptide of interest from the recombinant fusion polypeptide is greater than the cleavage of the same polypeptide of interest from an equivalent recombinant fusion polypeptide lacking the KEX2 site pre- and / or post-sequences disclosed herein. In some embodiments, the KEX2 site pre- and / or post-sequences can result in cleavage of the fusion protein to an efficiency of at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%, wherein 100% efficiency results in complete cleavage of the polypeptide of interest from the fusion polypeptide.

[0151] In certain embodiments, the efficiency of protein cleavage can be calculated by determining the amount of cleavage that has occurred, for example, by determining the amount of cleavage versus the amount of uncleaved protein. In one embodiment, the amount of protein cleavage can be calculated by determining the ratio of the amount of cleaved protein in the growth medium per volume of cell culture to the amount of uncleaved fusion protein in the growth medium.

[0152] In certain embodiments, a fusion polypeptide containing a KEX2 site pre-sequence and / or post-sequence or an optimized KEX2 site pre-sequence and / or post-sequence can result in cleavage of the fusion polypeptide to an efficiency of at least about 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, wherein 100% efficiency is complete cleavage of the target polypeptide.

[0153] In other embodiments, the secretion efficiency of the subject fusion polypeptide can be calculated by determining the amount of the secretory part of the fusion polypeptide in the growth medium of the cell that secretes the protein. This determination can be quantitative, qualitative, relative or absolute. In one embodiment, the amount of the secretory protein in the growth medium of the cell that secretes the subject fusion can be at least about 10%, at least about 30%, at least about 50%, at least about 70%, at least about 90%, at least twice, at least five times, or at least ten times greater than the amount of the secretory protein secreted by the cell that produces the equivalent fusion polypeptide that does not contain the optimized KEX2 presequence and / or postsequence.

[0154] In some embodiments, the increase in secretion and / or cleavage can be measured against a canonical KEX2 region defined as GGGB1B2, wherein B1B2 is KK, KR, RK, or RR, and preferably KR. In embodiments, the amount of secreted protein or polypeptide of interest in the growth medium of cells secreting the subject fusions can be at least about 10%, at least about 30%, at least about 50%, at least about 70%, at least about 80%, at least about 90%, at least about 100%, at least 2-fold, at least 3-fold, at least 5-fold, and at least 10-fold greater than the amount of secreted protein or polypeptide of interest secreted by the equivalent fusion polypeptide in an equivalent host under essentially the same conditions.

[0155] IV. Kit

[0156] Further provided herein are kits comprising one or more written instructions for producing any of the fusion polypeptides disclosed herein; one or more nucleic acids disclosed herein; any vector disclosed herein; and any host cell disclosed herein. The kits may further comprise one or more compositions comprising a Kex2 serine peptidase and / or a nucleic acid encoding a Kex2 serine peptidase. Additionally, the kits may further comprise a host cell expressing the Kex2 serine peptidase; one or more compositions comprising an additional protease; and / or a nucleic acid encoding one or more additional proteases.

[0157] The present invention may be further understood with reference to the following examples, which are provided by way of illustration and not limitation.

[0158] Examples

[0159] Example 1: Determination

[0160] In the following examples, for ease of reading, various assays as described below have been used. Any deviations from the protocols provided below are noted in the relevant sections. In these experiments, a spectrophotometer was used to measure the absorbance of the product formed after the reaction was complete.

[0161] Protein secretion assay: This method measures the amount of secreted protein (polypeptide of interest) released or cleaved from the fusion polypeptide in the growth medium relative to the amount of secreted protein retained as a fusion polypeptide (e.g., attached to a carrier protein). The fusion polypeptide and polypeptide of interest were purified from a crude broth of T. reesei using Protein A resin. A 96-well plate with Protein A resin was first equilibrated in PBS, the pH of the culture broth was adjusted to pH 7 using 1 M sodium phosphate, the broth was then filtered, and the filtrate was incubated with the resin in a 96-well plate with shaking for 5 min. Any protein not bound to the Protein A resin was washed off with PBS, and the bound protein was eluted from the resin using 100 mM glycine pH 2.7, which was then neutralized with 1 M Tris pH 9. The purified protein of each variant was run on an SDS-PAGE gel ( Figure 2 Based on the molecular weights of the fusion polypeptide and the target polypeptide, the band intensities of these proteins can be quantified using image quantification software. The ratio of the fusion polypeptide to the target polypeptide can be calculated.

[0162] CBH1 Hydrolysis: To compare the ratio of fusion polypeptides to the target polypeptide of different variants, the carrier protein CBH1 in the fusion polypeptide was quantified by measuring the hydrolysis of CBH1 on its substrate 4-nitrophenyl β-D-pyranolactoside (pNP, Sigma). In a 384-well plate, 10 μl of purified protein was incubated with 40 μl of 2.5 mM pNP in 50 mM sodium acetate buffer, pH 5. The plate was sealed and incubated at 50°C for 1 hour with shaking at 1400 rpm. After 1 hour of incubation, 20 μl of 500 mM sodium carbonate buffer, pH 10, was added to each well of the 384-well Greiner plate to stop the reaction. The OD405 was measured to quantify the relative CBH1 concentration in the polypeptide. Total polypeptide and target polypeptide concentrations were measured using a protein A probe on the Octet system (ForteBio). CBH1 activity was normalized to total polypeptide and target polypeptide concentrations. The performance index (PI) of each variant was calculated by dividing the normalized CBH1 activity of the unaltered control by the normalized CBH1 activity of the variant. Variants with a PI of 8 or higher indicated that CBH1 was cleaved from the fusion polypeptide by >90%. Variants without any CBH1 activity indicated that CBH1 was completely cleaved from the fusion polypeptide.

[0163] Example 2: Generation and evaluation of site evaluation libraries

[0164] A. Plasmid and site evaluation construction for anti-RSV HC heavy chain

[0165] The sequence of the monoclonal antibody (palivizumab or Synagis) heavy chain for respiratory syncytial virus is codon optimized and synthesized by GeneArt GmH (Germany). In order to prevent the potential degradation of Kex2 furin-like protease during expression in fungal cells, the lysine at position 251 in the heavy chain is mutated to threonine (K251T). The initially synthesized anti-RSV HC sequence is cloned separately into the catalytic core and joint region (1-479aa) of Trichoderma reesei natural cellobiohydrolase 1 (CBH1) and behind it. In order to release the mature antibody chain from the carrier partner, a Kex2 cleavage site is introduced between the joint and HC.

[0166] The cbhI-HC_Synagis fusion construct was then amplified by PCR, where gene-specific primers were extended with attB1 and attB2 sites to allow BP was recombinantly cloned into the pDonor221 vector (Invitrogen, USA). The supplier, BaseClear (Netherlands), used Figure 3 The plasmid pEntry-SynagisHCK251T_Geneart_SEL shown was used as a template for the construction of a site evaluation (SEL) library at positions 466-725 aa (counted from CbhI Met). The average number of mutant variants / aa position was approximately 17. The mutant sequence was further cloned into the pTTTpyr2-ISceI target vector using recombination technology to generate the final expression plasmid pTTTpyr2-ISceI-SynagisHC_Geneart_SEL( Figure 3 ).

[0167] This expression vector contains the Trichoderma reesei cbhI promoter and terminator region that allow strong induction expression of the target gene, and the Trichoderma reesei pyr2 selective marker that gives transformants growth on minimal medium without the need for supplemental uridine. Due to the presence of the telomere region derived from Trichoderma reesei, the plasmid is maintained autonomously in the fungal cell. The plasmid is propagated in commercially available Escherichia coli TOP10 cells (Invitrogen, USA), purified, sequence-verified, individually arrayed in 96-well MTPs and used for fungal transformation as described below.

[0168] pEntry-Synagis_LC_Geneart plasmid was obtained by BP recombinant clones were constructed and further recombined with the pTrex6g destination vector in a similar manner as described above to generate the expression vector pTrex6g-Synagis_LC. This vector was used as a template to generate a PCR fragment expressing the light chain driven by the cbhI promoter and linked to the alS marker that confers resistance to chlorimuron-ethyl to fungal cells ( Figure 4 ).

[0169] B. Construction and transformation of fungal host strains

[0170] The expression cassette consists of the CBH1 promoter, the CBH1 core, the antibody HC and LC linked by a CBH1 linker and kex2 for CBH1 processing, the CBH1 terminator, and an alS marker that confers resistance to chlorimuron-ethyl to fungal cells. The alS marker is used to generate a host strain that allows the pyr2 marker to be used with SEL variants. The expression cassette is randomly integrated into the host Trichoderma reesei genome in multiple copies. The complete expression cassette is amplified by PCR. The PCR product is cleaned and concentrated to 500-1000 ng / μL.

[0171] The host Trichoderma reesei strain used for transformation was deleted for the major cellulase and xylanase. The strain was transformed using a standard PEG-protoplast transformation method. A mixture of approximately 10 μg DNA and 5×10 6 The transformation mixture of 10 protoplasts (total volume of 250 μl) was diluted with 2 volumes of 1.2M sorbitol / 10mM Tris, pH 7.5 / 10mM CaCl2 solution and mixed with 26mL of 2% low melting point agarose containing 1M sorbitol, 1g / L uridine, and 75mg / L chlorimuron-methyl in minimal medium and distributed on four 10cm culture dishes, which were previously poured with minimal medium containing 1.5% agarose and 1M sorbitol. After sufficient growth transformants from each plate were observed, individual colonies were picked onto fresh 10cm culture dishes containing 1.5% agar, 1g / L uridine, and 75mg / L chlorimuron-methyl, 4 per plate, to allow space for stability assessment. The stable colony phenotype was concentric circular growth with smooth edges. Once stable transformants were observed and sporulation was complete, the spores were harvested and used to inoculate liquid cultures.

[0172] All high-throughput transformations of Synagis HC variants were performed robotically in a 24-well MTP format using a Biomek robot (Beckman Coulter, USA). Plasmids carrying the variants were received from the supplier in a 96-well format arranged according to a predetermined layout. A 25% PEG solution containing approximately 1 mg of DNA and 5 x 106 The transformation mixture of 10 protoplasts (total volume 50 ml) was diluted with 1 volume of 1.2 M sorbitol / 10 mM Tris, pH 7.5 / 10 mM CaCl solution, robotically rearranged into a 24-well MTP, and filled with 1 ml of 3% low-melting point agarose containing 1 M sorbitol in minimal medium. Sufficient growth transformants from each well were pooled and plated onto a fresh 24-well agar plate with minimal medium. Once sporulated, the spores were harvested and used to inoculate liquid cultures.

[0173] C. Fungal Fermentation in a Slow-Release 24-Well MTP

[0174] In order to generate sufficiently high antibody titers, 10 5 -10 6 100 T. reesei spores were inoculated into a custom-made 24-well MTP consisting of Sylgard 170 elastomer (from Dow Corning, USA) premixed with lactose, which was slowly released into the medium during fermentation to ensure continuous production. The culture was grown in 1.25 ml of medium containing: 16 g / L glucose, 9 g / L casamino acids, 10 g / L (NH4)2SO4, 4.5 g / L KH2PO4, 1 g / L MgSO4*7H2O, 1 g / L CaCl2*2H2O, 33 g / L PIPPS buffer [pH 5.5], 0.25% T. reesei trace elements (100%: 175 g / L citric acid (anhydrous), 200 g / L FeSO4*7H2O, 16 g / L ZnSO4*7H2O, 3.2 g / L CuSO4*5H2O, 1.4 g / L MnSO4*H2O, 0.8 g / L H3BO3).

[0175] The plates were incubated in an Infors shaker at 200 rpm and 28° C., 80% humidity with an amplitude of 50 mm. After 5-6 days of growth, the cultures were returned to 96-well deep-well MTP format and filtered using 96-well microtiter filter plates (0.2 μm hydrophilic PVDF membrane, Corning, Tewksbury, MA). The plates were frozen in Axygen half-deep-well plates (P-DW-11-C).

[0176] D. Purification

[0177] The plate was moved from the refrigerator to a cold room and the samples were allowed to thaw gradually at 4°C overnight. Prior to purification, the grown WT samples were removed from the plate and these samples were combined. The pooled WT, pooled low binding control, pooled high binding control, and pooled vector-only (vector expressing CBH1 in the same strain) samples were added to the designated wells, 1 mL / well. The library plate was grown in duplicate, and these controls were added to both plates. The plate was gently shaken for 2 minutes to homogenize the liquid in the wells, and then centrifuged for 1 minute to precipitate any precipitate.

[0178] The centrifuged plate was then moved to a robot to remove 20 μL of crude material for Octet Protein A quantification. 20 μL was added to 80 μL of 1X PBS in a 384-well plate (Greiner Bio-One 781209). Four library plates went into one 384-well plate, and a separate 384-well plate was used for duplicate growth of four plates (plates Xa and Xb).

[0179] After removing the sample for Octet quantification, the purification plate is then followed. The robot processes four library plates at a time. The robot adds 50 μL 1M KPi pH 7 to increase the pH of the supernatant to improve the binding of the antibody to the protein A resin. Then, the robot transfers the crude material (up to 880 μL / well) from the four plates to a 2mL filter plate (Pall Corporation (Pall) 8275), which was previously filled with 220 μL protein A resin in PBS. These filter plates are then shaken on a shaker for 5 minutes. The plate is then filtered by centrifugation at 1000g for 2 minutes, and the effluent is collected in an empty harvest plate from which the sample is transferred. This material is stored until quantitative. The filter plate is placed back on the robot platform, and duplicate growth plates are added to the same filter plate. These plates are incubated and centrifuged as described above. The resin is then washed with 880 μL of PBS buffer. The plate is shaken for 1 minute, then centrifuged at 1000g for 2 minutes. The effluent is discarded, and the plate is returned to the robot for a second PBS wash. After the second wash, the plate is moved to the robot which runs the elution program.

[0180] The elution program processes four plates at a time. It adds 11 μL of neutralization buffer (1M Tris pH 9) to a clean half-deep well plate, into which the sample will be eluted. Then, the program adds 440 μL of elution buffer (100 mM glycine pH 2.7) to the filter plate. The plate is then vibrated at setting 7 for 1 minute and then filtered into a newly prepared recovery plate by centrifugation (1000 g, 2 minutes). After centrifugation, the sample plate is vibrated for 1 minute to ensure that the neutralization buffer is properly mixed.

[0181] E. Results

[0182] The variants were tested for cleavage efficiency as described in Example 1. Figure 2 Representative SDS-PAGE gels of unchanged and variant KEX2 cleavage sites are shown. The performance index (PI) of each variant relative to the unchanged cleavage domain is calculated. A PI of 8 or higher for a variant indicates complete or near-complete (>90%) cleavage relative to the unchanged amino acid sequence, as shown in Table 1.

[0183] Table 1: Single variant cleavage sites with improved cleavage efficiency

[0184] Variants CBH1 PI Location A482H 11.6 A3 A482K 12.0 A3 A482R 29.3 A3 T479A 13.4 T1 T479F 25.3 T1 E484F 9.3 E5 T479M 19.5 T1 T479Q 17.6 T1 T479R 10.2 T1 T479Y 22.1 T1 E484W 17.5 E5 Q487D 32.9 Q6 Q487G 21.7 Q6 S480F 37.7 S2 S480H 22.6 S2 S480K 24.6 S2 S480L 37.9 S2 S480M 28.0 S2 S480P 89.0 S2 S480Q 14.5 S2 S480R 57.1 S2 S480V 18.3 S2 S480W 10.8 S2 V488F 9.8 V7 V483L 35.7 V4 V488L 21.0 V7

[0185] Example 3: Generation and testing of combinatorial variants

[0186] Further variants were generated containing multiple substitutions in the linker site from those shown in Table 1 to test the combinatorial potential of the substitutions to achieve greater or complete cleavage of the fusion protein.

[0187] The heavy and light chains of Synagis antibodies are assembled into one vector for co-expression.

[0188] The synthesized DNA was cloned into expression vectors for conversion of single-chain variants into single-chain antibody host strains. These vectors had combinations of variants designed based on the SEL data shown in Table 1. A specific subset of variants was selected that would be combined with each other and expressed in a host Trichoderma reesei strain that had been deleted for the major cellulases and xylanases.

[0189] This expression vector pAS25 ( Figure 6 ) contains the Trichoderma reesei cbhI promoter and terminator regions that allow strong inducible expression of the target gene, and the Trichoderma reesei pyr2 selectable marker that confers growth on minimal medium in the absence of uridine. Due to the presence of the Trichoderma reesei-derived telomeric region, these plasmids are maintained autonomously in fungal cells. The expression vector also contains CBH1 core exons 1 and 2 and part 3, where it is cut and the sequences of ccdB and chloramphenicol resistance marker are between this and the partial CBH1 terminator. To place the target cassette into this vector, ccdB and chloramphenicol are cut using restriction enzymes and gel purified to achieve seamless assembly (Gene Art).

[0190] The HC fragment is filled with the cbh1 core, HC itself, CBH1 terminator, and 48mer adapter arms for connecting the LC box that are missing in the plasmid with homology at the 5' end. The LC box utilizes a 48mer linker for homology on the 5' end, the CBH1 promoter, the CBH1 core, the LC, the cbh1 terminator, and homology to the vector at the 3' end. These boxes are generated by PCR and treated with DpnI before purification. All three fragments are combined and seamlessly assembled (Geneart) and transformed into commercially available E. coli TOP10 cells (Invitrogen, USA). 4-6 colonies / variant combinations are selected, the plasmids are purified, diagnostically cut with the restriction enzyme NotI, and analyzed on a ZAG fragment analyzer (Advanced Analytical) to assess which clones are correctly assembled. The correctly assembled vectors are then sent to Sanger sequencing to ensure that the sequence is correct. Plasmids were propagated in commercially available E. coli TOP10 cells (Invitrogen, USA), purified, individually arrayed in 96-well MTPs and used for fungal transformation as described previously.

[0191] As shown in Table 2, these combinations also resulted in increased KEX2-mediated complete cleavage of the fusion polypeptide.

[0192] Table 2: Combination variant cleavage sites with improved cleavage efficiency

[0193]

[0194]

[0195] Additionally, SELs were created for two additional amino acids at both the KEX2 pre-cleavage site and the post-cleavage site, as shown below:

[0196] G -2 -P -1 -T1-S2-V-A3-V4-E5-X1-X2-Q6-V7-T8-L9(SEQ ID NO:65)

[0197] Surprisingly, certain substitutions at these additional positions resulted in improved or complete cleavage when used in combination with the substitutions shown in Table 1. In particular, position T8 substituted with alanine and position L9 substituted with isoleucine (when combined with one or more of the substitutions shown in Table 1), as well as position G substituted with tyrosine. -2 Position and P substituted by threonine -1 Positions resulted in complete cleavage (Table 3). Additional highly combinable substitutions were found at S2 (His) and V4 (Leu) (Table 3).

[0198] Table 3: Combinatorial variants with improved cleavage efficiency.

[0199]

[0200]

[0201] Example 4: Generation and testing of additional antibody variants

[0202] The monoclonal anti-HER2 / neu antibody trastuzumab In the generation of , the improved linker variants identified in the previous examples were tested for cleavage efficiency.

[0203] Vector Construction: The expression vectors contain the Trichoderma reesei cbhI promoter and terminator regions, which provide strong inducible expression of the gene of interest, the CBH1 core, and either the light or heavy chain of CBH1, linked by a CBH1 linker and kex2. The light chain vector contains the Aspergillus nidulans amdS selectable marker, which confers growth on minimal medium with acetamide as the sole nitrogen source. The heavy chain vector contains the Trichoderma reesei pyr2 selectable marker, which confers growth on minimal medium without uridine. Vector construction was completed and delivered by Twist Biosciences (San Francisco, CA). In total, four vectors were constructed: two containing the light chain of the antibody trastuzumab and two containing the heavy chain of the antibody trastuzumab. For each chain, the linker portion contained or did not contain the S480K mutation, enabling evaluation of the effectiveness of processing the antibody chain from the fusion partner CBH1.

[0204] Fungal strains and transformation: To evaluate the effectiveness of the S480K linker mutation, all combinations of heavy and light chains with or without the S480K mutation were tested. Since the heavy and light chains are located on separate vectors with different selectable markers, the heavy and light chain vectors were co-transformed into the host Trichoderma reesei strain. Due to the presence of the T. reesei-derived telomeric region, these plasmids are autonomously maintained in the fungal cells. The use of replicating plasmids results in increased transformation frequency and avoids the problem of locus-dependent expression observed with integrative fungal transformations.

[0205] The host Trichoderma reesei strain used for transformation was deleted for the major cellulase and xylanase enzymes. The strain was transformed using a standard PEG-protoplast transformation method. A mixture containing approximately 1 μg of each vector DNA and 5 x 10 6The transformation mixture of 10 protoplasts (total volume of 250 μL) was diluted with 2 volumes of 1.2 M sorbitol / 10 mM Tris, pH 7.5 / 10 mM CaCl2 solution and mixed with 8 mL of 2% low melting point agarose in minimal medium containing 1 M sorbitol and 20 mM acetamide. The mixture was then distributed on 10 cm Petri dishes that had been previously filled with minimal medium containing 1.5% agarose, 1 M sorbitol, and 20 mM acetamide. After sufficient growth, the transformants were pooled and plated on fresh agar plates containing minimal medium containing 10 mM acetamide as the sole nitrogen source. Once sporulated, the spores were harvested and used to inoculate liquid cultures.

[0206] In order to generate sufficiently high antibody titers, 10 5 -10 6 100 T. reesei spores were inoculated into a custom 24-well MTP consisting of Sylgard 170 elastomer (from Dow Corning, USA) premixed with lactose, which was slowly released into the culture medium during fermentation to ensure continuous production. The culture was grown in 1 ml of medium containing: 16 g / L glucose, 9 g / L casamino acids, 10 g / L (NH4)2SO4, 4.5 g / L KH2PO4, 1 g / L MgSO4*7HO, 1 g / L CaCl2*2HO, 33 g / L PIPPS buffer [pH 5.5], and 0.25% T. reesei trace elements.

[0207] The plate was incubated in an Infors shaker at 200 rpm and 28° C., 80% humidity with an amplitude of 50 mm. After 5-6 days of growth, the culture was returned to the 96-well deep-well MTP mode and filtered using a 96-well microtiter filter plate (0.2 μm hydrophilic PVDF membrane, Corning, Tewksbury, Massachusetts). The antibody expression of the sample was clarified by Western blot analysis using premixed heavy and light chain-specific antibody peroxidase conjugates from Promega (Madison, Wisconsin).

[0208] Results: Complete CBH1 processing of trastuzumab light and heavy chains expressed with or without the S480K mutation was assessed by Western blotting. Figure 9 As shown, when the S480K mutation is present in either the heavy or light chain, it is almost fully processed from CBH1. In contrast, if the mutation is absent, a poorly processed CBH1 signal is evident from either the heavy or light chain. Thus, this mutation is essential for improved to complete CBH1 processing from the trastuzumab antibody chain.

Claims

1. A KEX2 region linker, characterized in that the amino acid sequence of the KEX2 region is T1-S2-V-A3-V4-E5-X1-X2-Q6-V7, wherein S2 is substituted with an amino acid selected from the group consisting of F, H, K, L, M, P, Q, R, N, and V, and X1 and X2 are KR.

2. The KEX2 region linker of claim 1, wherein the amino acid sequence of the KEX2 region is selected from the group consisting of TFVAVEKRQV, THVAVEKRQV, TKVAVEKRQV, TLVAVEKRQV, TMVAVEKRQV, TPVAVEKRQV, TQVAVEKRQV, TRVAVEKRQV, and TVVAVEKRQV.

3. The KEX2 region linker of claim 1, wherein the KEX2 region amino acid sequence is TKVAVEKRQV.

4. A KEX2 region linker, characterized in that the amino acid sequence of the KEX2 region is T1-S2-V-A3-V4-E5-X1-X2-Q6-V7, wherein X1 and X2 are KR, and wherein the amino acid sequence is substituted, and the substitution is selected from the following substitutions: S480H-Q487D; S480H-V483L; S480H-V483L-E484W; S480K-A482R-Q487D-V488L; S480K-Q487D-V488L; S480N-Q487D; T479E-S480H; T 479E-S480H-V483L-E484W; T479E-S480P-V483L-E484W; T479N-S480H-V483L-E484W; T479E-S480H-E484W; T479E-S480H-V483L; T479R-S480K-A482R-Q487D; T479R-S480K-A482R-Q487D-V488L; T479R-S480K-A482R-V488L; or T479R-S480K-Q487D-V488L.

5. A KEX2 region linker, characterized in that the amino acid sequence of the KEX2 region is an amino acid sequence selected from the group consisting of: THVAVEKRQVTI, THVAVEKRQVAL, TNVAVEKRQVAL and TNVALEKRDVAL.

6. Use of the KEX2 region linker according to any one of claims 1 to 5 in preparing a fusion polypeptide.

7. The method according to claim 6, wherein the amino acid sequence of the KEX2 region is TKVAVEKRQV.

8. The use according to claim 6, wherein the amino acid sequence of the KEX2 region is completely cleaved by one or more proteases.

9. The use according to claim 8, wherein the protease is Kex2 serine peptidase.

10. The use according to claim 6, wherein the fusion polypeptide further comprises a polypeptide encoding a signal sequence. The use according to claim 10 , wherein the polypeptide encoding the signal sequence is located at the N-terminus or C-terminus of the amino acid sequence of the KEX2 region.

12. The method of claim 6, wherein the fusion polypeptide further comprises a polypeptide encoding a carrier protein.

13. The use according to claim 12, wherein the polypeptide encoding the carrier protein is located at the N-terminus or C-terminus of the amino acid sequence of the KEX2 region.

14. The use according to claim 12, wherein the polypeptide encoding the carrier protein is adjacent to the polypeptide encoding the signal sequence. The use according to claim 12 , wherein the carrier protein comprises CBH1 or a fragment thereof.

16. The method of claim 6, wherein the fusion polypeptide further comprises a polypeptide of interest.

17. The use according to claim 16, wherein the polypeptide encoding the polypeptide of interest is located at the N-terminus or C-terminus of the amino acid sequence of the KEX2 region.

18. The use according to claim 16, wherein the polypeptide of interest is an enzyme.

19. The use according to claim 18, wherein the enzyme is an enzyme selected from the group consisting of active or inactive carbohydrate degrading enzymes, proteases, lipases, and cell lytic enzymes.

20. The use according to claim 16, wherein the polypeptide of interest is a therapeutic protein. The use according to claim 20 , wherein the therapeutic protein is an antibody or a functional fragment thereof.

22. The method of claim 21, wherein the antibody is a light chain or heavy chain monoclonal antibody.

23. The use according to claim 6, wherein the fusion polypeptide further comprises a signal sequence, a polypeptide encoding a carrier protein and a polypeptide of interest. The use according to claim 23 , wherein the signal sequence is a CBH1 signal sequence, the carrier protein is a carrier protein containing CBH1, and the target polypeptide is an antibody light chain or a functional fragment thereof.

25. The use according to claim 23, wherein the signal sequence is a CBH1 signal sequence, the carrier protein is a carrier protein containing CBH1, and the target polypeptide is an antibody heavy chain or a functional fragment thereof. The use according to claim 23 , wherein the signal sequence is a CBH1 signal sequence, the carrier protein is a carrier protein containing CBH1, and the target polypeptide is an antibody heavy chain or a fragment thereof and an antibody light chain or a functional fragment thereof.

27. The use according to claim 21, wherein the antibody or functional fragment thereof is a single domain antibody (sdAb).

28. The use according to claim 21, wherein the antibody or functional fragment thereof is selected from the group consisting of: Fv, Fab, Fab', Fab'-SH, F(ab')2, diabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

29. The use of any one of claims 21-22 and 24-28, wherein the antibody or functional fragment thereof is an anti-respiratory syncytial virus (RSV) antibody, an anti-Ebola virus antibody, an anti-aggregated β-amyloid protein (Aβ) antibody, an anti-human immunodeficiency virus (HIV) antibody, an anti-herpes simplex virus (HSV) antibody, an anti-sperm antibody, and an anti-HER2 / neu antibody.

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