Methods and compositions for protein synthesis and secretion

Abstract

To provide novel secretion signal peptides effective in improving extracellular production of proteins, including mammalian proteins such as human milk proteins.SOLUTION: The present invention provides isolated nucleic acids encoding such secretion signal peptides linked to a recombinant protein such as a human milk protein, as well as cells comprising such nucleic acids, and methods for producing and collecting recombinant proteins from such cells.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and the benefit of U.S. Provisional Application No. 63 / 227,820, filed July 30, 2021, and U.S. Provisional Application No. 63 / 273,858, filed October 21, 2021, which are incorporated by reference in their entirety into this specification.

[0002] Array List

[0002] This application contains a Sequence Listing that has been submitted in XML format, which is hereby incorporated by reference in its entirety. Said XML copy, created on July 76, 2022, is named HELA_P0005WO_Sequence_Listing.xml and is 61,471 bytes in size. [Background technology]

[0003] I.Technical field Aspects of the present invention relate to at least the fields of microbiology, genetics and biotechnology.

[0004] II. Background Yeast is a desirable host for the production of recombinant proteins due to its rapid growth, its ability to reach high cell densities, its ability to grow on defined minimal media, achieve high protein yields, and its ability to perform eukaryotic post-translational modifications. The most suitable yeast for protein production is Pichia pastoris (Komagataella pastoris, Komagataella phaffii) because of the widespread availability of genome information and molecular tools for genome manipulation. These have enabled the use of Pichia pastoris for the production of GRAS ingredients according to FDA standards.

[0005] For a variety of biotechnological applications, it is often preferred to produce proteins that are secreted into the growth medium for ease of recovery. Pichia pastoris is capable of secreting active recombinant proteins while maintaining low levels of secretion of endogenous proteins.

[0006] In eukaryotes, secreted proteins are first targeted from the cytoplasm to the lumen of the endoplasmic reticulum (ER) by translocation. Translocation into the ER can occur either post-translationally (i.e., once the polypeptide chain has been synthesized) or co-translationally (i.e., while the mRNA is being translated into its amino acid sequence). Post-translational translocation requires the action of chaperones that maintain the polypeptide chain in a loose conformation in the cytoplasm as well as the ER-resident chaperone Kar2, which acts as a molecular ratchet. As a result, this process can be hindered by partially folded domains and / or aggregation in the cytoplasm. Therefore, for biotechnological applications, it is desirable to promote co-translational translocation. Once in the ER, proteins are glycosylated, their disulfide bonds are isomerized, and they fold to their native state. Well-folded proteins then translocate to the Golgi complex where, after further glycosylation, they are loaded into secretory granules, which fuse to the cell membrane and release the proteins into the extracellular environment.

[0007] Targeting of proteins to the secretory pathway is mediated by secretory peptides. The most widely used in Pichia pastoris is the leader peptide of mating factor alpha from Saccharomyces cerevisiae. It is composed of two distinct regions: ii) a pre-region of the first 19 amino acids that facilitates post-translational translocation and is cleaved upon ER entry, and ii) a pro-segment of 70 amino acids that functions as an export signal from the ER to the Golgi apparatus, where it is cleaved at a dibasic amino acid cleavage site, KR.

[0008]

[0008] There is a need for synthetic secretory signal peptides that lead to higher extracellular production of proteins. Summary of the Invention

[0009] Aspects of the present disclosure address a particular need by providing novel secretory signal peptides that are effective in improving the extracellular production of proteins, including mammalian proteins such as human milk proteins. Certain aspects of the present disclosure are based, at least in part, on the development of signal peptides generated from an in-frame fusion of 1) a P. pastoris pre-secretory peptide from either i) the alpha subunit of the oligosaccharyltransferase complex of the ER lumen (Ost1) or ii) the GPI-anchored protein Pst1 with 2) a proregion of either i) the Saccharomyces cerevisiae mating factor or ii) the proregion of P. pastoris Epx1. Thus, described herein are isolated nucleic acids encoding such secretory signal peptides, in some cases linked to recombinant proteins such as human milk proteins, as well as cells containing such nucleic acids and methods for producing and recovering recombinant proteins from such cells.

[0010]

[0010] In one embodiment, described herein is an isolated nucleic acid encoding a polypeptide comprising a sequence having at least 90% sequence identity to SEQ ID NO:1, 2, 3 or 4. In one embodiment, the sequence comprises SEQ ID NO:1, 2, 3 or 4. In one embodiment, the polypeptide further comprises a sequence of a mammalian protein. In one embodiment, the mammalian protein is a human milk protein. In one embodiment, the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme or α-lactalbumin. In one embodiment, the human milk protein is human lactoferrin.

[0011] In some embodiments, the sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:1. In some embodiments, the sequence comprises SEQ ID NO:1. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:41. In some embodiments, the nucleic acid sequence comprises SEQ ID NO:41. In some embodiments, the polypeptide comprises SEQ ID NO:5. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:46. In some embodiments, the nucleic acid sequence comprises SEQ ID NO:46.

[0012] In some embodiments, the sequence is at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO:2. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:42. In some embodiments, the nucleic acid sequence comprises SEQ ID NO:42. In some embodiments, the polypeptide comprises SEQ ID NO:6. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity to SEQ ID NO:47. In some embodiments, the nucleic acid sequence comprises SEQ ID NO:47.

[0013] In some embodiments, the sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:3. In some embodiments, the sequence comprises SEQ ID NO:3. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:43. In some embodiments, the nucleic acid sequence comprises SEQ ID NO:43. In some embodiments, the polypeptide comprises SEQ ID NO:7. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:48. In some embodiments, the nucleic acid sequence comprises SEQ ID NO:48.

[0014] In some embodiments, the sequence has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO:4. In some embodiments, the sequence comprises SEQ ID NO:4. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:44. In some embodiments, the nucleic acid sequence comprises SEQ ID NO:44. In some embodiments, the polypeptide comprises SEQ ID NO:8. In some embodiments, the isolated nucleic acid comprises a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:49. In some embodiments, the nucleic acid sequence comprises SEQ ID NO:49.

[0015] Also disclosed herein, in certain aspects, are vectors that include a nucleic acid disclosed herein (eg, an isolated nucleic acid or a sequence or portion thereof).

[0016]

[0016] In some aspects, further disclosed are engineered eukaryotic cells comprising the nucleic acids disclosed herein. In some embodiments, the cells are fungal cells. In some embodiments, the fungal cells are selected from the group consisting of Arxula, Aspergillus, Aurantiochytrium, Candida, Claviceps, Cryptococcus, Cunninghamella, Geotrichum, and the like. The genera Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidiella, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus pus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, or Yarrowia. In some embodiments, the cell is a yeast cell. In some embodiments, the yeast cell is a Komagataella cell. In some embodiments, the yeast cell is a Komagataella puffii, Komagataella pastoris, or Komagataella pseudopastoris cell. In some aspects, the nucleic acid is integrated into the genome of the cell. In some aspects, the nucleic acid is not integrated into the genome of the cell.

[0017]

[0017] Also disclosed in one aspect is a method for producing a secreted protein, the method comprising growing an engineered eukaryotic cell of the present disclosure under conditions sufficient to secrete the polypeptide from the cell. In one aspect, the method further comprises recovering the secreted protein. In one aspect, the secreted protein is a human milk protein. In one aspect, the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin. In one aspect, the human milk protein is human lactoferrin. In one aspect, the human milk protein comprises one or more human-like N-glycans. In one aspect, the method further comprises producing a mixture comprising the human milk protein and one or more components of an infant formula.

[0018]

[0018] Further disclosed herein, in one aspect, is an engineered yeast cell comprising a nucleic acid encoding a polypeptide comprising a sequence having at least 90% sequence identity to SEQ ID NO:1, 2, 3 or 4. In one embodiment, the sequence comprises SEQ ID NO:1. In one embodiment, the sequence comprises SEQ ID NO:2. In one embodiment, the sequence comprises SEQ ID NO:3. In one embodiment, the sequence comprises SEQ ID NO:4. In one embodiment, the polypeptide further comprises a sequence of a mammalian protein. In one embodiment, the mammalian protein is a human milk protein. In one embodiment, the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, beta-casein, kappa-casein, leptin, lysozyme, or alpha-lactalbumin. In some embodiments, the human milk protein is human lactoferrin.

[0019] In one aspect, described herein is an engineered yeast cell that includes: (a) a first nucleic acid encoding a polypeptide comprising (i) a sequence having at least 90% sequence identity to SEQ ID NO:1, 2, 3, or 4, and ii) the sequence of a human milk protein; and (b) a second nucleic acid encoding an alpha-1,2-mannosidase (Man-I) protein, wherein the cell does not express a functional OCH1 protein. In one aspect, the sequence of (i) comprises SEQ ID NO:1, 2, 3, or 4. In one embodiment, the human milk protein is secretory IgA (sIgA), xanthene, or eosinophilic IgA (eosinophilic IgA). In some embodiments, the human milk protein is human lactoferrin, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin. In some embodiments, the human milk protein is human lactoferrin. In some embodiments, the human milk protein is human α-lactalbumin. In some embodiments, the Man-I protein is fused to a HDEL C-terminal tag. In some embodiments, the cell further comprises a third nucleic acid encoding one or more of: (a) N-acetylglucosaminyltransferase I (GnT-I) protein; (b) α-1,3 / 6-mannosidase (Man-II) protein; (c) β-1,2-acetylglucosaminyltransferase (GnT-II) protein; and (d) β-1,4-galactosyltransferase (GalT) protein. In some embodiments, the yeast cell is a cell of the genus Komagataella. In some embodiments, the yeast cell is a Komagataella puffii, Komagataella pastoris, or Komagataella pseudopastoris cell. In some aspects, the nucleic acid is integrated into the genome of the cell. In some aspects, the nucleic acid is not integrated into the genome of the cell.

[0020]

[0020] It is contemplated that any embodiment discussed herein can be implemented with respect to any method or composition of the disclosed embodiments, and vice versa. Furthermore, the compositions of the embodiments disclosed herein can be used to achieve the methods of those embodiments.

[0021]

[0021] Other objects, features and advantages of the present embodiments disclosed herein will become apparent from the following detailed description. However, it should be understood that the detailed description and specific examples, while showing particular embodiments, are given by way of illustration only, since various changes and modifications within the spirit and scope of the embodiments disclosed herein will become apparent to those skilled in the art from this detailed description.

[0022] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein. [Brief description of the drawings]

[0023] [Figure 1]

[0023] Figure 1 is an image of a Western blot of the supernatant. Lane 1 is loaded with protein standard, Genscript, M00624 (ThermoFisher Scientific, Waltham, Massachusetts, USA). Lane 2 is loaded with human milk lactoferrin, Sigma Aldrich, SRP6519 (Sigma Aldrich, St. Louis, Missouri, USA). Lane 3 is loaded with a control (Saccharomyces cerevisiae pre-pro-MFα). Lane 4 is loaded with a negative control (supernatant of untransformed yeast cells). Lanes 5-6 are loaded with supernatant from SP2-lactoferrin transformed yeast cells. Lanes 7-8 are loaded with supernatant from SP3-lactoferrin transformed yeast cells. Lanes 9-10 are loaded with SP1-lactoferrin transformed yeast cells.

[0024] [Diagram 2]

[0024] Figure 2 is a bar graph showing protein expression levels. Quantification of extracellular proteins was performed by ELISA. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025]

[0025] Described herein is the generation of novel synthetic secretion signal peptides. Also disclosed are cells (e.g., fungal cells, such as yeast cells) engineered to express one or more exogenous proteins (e.g., human milk proteins) that contain such signal peptides. As disclosed herein, the "protease inhibitors" derived from P. pastoris Ost1 or Pst1 are In-frame fusion of the "le domain" sequence and the "pro domain" sequence from Saccharomyces cerevisiae mating factor alpha or P. pastoris Epx1 can facilitate increased extracellular protein production compared to previously used signal peptides. The disclosed signal peptides include, for example, peptides comprising SEQ ID NO:1, 2, 3 or 4, as well as peptides comprising 1, 2, 3, 4 or 5 amino acid substitutions (or more) relative to SEQ ID NO:1, 2, 3 or 4. As described herein, in-frame fusion of these hybrid signal peptides to the N-terminus of a mammalian protein (e.g., a human milk protein such as lactoferrin or alpha-lactalbumin) facilitates highly efficient protein secretion.

[0026] I. Definition

[0026] The term "biologically active portion" refers to an amino acid sequence that is less than the full-length amino acid sequence but that exhibits at least one activity of the full-length sequence. For example, a biologically active portion of an enzyme can refer to one or more domains of the enzyme that have the catalytic activity of the enzyme (i.e., the catalytic domain). In one aspect, a biologically active portion of an enzyme is a portion of the enzyme that includes the catalytic domain of the enzyme. A biologically active portion of a protein includes a peptide or polypeptide that includes an amino acid sequence that is sufficiently identical to or derived from the amino acid sequence of the protein, which includes fewer amino acids than the full-length protein, and which exhibits at least one activity of the protein (e.g., enzymatic activity, functional activity, etc.).

[0027] The term "exogenous" refers to anything that is introduced or has been introduced into a cell. An "exogenous nucleic acid" is a nucleic acid that enters or has entered a cell through a cell membrane. An "exogenous nucleic acid sequence" is a nucleic acid sequence of an exogenous nucleic acid. An exogenous nucleic acid can contain a nucleotide sequence that is present in the native genome of the cell and / or a nucleotide sequence that was not previously present in the genome of the cell. An exogenous nucleic acid includes an exogenous gene. An "exogenous gene" is a nucleic acid that codes for the expression of an RNA and / or protein that has been introduced into a cell (e.g., by transformation / transfection), also called a "transgene". A cell that contains an exogenous nucleic acid may be called a recombinant cell, into which additional exogenous gene(s) may be introduced. An exogenous gene may be from the same or a different species relative to the cell being transformed. Thus, an exogenous gene may include a native gene that occupies a different location in the genome of the cell or is under different control relative to the endogenous copy of that gene. An exogenous gene may be present in a cell in more than one copy number. The exogenous gene may be maintained in the cell either as an insertion in the genome (nuclear, mitochondrial, or plastid) or as an episomal molecule.

[0028]

[0028] "Operable linkage" (or "operably linked") refers to a functional linkage between two nucleic acid sequences, such as a regulatory sequence (typically a promoter) and a linked sequence (typically a sequence that encodes a protein, also called a coding sequence). A promoter is operably linked to a gene if it is capable of mediating transcription of the gene.

[0029] The term "native" refers to the composition of a cell or parent cell prior to a transformation event. A "native gene" (also called an "endogenous gene") refers to a nucleotide sequence that encodes a protein that has not been introduced into a cell by a transformation event. A "native protein" (also called an "endogenous protein") refers to an amino acid sequence encoded by a native gene.

[0030] "Recombinant" refers to a cell, nucleic acid, protein, or vector that has been modified by the introduction of an exogenous nucleic acid or the alteration of a native nucleic acid. A recombinant cell is a gene that is a nucleic acid that is generated by a host cell or vector, and any progeny, descendants, duplicates, or copies of the gene are also considered recombinant. Thus, for example, a recombinant cell can express genes not found in the native (non-recombinant) form of the cell, or express native genes differently than those same genes expressed by non-recombinant cells. A recombinant cell can include, but is not limited to, a recombinant nucleic acid that encodes a gene product, or an inhibitory element that reduces the level of an active gene product in a cell, such as a mutation, knockout, antisense, interfering RNA (RNAi), or dsRNA. A "recombinant nucleic acid" is generally derived from a nucleic acid originally formed in vitro, for example, by manipulating the nucleic acid with polymerases, ligases, exonucleases, endonucleases, or in a form that is not normally found in nature otherwise. Once a recombinant nucleic acid is made and introduced into a host cell or organism, it can be replicated using the in vivo cellular machinery of the host cell; however, such a nucleic acid, once produced recombinantly, is still considered recombinant for the purposes of this disclosure, even though it is subsequently replicated within the cell. In addition, recombinant nucleic acid refers to a nucleotide sequence that comprises an endogenous nucleotide sequence and an exogenous nucleotide sequence; thus, an endogenous gene recombined with an exogenous promoter is a recombinant nucleic acid. A "recombinant protein" is a protein made using recombinant techniques, i.e., by expression of a recombinant nucleic acid.

[0031]

[0031] "Transformation" refers to the transfer of a nucleic acid into a host organism or the genome of a host organism. Host organisms (and their progeny) containing the transformed nucleic acid fragments are referred to as "recombinant", "transgenic" or "transformed" organisms. Thus, the isolated polynucleotides of the present disclosure can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell. Such constructs can be vectors that contain a replication system and sequences that allow transcription and translation of a polypeptide coding sequence in a given host cell. Typically, an expression vector contains one or more cloned genes and a selectable marker, for example, under the transcriptional control of 5' and 3' regulatory sequences. Such vectors can also contain a promoter control region (e.g., a control region that controls inducible or constitutive, environmentally or developmentally regulated, or site-specific expression), a transcription initiation start site, a ribosome binding site, a transcription termination site, and / or a polyadenylation signal. Alternatively, cells can be transformed with a single genetic element, such as a promoter, which upon integration into the genome of the host organism, such as by homologous recombination, can result in genetically stable inheritance.

[0032]

[0032] The term "transformed cell" refers to a cell that has undergone transformation. Thus, a transformed cell contains the parent genome and the inheritable genetic alteration. Embodiments include the progeny and descendants of such transformed cells.

[0033]

[0033] The term "vector" refers to a means by which a nucleic acid can be propagated and / or transferred between organisms, cells, or cellular components. Vectors include plasmids, linear DNA fragments, viruses, bacteriophages, proviruses, phagemids, transposons, artificial chromosomes, and the like, whether or not they can replicate autonomously or can integrate into the chromosome of a host cell.

[0034]

[0034] "Individual," "subject," and "patient" are used interchangeably and can refer to a human or a non-human.

[0035]

[0035] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error in the measurement or quantification method.

[0036]

[0036] When used in conjunction with the term "including," the use of the words "a" or "an" , "1," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one."

[0037] The phrase "and / or" means "and" or "or." Depicting A, B and / or C includes: A only, B only, C only, a combination of A and B, a combination of A and C, a combination of B and C, or a combination of A, B and C. In other words, "and / or" operates as inclusive.

[0038]

[0038] The words "comprising" (and all forms of comprising, e.g., "comprise" and "comprises"), "having" (and all forms of having, e.g., "have" and "has"), "including" (and all forms of including, e.g., "includes" and "include") or "containing" (and all forms of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0039]

[0039] The compositions and methods for their use may "comprise," "consist essentially of," or "consist" of any of the components or steps disclosed throughout this specification. Compositions and methods "consisting essentially of" any of the disclosed components or steps limit the scope of the claims to the specified materials or steps that do not materially affect the basic and novel characteristics of the claimed embodiment.

[0040] II. Proteins and Nucleic Acids

[0040] As used herein, "protein" or "polypeptide" refers to a molecule that comprises at least five amino acid residues. As used herein, the term "wild type" refers to the endogenous version of a molecule that occurs naturally in an organism. In some embodiments, a wild type version of a protein or polypeptide is employed, but in many embodiments of the present disclosure, a modified protein or polypeptide is employed. The above terms may be used interchangeably. "Modified protein" or "modified polypeptide" or "variant" refers to a protein or polypeptide whose chemical structure, particularly its amino acid sequence, has been altered relative to a wild type protein or polypeptide. In some embodiments, a modified / variant protein or polypeptide has at least one modified activity or function (recognizing that a protein or polypeptide may have multiple activities or functions). It is specifically contemplated that a modified / variant protein or polypeptide can be altered with respect to one activity or function yet retain a wild type activity or function in other respects.

[0041]

[0041] When a protein is specifically mentioned herein, it is generally a reference to a natural (wild type) or recombinant (modified) protein, or a protein with any signal sequence removed, if necessary. A protein can be directly isolated from an organism in which it is natural, can be produced by recombinant DNA / exogenous expression methods, or can be produced by solid phase peptide synthesis (SPPS) or other in vitro methods. In certain embodiments, there are isolated nucleic acid segments and recombinant vectors incorporating nucleic acid sequences encoding polypeptides. The term "recombinant" can be used in conjunction with the name of a polypeptide or a particular polypeptide, and generally refers to a polypeptide produced from a nucleic acid molecule manipulated in vitro, or a product of replication of such a molecule.

[0042] In certain embodiments, the protein or polypeptide (wild-type or modified) Available in sizes 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120 , 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850 , 875, 900, 925, 950, 975, 1000, 1100, 1200, 1300, 1400, 1500, 1750, 2000, 2250, 2500 amino acid residues or more, and any range derivable therein, or derivatives of the corresponding amino acid sequences described or referenced herein. Polypeptides can be mutated by truncation to make them shorter than their corresponding wild-type forms, and they can be modified by fusing or conjugating heterologous protein or polypeptide sequences having specific functions (e.g., for targeting or localization, for enhanced immunogenicity, for purification purposes, etc.). As used herein, the term "domain" refers to any discrete functional or structural unit of a protein or polypeptide, and generally refers to a sequence of amino acids having a structure or function recognizable to one of skill in the art.

[0043]

[0043] The term "polynucleotide" refers to a nucleic acid molecule that is either recombinant or isolated from total genomic nucleic acid. Included in the term "polynucleotide" are oligonucleotides (nucleic acids of 100 residues or less in length), recombinant vectors, including plasmids, cosmids, phages, viruses, and the like. Polynucleotides, in certain aspects, include regulatory sequences that are isolated substantially away from their naturally occurring gene or protein-coding sequences. Polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be RNA, DNA (genomic, cDNA, or synthetic), analogs thereof, or combinations thereof. Additional coding or non-coding sequences can be, but need not be, present within a polynucleotide.

[0044] In this regard, the terms "gene", "polynucleotide" or "nucleic acid" are used to refer to a nucleic acid that encodes a protein, polypeptide or peptide (including any sequences required for proper transcription, post-translational modification or localization). As will be understood by those skilled in the art, this term encompasses genomic sequences, expression cassettes, cDNA sequences and smaller engineered nucleic acid segments that express or can be adapted to express proteins, polypeptides, domains, peptides, fusion proteins and variants. A nucleic acid that encodes all or a portion of a polypeptide may contain a contiguous nucleic acid sequence that encodes all or a portion of such a polypeptide. It is also contemplated that a particular polypeptide may be encoded by a nucleic acid that contains variations having slightly different nucleic acid sequences, but still encodes the same or substantially similar protein.

[0045] In certain embodiments, polynucleotide variants having substantial identity to the sequences disclosed herein; at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% or more, including all values ​​and ranges therebetween, compared to the polynucleotide sequences provided herein using the methods described herein (e.g., BLAST analysis using standard parameters). In certain aspects, the isolated polynucleotide will comprise a nucleotide sequence that encodes a polypeptide having at least 90%, and in some cases 95% or more identity over the entire length of the sequence, to an amino acid sequence described herein; or a nucleotide sequence that is complementary to the isolated polynucleotide.

[0046]

[0046] Regardless of the length of the coding sequence itself, nucleic acid segments can be combined with other nucleic acid sequences, such as promoters, polyadenylation signals, additional restriction enzyme sites, multiple cloning sites, other coding segments, etc., and therefore their overall length can vary considerably. Nucleic acids can be of any length. They can be, for example, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, 100, 125, 175, 200, 250, 300, 350, 400, 450, 500, 750, 1000, 1500, 3000, 5000 or more nucleotides in length, and / or can include one or more additional sequences, such as regulatory sequences, and / or can be part of a larger nucleic acid, such as a vector. Thus, it is contemplated that nucleic acid fragments of almost any length can be employed, with the total length preferably being limited by the ease of preparation and use in the intended recombinant nucleic acid protocol. In some cases, the nucleic acid sequence may encode a polypeptide sequence with additional heterologous coding sequences, for example to allow purification, transport, secretion, post-translational modification of the polypeptide, or for therapeutic benefits such as targeting or efficacy. As discussed above, tags or other heterologous polypeptides can be added to the modified polypeptide-encoding sequence, where "heterologous" refers to a polypeptide that is not the same as the modified polypeptide.

[0047]

[0047] The polypeptides, proteins, or polynucleotides encoding such polypeptides or proteins of the present disclosure can include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 (or any derivable range therein) or more variant amino acid or nucleic acid substitutions, or can include at least or up to the sequence represented by SEQ ID NO: NO: 1-49, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126 , 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176 , 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226 , 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 300, 400, 500, 550, 1000 or more consecutive amino acids or nucleic acids, or any range derivable therein and at least 60%, 61%, 62%, 63%, 64%, 65%, 66%, It can be 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any derivable range therein) similar, identical or homologous.

[0048] In one embodiment, the protein or polypeptide has the sequence represented by SEQ ID NO: NO:1-14 or 34-40 amino acids 1-2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 7, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262,263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、 、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699, or 700 (or any range derivable therein).

[0049] In one embodiment, the protein, polypeptide or nucleic acid is one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, fifteen, six, seven, eight, nine, nine, twenty, twenty-one, twenty-two, twenty-three, twenty-four, twenty-five, twenty-six, twenty-seven, twenty-eight, twenty-nine ... 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 20A, 201, 209, 3, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196、197、198、199、200、201、202、203、204、205、206、207、208、209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、525、526、527、528、529、530、531、532、533、534、535、536、537、538、539、540、541、542、543、544、545、546、547、548、549、550、551、552、553、554、555、556、557、558、559、560、561、562、563、564、565、566、567、568、569、570、571、572、573、574、575、576、577、578、579、580、581、582、583、584、585、586、587、588、589、590、591、592、593、594、595、596、597、598、599、600、601、602、603、604、605、606、607、608、609、610、611、612、613、614、615、616、617、618、619、620、621、622、623、624、625、626、627、628、629、630、631、632、633、634、635、636、637、638、639、640、641、642、643、644、645、646、647、648、649、650、651、652、653、654、655、656、657、658、659、660、661、662、663、664、665、666、667、668、669、670、671、672、673、674、675、676、677、678、679、680、681、682、683、684、685、686、687、688、689、690、691、692、693、694、695、 It may comprise 696, 697, 698, 699 or 700 contiguous amino acids (or any derivable range therein).

[0050] In certain embodiments, a polypeptide, protein, or nucleic acid has at least, at most, or exactly the same sequence as SEQ ID NO: NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79 , 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185 3, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 2 02, 203, 204, 205, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260,261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、 , 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 521, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 59 6, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699 or 700 contiguous amino acids (or any derivable range therein), which areis at least, at most, or exactly 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% (or any range derivable therein) similar, identical, or homologous to one of NO:1-49.

[0051] In one aspect, the amino acid sequence of any one of positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 6 0, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 11 6, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162 , 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209、210、211、212、213、214、215、216、217、218、219、220、221、222、223、224、225、226、227、228、229、230、231、232、233、234、235、236、237、238、239、240、241、242、243、244、245、246、247、248、249、250、251、252、253、254、255、256、257、258、259、260、261、262、263、264、265、266、267、268、269、270、271、272、273、274、275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459, 460, 461, 462, 463, 464, 465, 466, 467, 468, 469, 470, 471, 472, 473, 474, 475, 476, 477, 478, 479, 480, 481, 482, 483, 484, 485, 486, 487, 488, 489, 490, 491, 492, 493, 494, 495, 496, 497, 498, 499, 500, 501, 502, 503, 504, 505, 506, 507, 508, 509, 510, 511, 512, 513, 514, 515, 516, 517, 518, 519, 520, 5 21, 522, 523, 524, 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 5 52, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572, 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 58 3, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 6 4, 615, 616, 617, 618, 619, 620, 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645 , 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 668, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699 or 700 andNO: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 8 3, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118 , 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 1 81, 182, 183, 184, 185, 186, 187, 188, 189, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 205, 206, 207, 208, 209, 210, 211, 21 2, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243 , 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, 258, 259, 260, 261, 262, 263, 264, 265, 266, 267, 268, 269, 270, 271, 272, 273, 274,275、276、277、278、279、280、281、282、283、284、285、286、287、288、289、290、291、292、293、294、295、296、297、298、299、300、301、302、303、304、305、306、307、308、309、310、311、312、313、314、315、316、317、318、319、320、321、322、323、324、325、326、327、328、329、330、331、332、333、334、335、336、337、338、339、340、341、342、343、344、345、346、347、348、349、350、351、352、353、354、355、356、357、358、359、360、361、362、363、364、365、366、367、368、369、370、371、372、373、374、375、376、377、378、379、380、381、382、383、384、385、386、387、388、389、390、391、392、393、394、395、396、397、398、399、400、401、402、403、404、405、406、407、408、409、410、411、412、413、414、415、416、417、418、419、420、421、422、423、424、425、426、427、428、429、430、431、432、433、434、435、436、437、438、439、440、441、442、443、444、445、446、447、448、449、450、451、452、453、454、455、456、457、458、459、460、461、462、463、464、465、466、467、468、469、470、471、472、473、474、475、476、477、478、479、480、481、482、483、484、485、486、487、488、489、490、491、492、493、494、495、496、497、498、499、500、501、502、503、504、505、506、507、508、509、510、511、512、513、514、515、516、517、518、519、520、521、522、523、524、 525, 526, 527, 528, 529, 530, 531, 532, 533, 534, 535, 536, 537, 538, 539, 540, 541, 542, 543, 544, 545, 546, 547, 548, 549, 550, 551, 552, 553, 554, 555, 556, 557, 558, 559, 560, 561, 562, 563, 564, 565, 566, 567, 568, 569, 570, 571, 572 , 573, 574, 575, 576, 577, 578, 579, 580, 581, 582, 583, 584, 585, 586, 587, 588, 589, 590, 591, 592, 593, 594, 595, 596, 597, 598, 599, 600, 601, 602, 603, 604, 605, 606, 607, 608, 609, 610, 611, 612, 613, 614, 615, 616, 617, 618, 619, 620 , 621, 622, 623, 624, 625, 626, 627, 628, 629, 630, 631, 632, 633, 634, 635, 636, 637, 638, 639, 640, 641, 642, 643, 644, 645, 646, 647, 648, 649, 650, 651, 652, 653, 654, 655, 656, 657, 658, 659, 660, 661, 662, 663, 664, 665, 666, 667, 66 There are nucleic acid molecules or polypeptides that contain 8, 669, 670, 671, 672, 673, 674, 675, 676, 677, 678, 679, 680, 681, 682, 683, 684, 685, 686, 687, 688, 689, 690, 691, 692, 693, 694, 695, 696, 697, 698, 699 or 700 (or any derivable range therein) consecutive amino acids or nucleotides.

[0052] Nucleotide and protein, polypeptide and peptide sequences for various genes have been previously disclosed and can be found in recognized computerized databases. Two commonly used databases are the National Center for Biotechnology Information's Genbank and GenPept databases (on the World Wide Web at ncbi.nlm.nih.gov / ) and the Universal Protein Repositories. The source is UniProt; uniprot.org on the World Wide Web. The coding regions for these genes can be amplified and / or expressed using the techniques disclosed herein or as would be known to one of skill in the art.

[0053] It is contemplated that in the composition of the present disclosure, there is about 0.001mg to about 10mg of total polypeptide, peptide, and / or protein per ml.The concentration of protein in the composition can be about (at least about or at most about) 0.001, 0.010, 0.050, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0mg / ml or more (or any range derivable therein).

[0054]

[0054] For proteins with catalytic activity (e.g., enzymes), such proteins can be described using the Enzyme Classification (EC) nomenclature. The EC classification of various enzymes has been previously disclosed and can be found in recognized databases, such as the ENZYME database (Bairoch A. The ENZYME database in 2000. Nucleic Acids Res. 2000 Jan 1;28(1):304-5. doi: 10.1093 / nar / 28.1.304; incorporated herein by reference in its entirety).

[0055] A. Signal Peptides Aspects of the present disclosure are directed to synthetic signal peptides and polynucleotides and nucleic acids encoding such signal peptides. Also disclosed are cells containing such signal peptides and methods of using the cells in the production and secretion of proteins (e.g., mammalian proteins such as human milk proteins). As used herein, "signal peptide" (or "signal peptide sequence") describes any peptide that, when present at the N-terminus of a newly synthesized polypeptide, is capable of directing the polypeptide across or into a cell membrane (e.g., the plasma membrane, the endoplasmic reticulum membrane, etc.) of a cell. In certain aspects, a signal peptide of the disclosure is capable of directing a polypeptide into the secretory pathway of a cell and subsequent secretion of the polypeptide (referred to herein as a "secretory signal peptide").

[0056] As described herein, aspects of the present disclosure relate to a synthetic signal peptide comprising: (a) a preregion sequence from: (i) P. pastoris Ost1; or (ii) P. pastoris Pst1; and (b) the proregion sequence from (i) Saccharomyces cerevisiae mating factor α (MFα); or (ii) P. pastoris Epx1.

[0057]

[0057] Particular signal peptides of the present disclosure are set forth in Table 1 below.

[0058] [Table 1-1]

[0059] [Table 1-2]

[0058] In one aspect, a polypeptide comprising a signal peptide of the present disclosure is disclosed. Nucleic acids encoding such polypeptides are also disclosed. Further, cells expressing a polypeptide comprising a signal peptide of the present disclosure are also disclosed.

[0060] In some aspects, the polypeptides of the disclosure include SEQ ID NO:1. In some embodiments, the polypeptides of the disclosure include a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:1. In some aspects, the polypeptides of the disclosure include a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions (or more) to SEQ ID NO:1.

[0061] In some aspects, the polypeptide of the disclosure comprises SEQ ID NO:2. In some embodiments, the polypeptide of the disclosure comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:2. In some aspects, the polypeptide of the disclosure comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions (or more) to SEQ ID NO:2.

[0062] In some aspects, the polypeptide of the disclosure comprises SEQ ID NO:3. In some embodiments, the polypeptide of the disclosure comprises a sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:3. In some aspects, the polypeptide of the disclosure comprises a sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions (or more) to SEQ ID NO:3.

[0063] In some aspects, the polypeptide of the present disclosure comprises SEQ ID NO: 4. In some embodiments, the polypeptide of the present disclosure comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, 210%, 220%, 230%, 240%, 250%, 260%, 270%, 280%, 290%, 300%, 310%, 320%, 330%, 340%, 350%, 360%, 370%, 380%, 390%, 400%, 410%, 420%, 430%, 440%, 450%, 460%, 470%, 480%, 490%, 500%, 510%, 520%, 530%, 540%, 550%, 560%, 570%, 580%, 590%, 600%, 610%, 620%, 630%, 640%, 650%, 660%, 670%, 680%, 690%, 700%, 710%, 720%, 730%, 740%, 750%, 760%, 770%, 780%, 790%, 800%, 810%, 820%, 830%, 840%, 850%, 860%, 870%, 880%, 890%, 900%, 910%, 920%, 930%, 940%, 950%, 960%, 970 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO:4. In certain aspects, polypeptides of the disclosure include sequences having 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid substitutions (or more) to SEQ ID NO:4.

[0064] Any one or more of the signal peptides disclosed herein may be excluded from certain embodiments.

[0065] B. Secreted proteins Aspects of the present disclosure include secreted proteins (also referred to as "secreted proteins"), as well as compositions comprising secreted proteins, methods of expressing secreted proteins, and methods of their use. As used herein, "secreted protein" describes any protein that is secreted outside of a cell. In certain cases, the secreted proteins of the present disclosure are proteins present in human secretions, such as colostrum, milk, tears, semen, vaginal fluid, saliva, or other secretions. In some aspects, the secreted proteins of the present disclosure are human milk proteins. In some aspects, the secreted proteins of the present disclosure are not human milk proteins.

[0066] 1. Human milk proteins Aspects of the disclosure include human milk proteins, as well as compositions comprising human milk proteins (e.g., infant formula compositions), methods of producing human milk proteins, and methods of their use. In one aspect, cells are disclosed that express a human milk protein (e.g., comprising SEQ ID NO:1, 2, 3, or 4) linked to a signal peptide of the disclosure. As used herein, "human milk protein" describes any protein present in human breast milk. Human milk proteins include proteins derived from (e.g., isolated from) human breast milk, as well as any protein produced by other means (e.g., recombinant expression, chemical synthesis, etc.) that has the amino acid sequence of a protein present in human breast milk. A variety of human milk proteins are recognized in the art and are contemplated herein. The human milk protein contemplated herein includes, but is not limited to, secretory IgA (sIgA), human serum albumin, xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, and α-lactalbumin.In some embodiments, the human milk protein of the present disclosure is human whey protein.In some embodiments, the human milk protein of the present disclosure is recombinant human milk protein (e.g., produced by non-mammalian cells such as yeast cells).

[0067]

[0066] Certain aspects of the present disclosure are directed to human milk proteins having "human-like" glycans. Human-like glycans (also referred to as "human-like glycan structures") describe glycans having structures present on human glycoproteins. Such glycans include, for example, hybrid N-glycans, complex N-glycans, biantennary, triantennary and tetraantennary N-glycans, as well as glycans containing sialic acid, galactose, N-acetylgalactosamine or fucose. Human-like glycans include those having a Man3GlcNAc2 core structure. Thus, the human milk proteins of the present disclosure include those having one or more human-like glycans, such as hybrid N-glycans, complex N-glycans, biantennary N-glycans, triantennary N-glycans, tetraantennary N-glycans, and combinations thereof.

[0068]

[0067] Thus, in one embodiment, recombinant human milk proteins (e.g., recombinant human lactoferrin) are disclosed that contain one or more human-like glycans. Such recombinant proteins include those produced by engineered mammalian, fungal, yeast, bacterial, or other cells, including, for example, the engineered cells described elsewhere herein. In certain aspects, such recombinant proteins have a glycan pattern that differs from the glycan pattern of the corresponding native human milk protein. For example, in one embodiment, a recombinant human lactoferrin is disclosed that comprises one or more human-like glycans, wherein the lactoferrin has a glycan pattern that differs from the glycan pattern of any naturally occurring human lactoferrin (e.g., human lactoferrin in human breast milk).

[0069] Lactoferrin Aspects of the present disclosure are directed to lactoferrin and compositions comprising lactoferrin, including infant formula compositions. In one aspect, cells are disclosed that express human lactoferrin linked to a signal peptide of the present disclosure (e.g., comprising SEQ ID NO:1, 2, 3, or 4). Lactoferrin (also called "lactotransferrin") is a whey protein found in exocrine fluids, such as breast milk, and is encoded by the LTF gene. Without wishing to be bound by theory, lactoferrin is understood to have antimicrobial and anti-inflammatory properties. Certain aspects of the present disclosure are directed to human lactoferrin, including its isoforms (UniProtKB / Swiss-Prot Accession No. P02788). The full sequence of human lactoferrin, including the signal peptide, is provided as SEQ ID NO:34. The sequence of mature human lactoferrin after cleavage of the signal peptide is provided as SEQ ID NO:9.

[0070] [Table 2-1]

[0071] [Table 2-2]

[0072] [Table 2-3]

[0073] [Table 2-4]

[0074] [Table 2-5]

[0075] [Table 2-6]

[0076]

Table 2-7

[0077]

Table 2-8

[0078]

Table 2-9

[0079]

Table 2-10

[0080]

Table 2-11

[0069] In some aspects, the human lactoferrin of the present disclosure is recombinant human lactoferrin (rhLactoferrin). In some aspects, the recombinant human lactoferrin of the present disclosure is obtained from mammalian, fungal, yeast, bacterial or other cells. In some aspects, the recombinant human lactoferrin of the present disclosure is not obtained from mammalian cells. In certain aspects, the recombinant human lactoferrin of the present disclosure is obtained from fungal cells. The fungal cell can be, for example, a cell of the genera Arcthura, Aspergillus, Aurantiochytrium, Candida, Cryptococcus, Csudaema, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidia, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, or Yarrowia. In one aspect, the fungal cell is a yeast cell. In some aspects, the yeast cell is a Komagataella cell (Komagataella puffii, Komagataella pastoris, Komagataella pseudopastoris). Additional cells suitable for recombinant protein production are recognized in the art and are contemplated herein. In some aspects, the recombinant human lactoferrin of the present disclosure is obtained from a bacterial cell. In other aspects, the human lactoferrin of the present disclosure is isolated from a natural source.

[0081]

[0070] Certain aspects of the present disclosure are directed to human lactoferrin with at least one hybrid or complex N-glycan.In some aspects, human lactoferrin comprises glycan that comprises one or more of sialic acid, galactose, N-acetylgalactosamine, or fucose.In some aspects, human lactoferrin comprises biantennary, triantennary, or tetraantennary N-glycan.As disclosed herein, human lactoferrin with one or more hybrid, complex, biantennary, triantennary, or tetraantennary N-glycan can be useful, for example, in infant formula or other nutritional compositions or supplements.

[0082] B. Alpha-lactalbumin (α-lactalbumin) Aspects of the present disclosure are directed to alpha-lactalbumin and compositions comprising alpha-lactalbumin, including infant formula compositions. In one aspect, cells are disclosed that express human alpha-lactalbumin linked to a signal peptide of the present disclosure (e.g., comprising SEQ ID NO:1, 2, 3 or 4). Alpha-lactalbumin (also referred to as "α-lactalbumin") is a whey protein found in breast milk and is encoded by the LALBA gene. Certain aspects of the present disclosure are directed to human α-lactalbumin (UniProtKB / Swiss-Prot Accession No. P00709), including its isoforms. The full sequence of human α-lactalbumin, including the signal peptide, is provided as SEQ ID NO:36. The sequence of mature human α-lactalbumin after cleavage of the signal peptide is provided as SEQ ID NO:35.

[0083] [Table 3] In some aspects, the human alpha-lactalbumin of the present disclosure is recombinant human alpha-lactalbumin. In some aspects, the recombinant human alpha-lactalbumin of the present disclosure is obtained from mammalian, fungal, yeast, bacterial, or other cells. In some aspects, the recombinant human alpha-lactalbumin of the present disclosure is not obtained from mammalian cells. In a particular aspect, the recombinant human alpha-lactalbumin of the present disclosure is obtained from yeast cells. The yeast cell can be, for example, a cell of the genus Arcthura, Aspergillus, Aurantiochytrium, Candida, Cryptococcus, Csudaema, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidia, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, or Yarrowia. In one aspect, the yeast cell is a Komagataella cell (e.g., Komagataella paphii, Komagataella pastoris, Komagataella pseudopastoris). Additional yeast cells suitable for recombinant protein production are recognized in the art. and are contemplated herein. In one aspect, the recombinant human alpha-lactalbumin of the disclosure is obtained from a bacterial cell. In another aspect, the human alpha-lactalbumin of the disclosure is isolated from a natural source.

[0084]

[0073] Certain aspects of the present disclosure are directed to human α-lactalbumin having at least one hybrid or complex N-glycan.In some aspects, human α-lactalbumin comprises glycan comprising one or more of sialic acid, galactose, N-acetylgalactosamine or fucose.In some aspects, human lactoferrin comprises biantennary, triantennary or tetraantennary N-glycan.As disclosed herein, human α-lactalbumin having one or more hybrid, complex, biantennary, triantennary or tetraantennary N-glycan can be useful, for example, in infant formula or other nutritional compositions or supplements.

[0085] c. Additional human milk proteins

[0074] Additional human milk proteins contemplated in the compositions (e.g., infant formula compositions) and methods of the present disclosure include, but are not limited to, secretory IgA (sIgA), human serum albumin, xanthine dehydrogenase, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, osteopontin, bile salt-stimulated lipase (BSSL) and lysozyme. Any one or more of these human milk proteins may be included in the compositions (e.g., infant formulas) of the present disclosure. Any one or more of these human milk proteins may be excluded in certain embodiments.

[0086] CN-acetylglucosaminyltransferase Aspects of the present disclosure relate to N-acetylglucosaminyltransferase proteins. As used herein, "N-acetylglucosaminyltransferase protein" describes any polypeptide having N-acetylglucosaminyltransferase activity. N-acetylglucosaminyltransferase describes an enzyme that catalyzes the transfer of a monosaccharide from a specific sugar nucleotide donor to a specific hydroxyl position of the monosaccharide in a growing glycan chain in one of two possible anomeric bonds (either α or β).

[0087]

[0076] The N-acetylglucosaminyltransferase protein can be from any suitable organism. In one aspect, the N-acetylglucosaminyltransferase protein is a eukaryotic N-acetylglucosaminyltransferase protein. In one aspect, the N-acetylglucosaminyltransferase protein is a mammalian N-acetylglucosaminyltransferase protein.

[0088] 1. N-acetylglucosaminyltransferase I In some embodiments, the N-acetylglucosaminyltransferase protein is N-acetylglucosaminyltransferase I protein (EC 2.4.1.101). The systematic name of this enzyme class is alpha-1,3-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase. Other names include: GnT-I, N-acetylglucosaminyltransferase I, and uridine diphosphoacetylglucosamine-alpha-1,3-mannosylglycoprotein beta-1,2-N-acetylglucosaminyltransferase. In certain embodiments, the N-acetylglucosaminyltransferase I protein of the present disclosure is homo sapiens GnT-I, but any eukaryotic N-acetylglucosaminyltransferase I protein can be used as part of the methods and compositions of the present disclosure.

[0089] 2. β-1,2-N-acetylglucosaminyltransferase In some embodiments, the N-acetylglucosaminyltransferase protein is a β-1,2-N-acetylglucosaminyltransferase protein (EC 2.4.1.143). The systematic name for this enzyme class is alpha-1,6-mannosyl-glycoprotein 2-beta-N-acetylglucosaminyltransferase. Other names include: GnT-II, N-acetylglucosaminyltransferase II, and uridine diphosphoacetylglucosamine-alpha-1,6-mannosylglycoprotein beta-1-2-N-acetylglucosaminyltransferase. In certain embodiments, the β-1,2-N-acetylglucosaminyltransferase protein of the present disclosure is Rattus norvegicus GnT-II, although β-1,2-N-acetylglucosaminyltransferase proteins from any eukaryotic organism may be used as part of the methods and compositions of the present disclosure.

[0090] D. Alpha-1,3 / 6-mannosidase (α-1,3 / 6-mannosidase) Aspects of the present disclosure relate to α-1,3 / 6-mannosidase proteins (EC 3.2.114). As used herein, "α-1,3 / 6-mannosidase protein" (or "alpha-1,3 / 6-mannosidase protein") describes any polypeptide having α-1,3 / 6-mannosidase activity. Alpha-1,3 / 6-mannosidase describes an enzyme that catalyzes the removal of two mannosyl residues from an N-glycan. The systematic name for this enzyme class is mannosyl oligosaccharide 1,3-1,6-alpha-mannosidase. Other names include: Man-II and mannosidase II. Alpha-1,3 / 6-mannosidase proteins can be from any suitable organism. In some embodiments, the α-1,3 / 6-mannosidase protein is a eukaryotic α-1,3 / 6-mannosidase protein. In certain embodiments, the α-1,3 / 6-mannosidase protein is Drosophila Man-II, although any eukaryotic α-1,3 / 6-mannosidase protein can be used as part of the methods and compositions of the present disclosure.

[0091] E. Alpha-1,2-mannosidase (α-1,2-mannosidase) Aspects of the present disclosure relate to α-1,2-mannosidase proteins (EC 3.2.1.130). As used herein, "α-1,2-mannosidase protein" (or "alpha-1,2-mannosidase protein") describes any polypeptide having α-1,2-mannosidase activity. The systematic name for this enzyme class is glycoprotein endo-alpha-1,2-mannosidase. Other names include: endo-alpha-D-mannosidase and Man-I. In some embodiments, the α-1,2-mannosidase protein is fungal Man-I. In certain embodiments, the Man-I is Trichoderma reesei Man-I.

[0092] F. beta-1,4-galactosyltransferase (β-1,4-galactosyltransferase) Aspects of the present disclosure relate to β-1,4-galactosyltransferase proteins (EC 2.4.1.38). As used herein, "β-1,4-galactosyltransferase protein" (or "beta-1,4-galactosyltransferase protein") describes any polypeptide having β-1,4-galactosyltransferase activity. The systematic name for this enzyme class is beta-N-acetylglucosaminylglycopeptide beta-1,4-galactosyltransferase. Other names include: glycoprotein 4-beta-galactosyltransferase, UDP-galactose-glycoprotein galactosyltransferase, and GalT. In some embodiments, the β-1,4-galactosyltransferase protein is a mammalian GalT. In certain embodiments, the GalT is a Homo sapiens GalT.

[0093] G. Glycosylated Proteins Aspects of the present disclosure are directed to methods and compositions for producing glycosylated proteins (also referred to as "glycoproteins") that have glycosylation patterns similar to those of glycoproteins produced by human cells. In certain embodiments, the glycoproteins of the present disclosure are N-linked glycoproteins. N-linked glycoproteins contain N-acetylglucosamine residues attached to the amide nitrogen of an asparagine residue in the protein. The predominant sugars found on glycoproteins are glucose, galactose, mannose, fucose, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc) and sialic acids, such as N-acetylneuraminic acid (NANA). Processing of sugar groups occurs cotranslationally in the lumen of the ER and continues in the Golgi apparatus for N-linked glycoproteins.

[0094] H. Protein Targeting

[0083] Certain aspects of the disclosure include cells expressing one or more proteins from a nucleic acid molecule, where the protein is targeted to a desired subcellular location (e.g., an organelle such as the Golgi apparatus). In some cases, the protein is targeted to a subcellular location by forming a fusion protein that includes a portion of the protein (e.g., the catalytic domain of an enzyme) and a cellular targeting signal peptide, such as a heterologous signal peptide that is not normally ligated to or associated with a portion of the protein (e.g., a signal peptide comprising SEQ ID NO:1, 2, 3, or 4). A fusion protein can be encoded by ligating a polynucleotide encoding a cellular targeting signal peptide in the same translation reading frame ("in frame") to a nucleic acid fragment encoding a protein (e.g., an enzyme) or a catalytically active fragment thereof.

[0095] The targeting signal peptide component of the fusion construct or protein can be derived from ER or Golgi membrane-bound proteins, retrieval signals, type II membrane proteins, type I membrane proteins, membrane-spanning nucleotide sugar transporters, mannosidases, sialyltransferases, glucosidases, mannosyltransferases and phosphomannosyltransferases. In some aspects, the targeting signal peptide is a Golgi localization tag. Examples of Golgi localization tags include, but are not limited to, transmembrane domains from Saccharomyces cerevisiae Kre2p, Saccharomyces cerevisiae Mnn2p, Saccharomyces cerevisiae Mnn9, Komagataella puffii Bmt2, Komagataella puffii Bmt3 or Komagataella puffii Ktr2.

[0096] III. Sequence

[0085] Specific examples of polypeptide and nucleic acid sequences contemplated herein are shown in Table 4 below.

[0097] [Table 4-1]

[0098] [Table 4-2]

[0099] [Table 4-3]

[0100] [Table 4-4]

[0101] [Table 4-5]

[0102] [Table 4-6]

[0103] [Table 4-7]

[0104] [Table 4-8] IV. Genetic Engineering

[0086] Vectors for transforming microorganisms (e.g., fungal cells, yeast cells) according to the present disclosure can be prepared by known techniques well known to those skilled in the art in view of the disclosure herein. A vector typically contains one or more genes, where each gene codes for the expression of a desired product (gene product) and is operably linked to one or more control sequences that regulate gene expression or target the gene product to a specific location in the recombinant cell.

[0105]

[0087] Exogenous nucleic acid sequences, including, for example, nucleic acid sequences encoding fusion proteins, nucleic acid sequences encoding wild-type or mutant proteins, can be introduced into many different host cells. The nucleic acid sequence configured to promote genetic mutation in genes can also be introduced into various host cells, as described further herein.Suitable host cells are microbial hosts that can be found widely within the fungal family.Examples of suitable host strains include, but are not limited to, fungal or yeast species, such as Arcthura, Aspergillus, Aurantiochytrium, Candida, Bacillus subtilis, Cryptococcus, Csudaema, Hansenula, Kluyveromyces, Komagataella, Leucosporidia, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon and Yarrowia.In some embodiments, the host cell of the present disclosure is a Komagataella cell. In some embodiments, the host cell of the present disclosure is Komagataella puffii. In some embodiments, the host cell of the present disclosure is Komagataella pastoris. In some embodiments, the host cell of the present disclosure is Komagataella pseudopastoris.

[0106] Microbial expression systems and expression vectors are well known to those skilled in the art. Any such expression vector can be used to introduce instant genes and nucleic acid sequences into organisms. The nucleic acid sequence can be introduced into a suitable microorganism by transformation techniques. For example, the nucleic acid sequence can be cloned into a suitable plasmid, and the parent cell can be transformed with the resulting plasmid. The plasmid is not particularly limited, as long as it allows the desired nucleic acid sequence to be inherited by the progeny of the microorganism.

[0107]

[0089] The vector or cassette useful for transformation of suitable host cells is recognized in the art.Typically, vector or cassette contains gene, sequence that directs transcription and translation of related gene including promoter, selectable marker and sequence that allows autonomous replication or chromosomal integration.Suitable vector contains 5' region of gene with promoter and other transcription initiation control and 3' region of DNA fragment that controls transcription termination.

[0108]

[0090] The promoter, cDNA and 3'UTR, as well as other elements of the vector, can be generated by cloning techniques using fragments isolated from natural sources (Green & Sambrook, Molecular Cloning: A Laboratory Manual, (4th ed., 2012); US Pat. No. 6,333,621; and US Pat. No. 6,333,621). (U.S. Pat. No. 4,683,202; incorporated by reference). Alternatively, the elements can be produced synthetically using known methods (Gene 164:49-53 (1995)).

[0109] A. Vectors and Vector Components

[0091] Vectors for transforming microorganisms (e.g., yeast cells) according to the present disclosure can be prepared by known techniques well known to those skilled in the art in view of the disclosure herein. A vector typically contains one or more genes, where each gene codes for the expression of a desired product (gene product) and is operably linked to one or more control sequences (e.g., promoter sequences, signal peptide sequences) that regulate gene expression or target the gene product to a specific location in the recombinant cell.

[0110] 1. Control Arrays

[0092] A control sequence is a nucleic acid sequence that regulates the expression of a coding sequence or directs a gene product to a specific location inside or outside a cell. Control sequences that regulate expression include, for example, promoters that control the transcription of a coding sequence and terminators that terminate the transcription of a coding sequence. Another control sequence is the 3' untranslated sequence located at the end of a coding sequence that codes for a polyadenylation signal. Control sequences that direct a gene product to a specific location include those that code for signal peptides, which direct the proteins they are bound to to a specific location inside or outside a cell.

[0111] Thus, an example of vector design for expressing genes in microorganisms contains a coding sequence for a desired gene product (e.g., a selectable marker, an enzyme, a fusion protein, etc.) operably linked to a promoter active in yeast. Alternatively, if the vector does not contain a promoter operably linked to the coding sequence of interest, the coding sequence can be transformed into the cell so that it is operably linked to an endogenous promoter at the time of vector integration. Examples of promoters contemplated herein include, but are not limited to, AOX1, GAP, TEF1, TPI1, DAS1, DAS2, CAT1 and FMD promoters.

[0112]

[0094] The promoter used to express a gene can be the promoter naturally linked to that gene or a different promoter.

[0113]

[0095] Promoters are generally characterized as constitutive or inducible. Constitutive promoters are generally active or function to drive expression at the same level at all times (or at certain times in the cell's life cycle). Inducible promoters, on the other hand, are active (or inactivated) or are significantly up-regulated or down-regulated only in response to a stimulus. Both types of promoters are applicable to the disclosed methods. Useful inducible promoters include those that mediate transcription of an operably linked gene in response to a stimulus such as an exogenously provided small molecule, temperature (heat or cold), or nitrogen deficiency in the culture medium. A suitable promoter can activate transcription of an essentially inactive gene or up-regulate transcription of an operably linked gene that is transcribed at a low level.

[0114]

[0096] The insertion of a termination region control sequence is optional. The termination region may be native to the transcription initiation region (promoter), may be native to the DNA sequence of interest, or may be available from another source (see, for example, Chen & Orozco, Nucleic Acids Research 16:8411 (1988)).

[0115] In some cases, the entire nucleotide sequence of the promoter is not necessary to drive transcription, and a sequence shorter than the entire nucleotide sequence of the promoter can drive the transcription of an operably linked gene. The smallest part of the promoter is called the core promoter and includes the transcription initiation site, the binding site for RNA polymerase, and the binding site for transcription factors.

[0116] The promoter can be linked to the target by introducing the promoter and the target into a nucleic acid molecule, for example, a vector.The vector is introduced into a cell, thereby expressing the promoter and the target.In one embodiment, the promoter is linked to the target by, for example, introducing the promoter into the DNA of the cell via homologous recombination, thereby integrating the promoter into the genome of the cell.

[0117] B. Gene and Codon Optimization Typically, a gene comprises a promoter, a coding sequence and a termination control sequence. When assembled by recombinant DNA technology, the gene can be called an expression cassette, and can be flanked by restriction sites for convenient insertion into a vector used to introduce the recombinant gene into a host cell. The expression cassette can be flanked by DNA sequences from a genome or other nucleic acid target to facilitate stable integration of the expression cassette into the genome by homologous recombination. Alternatively, the vector and its expression cassette can remain unintegrated (e.g., episome), in which case the vector typically comprises an origin of replication that can provide for replication of vector DNA.

[0118]

[0100] A typical gene present on a vector is a gene that codes for a protein. A gene that expresses a selectable marker is a gene whose expression allows recombinant cells containing the protein to be differentiated from cells that do not express the protein. Such a gene and its corresponding gene product are called selectable markers or selection markers. Any of a wide variety of selectable markers can be utilized in transgene constructs useful for transforming organisms covered in the disclosed embodiments.

[0119] For optimal expression of a recombinant protein, the expression level should be determined according to the host cell to be transformed. It may be beneficial to utilize a coding sequence that produces an mRNA with optimal codon usage. Thus, proper expression of a transgene may require that the codon usage of the transgene matches the specific codon bias of the organism in which the transgene is expressed. The exact mechanisms underlying this effect are many, but include a proper balance between the available aminoacylated tRNA pool and the proteins synthesized in the cell, coupled with more efficient translation of the transgenic messenger RNA (mRNA) when this need is met. If the codon usage in the transgene is not optimized, the available tRNA pool may not be sufficient to allow efficient translation of the transgenic mRNA, resulting in ribosome stalling and termination, as well as possible instability of the transgenic mRNA.

[0120]

[0102] The coding sequences of the present disclosure may be modified to convert one or more rare codons to more frequent codons in a host cell. The codons can be optimized for a particular host cell by replacing one or more codons found in the host cell with those found in the host cell. A rare codon in a host cell describes a codon found in less than 5%, less than 10%, or less than 20% of the coding sequences in the host cell. A rare codon can be identified using methods known to those skilled in the art.

[0121] Aspects of the present disclosure include methods for producing microorganisms using nucleic acid sequences that include genes encoding proteins. This includes transformation of organisms. The gene can be native to the cell or from a different species. The gene can be from a different species but modified (e.g., codon-optimized) for optimal expression in the microorganism. In certain embodiments, the gene is heritable to the progeny of the transformed cell. In some embodiments, the gene is heritable because it is present on a plasmid. In certain embodiments, the gene is heritable because it is integrated into the genome of the transformed cell.

[0122] A further aspect of the present disclosure is a method for generating mutations in a gene of a microorganism. It may include transformation of a microorganism with a constructed nucleic acid sequence. For example, aspects of the present disclosure may include transformation of a microorganism with a nucleic acid sequence that includes sequences upstream and downstream of a gene (e.g., the OCH1 gene), thereby facilitating the reduction or deletion of expression of the gene via homologous recombination. A variety of methods for generating mutations in a gene of a microorganism (including deletion or knockout mutations, as well as mutations that reduce expression of a gene) are recognized in the art and are contemplated herein. A microorganism with a deletion or knockout mutation of a gene does not produce a functional copy of the protein. For example, a recombinant yeast cell of the present disclosure may include a deletion of the endogenous OCH1 gene, such that the recombinant yeast cell does not express the endogenous functional OCH1 protein. A microorganism with reduced expression of a gene or protein produces a functional copy of the protein, but in a reduced amount compared to a wild-type (i.e., non-recombinant or non-genetically modified) microorganism of the same species. Methods for reducing expression of a protein are recognized in the art and include, for example, replacement of the endogenous promoter and / or modification of one or more regulatory elements.

[0123] C. Transformation

[0105] Cells may be transformed by any suitable technique, including, for example, gene gun, electroporation, glass bead transformation, and silicon carbide whisker transformation. Any convenient technique for introducing transgenes into microorganisms can be employed in the embodiments disclosed herein.

[0124]

[0106] Vectors for transformation of microorganisms can be prepared by known techniques well known to those skilled in the art. It can be made. In one embodiment, an exemplary vector design for the expression of a gene in a microorganism contains a gene encoding an enzyme operably linked to a promoter active in the microorganism. Alternatively, if the vector does not contain a promoter operably linked to the gene of interest, the gene can be transformed into the cell so that it is operably linked to the native promoter at the time of vector integration. The vector can also contain a second gene encoding a protein. Optionally, one or both gene(s) are followed by a 3' untranslated sequence containing a polyadenylation signal. The expression cassettes encoding the two genes can be physically linked in the vector or on separate vectors. Co-transformation of microorganisms can also be used, where separate vector molecules are used simultaneously to transform the cell (Protist 155:381-93 (2004)). Transformed cells can optionally be selected based on their ability to grow in the presence of an antibiotic or other selectable marker under conditions in which cells lacking the resistance cassette will not grow.

[0125] D. Genetically engineered cells Aspects of the present disclosure include genetically engineered cells ("engineered cells" or "recombinant The present invention includes recombinant cells (also referred to as "cells") containing one or more exogenous nucleic acid sequences, as well as methods for making and using such cells. In certain embodiments, recombinant cells containing one or more exogenous nucleic acid sequences are disclosed. Methods for generating such recombinant cells comprising introducing one or more exogenous nucleic acid sequences into a host cell are also disclosed. Further described are methods for harvesting one or more products (e.g., mammalian proteins) from such recombinant cells comprising culturing the cells and harvesting the products.

[0126] In some embodiments, the recombinant cell is a prokaryotic cell, such as a bacterial cell. In some embodiments, the recombinant cell is a eukaryotic cell, such as a mammalian cell, a yeast cell, a filamentous fungal cell, a protist cell, an algae cell, an avian cell, a plant cell, or an insect cell. In some embodiments, the cell is a yeast cell. Those skilled in the art will recognize that many forms of filamentous fungi provide yeast-like growth, and the definition of yeast herein encompasses such cells. The recombinant cell of the present disclosure may be selected from the group consisting of algae, bacteria, mold, fungi, plants, and yeast. In some embodiments, the recombinant cell of the present disclosure is a bacterial cell (e.g., E. coli), a fungal cell, or a yeast cell.

[0127] In some embodiments, the recombinant cell of the present disclosure is a recombinant fungal cell. The fungal cell may be any suitable fungal cell recognized in the art. In one aspect, the fungal cell is a cell of the genus Arcthura, Aspergillus, Aurantiochytrium, Candida, Cryptococcus, Csudaema, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidia, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, or Yarrowia. In certain embodiments, the fungal cell is selected from the group consisting of Arxula adeninivorans, Aspergillus niger, Aspergillus orzyae, Aspergillus terreus, Aurantiochytrium limacinum, Candida utilis, Cladophora cerevisiae, and the like. viceps purpurea, Cryptococcus albidus, Cryptococcus curvatus, Cryptococcus ramirezgomezianus, Cryptococcus terreus, Cryptococcus wieringae, Cunninghamella echinulata, Cunninghamella japonica, Geotrichum fermentans, Hansenula polymorpha, Kluyveromyces lactis lactis, Komagataella puffii, Komagataella pastoris, Komagataella pseudopastoris, Kluyveromyces marxianus, Kodamaea ohmeri, Leucosporidiella creatinivora, Lipomyces lipofer, Lipomyces starkeyi, Lipomyces tetrasporus, Mortierella isabellina, Mortierella alpina, Ogataea polymorpha, Pichia ciferi ciferrii, Pichia guilliermondii, Pichia pastoris, Pichia stipites, Prototheca zopfii, Rhizopus arrhizus, Rhodosporidium babjebaebabjevae, Rhodosporidium toruloides, Rhodosporidium paludigenum, Rhodotorula glutinis, Rhodotorula mucilaginosa, Saccharomyces cerevisiae, Schizosaccharomyces cerevisiae, Tremella enchepala, Trichosporon cutaneum, Trichosporon fermentans, Wickerhamomyces ciferrii or Yarrowia lipolytica.

[0128] In one aspect, the fungal cell is a yeast cell. The yeast cell is a Komagataella cell. In some embodiments, the yeast cell is Kluyveromyces puffii, Komagataella pastoris, or Komagataella pseudopastoris. In certain embodiments, the yeast cell is Kluyveromyces puffii.

[0129] In some embodiments, the engineered cells of the present disclosure comprise N-glycans that contain human-like N-glycans. - A yeast cell comprising one or more modifications for improving the production of glycans. Examples of such cells and modifications are described, for example, in U.S. Patent No. 9,617,550, which is incorporated herein by reference in its entirety.

[0130] E. Gene Editing Systems

[0112] Certain aspects of the present disclosure relate to knockout or other induction of genes in a population of cells. The present invention is directed to the use of gene editing techniques to generate mutations in a gene. Various methods and systems for gene editing are known in the art, such as zinc finger nuclease (ZFN)-based gene editing, transcription activator-like effector nuclease (TFA)-based gene editing, transcription activator-like effector nuclease (TFA)-based gene editing, and gene editing systems using gene editing techniques. (TALEN)-based gene editing, and CRISPR / Cas-based gene editing.Various methods and systems for gene editing are recognized in the art and are contemplated herein.In some embodiments, the method of the present disclosure includes CRISPR / Cas-based gene editing, which includes the use of CRISPR system components such as guide RNA (gRNA) and Cas nuclease.In some embodiments, the method of the present disclosure does not include CRISPR / Cas-based gene editing (including, for example, ZFN-based, TALEN-based, or any other gene editing method or system).

[0131] Generally, a "CRISPR system" includes sequences encoding Cas genes. , transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes, tracr (trans-activating CRISPR) sequences (e.g., The term "tracrRNA" refers collectively to the tracrRNA or active partial tracrRNA), tracr-mate sequences (including "direct repeats" and partial direct repeats in the context of an endogenous CRISPR system processed by tracrRNA), guide sequences (also referred to as "spacers" in the context of an endogenous CRISPR system), and / or other sequences and transcripts derived from the CRISPR locus.

[0132] CRISPR / Cas nuclease or CRISPR / Cas nuclease The system uses a non-coding RNA molecule (guide RNA) that binds to DNA in a sequence-specific manner, and a nucleic acid The CRISPR system may include a Cas protein (e.g., Cas9) having nuclease functionality (e.g., two nuclease domains). One or more elements of the CRISPR system may be derived from a type I, type II, or type III CRISPR system, and may be derived from a particular organism that contains an endogenous CRISPR system, such as, for example, Streptococcus pyogenes.

[0133] In one aspect, Cas nuclease and gRNA (comprising a fusion of target sequence-specific crRNA and fixed tracrRNA) are introduced into cells. Cas nuclease and gRNA can be introduced into cells indirectly through the introduction of one or more nucleic acids (e.g., vectors) encoding Cas nuclease and / or gRNA. Cas nuclease and gRNA can be introduced into cells directly by the introduction of Cas nuclease protein and gRNA molecule. In general, the target site at the 5' end of gRNA targets Cas nuclease to the target site, e.g., gene, using complementary base pairing. Target site can be selected based on its immediate 5' position of protospacer adjacent motif (PAM) sequence, e.g., typically NGG or NAG. In this regard, gRNA can be targeted to a desired sequence by modifying the first 20, 19, 18, 17, 16, 15, 14, 14, 12, 11, or 10 nucleotides of the guide RNA to correspond to the target DNA sequence. In general, CRISPR systems are characterized by elements that promote the formation of CRISPR complexes at the site of the target sequence. Typically, "target sequence" generally refers to the sequence that the guide sequence is designed to have complementarity with, where hybridization between the target sequence and the guide sequence promotes the formation of CRISPR complexes. Full complementarity is not necessary, provided there is sufficient complementarity to cause hybridization and promote the formation of CRISPR complexes.

[0134]

[0116] The CRISPR system can induce double-strand breaks (DSBs) at target sites; The break can then be induced as discussed herein. In other embodiments, a Cas9 variant considered a "nickase" is used to nick the single strand at the target site. Paired nickases can be used to improve the specificity directed by a pair of distinct gRNA targeting sequences, for example, such that a 5' overhang is introduced simultaneously upon introduction of a nick. In other embodiments, catalytically inactive Cas9 can be used to affect gene expression by using a transcriptional repressor or activator, such as a transcriptional repressor or activator. fused to a heterologous effector domain.

[0135]

[0117] The target sequence may be any polynucleotide, such as a DNA or RNA polynucleotide. The target sequence may be located in the nucleus or cytoplasm of a cell, such as in an organelle of a cell. In general, a sequence or template that can be used for recombination into a target locus that contains a target sequence is referred to as an "editing template" or "editing polynucleotide" or "editing sequence." In some aspects, an exogenous template polynucleotide may be referred to as an editing template. In some aspects, the recombination is homologous recombination.

[0136] Typically, in the context of an endogenous CRISPR system, a CRISPR complex (target Formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near the target sequence (e.g., within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from the target sequence). A tracr sequence that may comprise or consist of all or a portion of a wild-type tracr sequence (e.g., about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of the wild-type tracr sequence) may also form part of a CRISPR complex, for example, by hybridization to all or a portion of a tracr mate sequence operably linked to the guide sequence along at least a portion of the tracr sequence. The tracr sequence has sufficient complementarity to the tracr mate sequence to hybridize and participate in the formation of a CRISPR complex, such as at least 50%, 60%, 70%, 80%, 90%, 95% or 99% sequence complementarity along the length of the tracr mate sequence when optimally aligned.

[0137]

[0119] The one or more vectors driving expression of one or more elements of the CRISPR system can be The expression of the elements of the RISPR system can be introduced into a cell to direct the formation of a CRISPR complex at one or more target sites. The components can also be delivered to the cell as proteins and / or RNA. For example, the Cas enzyme, the guide sequence linked to the tracr-mate sequence, and the tracr sequence can each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements can be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system that are not included in the first vector. The vector can include one or more insertion sites, such as restriction endonuclease recognition sequences (also called "cloning sites"). In some embodiments, the one or more insertion sites are located upstream and / or downstream of one or more sequence elements of one or more vectors. When multiple different guide sequences are used, a single expression construct can be used to target CRISPR activity to multiple different corresponding target sequences in a cell.

[0138]

[0120] The vector encodes a Cas protein (also called a "Cas nuclease"). Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12a (Cpf1), Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, The enzymes include Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csfl, Csf2, Csf3, Csf4, their homologs, or modified versions thereof. These enzymes are known; for example, the amino acid sequence of the Streptococcus pyogenes Cas9 protein can be found in the SwissProt database under the accession number Q99ZW2.

[0139]

[0121] Cas nucleases include Cas9 (e.g., Streptococcus pyogenes or Streptococcus pneumoniae ( The Cas nuclease can be derived from S. pneumoniae). The Cas nuclease can be Cas12a. The Cas nuclease can direct the cleavage of one or both strands at the location of a target sequence, such as within the target sequence and / or within the complementary sequence of the target sequence. The vector can encode a Cas nuclease that is mutated with respect to the corresponding wild-type enzyme, such that the mutated Cas nuclease lacks the ability to cleave one or both strands of a target polynucleotide that contains the target sequence. In some embodiments, the Cas9 nickase can be used in combination with a guide sequence(s), for example, two guide sequences that target the sense and antisense strands, respectively, of a DNA target. This combination allows both strands to be nicked and used to induce NHEJ or HDR.

[0140] In some embodiments, the enzyme coding sequence encoding the CRISPR enzyme is The gene is codon-optimized for expression in a cell, such as a yeast cell.

[0141] Generally, a guide sequence hybridizes to a target sequence and binds to the CRISPR complex. Any polynucleotide sequence that has sufficient complementarity with a target polynucleotide sequence to direct sequence-specific binding to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence is 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more, when optimally aligned using a suitable alignment algorithm.

[0142]

[0124] Optimal alignment can be performed using any suitable algorithm for aligning sequences. The alignment can be determined using any of a number of algorithms, non-limiting examples of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g., the Burrows Wheeler Aligner), Clustal W, Clustal X, BLAST, Novoalign (Novocraft Technologies, ELAND (Illumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).

[0143]

[0125] Cas nucleases are fusion proteins that contain one or more heterologous protein domains. Cas nuclease fusion proteins can be part of a protein. Cas nuclease fusion proteins can include any additional protein sequences and optionally linker sequences between any two domains. Examples of protein domains that can be fused to Cas nuclease include, but are not limited to, epitope tags, reporter gene sequences, and protein domains with one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity, and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tag, V5 tag, FLAG tag, influenza hemagglutinin (HA) tag, Myc tag, VSV-G tag, and thioredoxin (Trx) tag. Examples of reporter genes include, but are not limited to, glutathione-5 transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, autofluorescent proteins including green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP). Cas nucleases bind to DNA molecules or are useful for binding to maltose binding protein (MBP), S-tag, Lex A, and other proteins. Genetic sequences encoding proteins or protein fragments that bind to other cellular molecules, including, but not limited to, DNA binding domain (DBD) fusions, GAL4A DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of fusion proteins containing Cas nucleases are described in US 20110059502, which is incorporated herein by reference. EXAMPLES

[0144]

[0126] The following examples are included to demonstrate certain aspects disclosed herein. It should be understood by those skilled in the art that the techniques disclosed in the following examples represent techniques discovered by the inventors to function well in the practice of the disclosed embodiments, and therefore can be considered to constitute specific modes for the practice thereof. However, those skilled in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments disclosed and still obtain a like or similar result without departing from the spirit and scope of the embodiments disclosed herein.

[0145] Example 1 - Novel signal peptides increase extracellular protein levels To determine the effect of novel signal peptides on extracellular protein levels DNA encoding SEQ ID NO:1 (“SP1”), SEQ ID NO:2 (“SP2”) and SEQ ID NO:4 (“SP4”) were cloned in frame at the 5′ end of DNA encoding the protein of interest (POI), i.e., Pichia pastoris codon-optimized human lactoferrin, resulting in the replacement of pre-pro-MFα from Saccharomyces cerevisiae, which is the most widely used signal peptide in yeast and serves as a control. Single copies of the resulting sequences and controls were integrated into the AOX1 locus via double crossover. Multiple colonies from each transformation plate were grown in 96-deep well plates.

[0146] To establish the presence of the protein of interest, a Western blot was performed on the supernatant. As shown in Figure 1, when a single copy of human lactoferrin is integrated and secretion is driven by the widely used preproMFα from Saccharomyces cerevisiae, no protein is detected in the supernatant. Conversely, when secretion is driven by SEQ ID NO:1 ("SP1"), SEQ ID NO:2 ("SP2"), and SEQ ID NO:3 ("SP3"), extracellular protein is detected.

[0147] To assess the magnitude of the improvement in secretion, the extracellular protein was analyzed by ELISA. Quantification was performed. As can be seen in FIG. 2, the novel engineered signals were identified as SEQ ID NO:1 (“SP1”), SEQ ID NO:2 (“SP2”), and SEQ For ID NO:3 ("SP3"), extracellular protein levels were enhanced 2.38-fold, 2.41-fold, and 2.20-fold over the control (preproMFα).

[0148] Materials and Methods Vector and strain construction. Oligonucleotides and gBlocks were The materials were ordered from Integrated DNA Technologies (San Diego, CA, USA) and are listed in Table 5. NEBuilder® HiFi DNA Assembly Master Mix, OneTaq® Quickload® DNA polymerase, and E. coli DH5α cells were from New England Biolabs. All polymerase chain reaction (PCR) amplified sequences were confirmed by sequencing at Genewiz.

[0149] [Table 5] Transformation of linear dsDNA for integration was performed using the method described by Madden, Tolstorukov, & Cregg (2014) Fungi, Volume 1, Fungal Biology. Total yeast genomic DNA extraction was performed using the kit Easy DNA from Invitrogen ((ThermoFisher, Applied Biosystems™, PrepSEQ™ 1-2-3 Nucleic Acid Extraction Kit, Catalog number: 4452222)). The resulting plasmids are summarized in Table 6.

[0150] [Table 6] Leaders from Pichia pastoris endogenous proteins Ost1 and Pst1 Peptide sequences were determined using publicly available SignalP-5.0 bioinformatics software from the Center for Biological Sequence Analysis (CBS). The pro-region of Epx1 was described by Heiss et al. (2015) Microbiology, 161(7).

[0151] Plasmid P1 containing a gene encoding human lactoferrin lacking its native secretory peptide fused in frame with the pre-pro leader peptide of mating factor alpha from Saccharomyces cerevisiae was synthesized by Genscript. The human lactoferrin gene was codon optimized for expression in Pichia pastoris.

[0152] Plasmid P2 containing the signal sequence SP1 (SEQ ID NO:1) was To generate this, primers PMR1 (SEQ ID NO:16) and PMR2 (SEQ ID NO:17) were used to amplify the Ost1 leader sequence using gBLOCK1 as a template. A backbone containing human lactoferrin, yeast HIS4 auxotrophic marker, and E. coli antibiotic resistance and replication origin was obtained by polymerase chain reaction (PCR) of the P1 plasmid using primers PMR3 (SEQ ID NO:18) and PMR4 (SEQ ID NO:19). The two resulting fragments were assembled using NEBuilder® HiFi DNA Assembly Master Mix according to the manufacturer's instructions.

[0153] Plasmid P3 containing the signal sequence SP2 (SEQ ID NO:2) was To generate this, gBLOCK1 (SEQ ID NO:15) was used as template with primers PMR5 (SEQ ID NO:20) and PMR6 (SEQ ID NO:21) used for amplification. A backbone containing human lactoferrin, yeast HIS4 auxotrophic marker, and E. coli antibiotic resistance and replication origin was obtained by PCR of the P1 plasmid with primers PMR7 (SEQ ID NO:22) and PMR8 (SEQ ID NO:23). The two resulting fragments were assembled using NEBuilder® HiFi DNA Assembly Master Mix according to the manufacturer's instructions.

[0154] Plasmid P4 containing the signal sequence SP3 (SEQ ID NO:3) was To generate the cDNA, primers PMR9 (SEQ ID NO:24) and PMR10 (SEQ ID NO:25) were used for amplification using gBLOCK1 as a template. The backbone containing the resistance and replication origin was obtained by PCR of the P1 plasmid with primers PMR11 (SEQ ID NO:26) and PMR12 (SEQ ID NO:27). The two resulting fragments were amplified using the NEBuilder® HiFi DNA Assembled using Assembly Master Mix according to manufacturer's instructions.

[0155] Plasmid P5, containing the signal sequence SP4 (SEQ ID NO:4), To generate this, primers PMR13 (SEQ ID NO:28) and PMR14 (SEQ ID NO:29) were used for amplification using gBLOCK1 (SEQ ID NO:15) as template. A backbone containing human lactoferrin, yeast HIS4 auxotrophic marker, and E. coli antibiotic resistance and replication origin was obtained by PCR of the P1 plasmid using primers PMR15 (SEQ ID NO:30) and PMR16 (SEQ ID NO:31). The two resulting fragments were assembled using NEBuilder® HiFi DNA Assembly Master Mix according to the manufacturer's instructions.

[0156] The assembly mixture was incubated in E. coli DH5α cells as directed by the manufacturer. and plated on Luria Broth (LB) agar plates containing 100 μg / mL ampicillin. Positive clones were selected by colony polymerase chain reaction (PCR) and inoculated overnight into 5 mL of liquid Luria Broth medium supplemented with 100 μg / mL ampicillin. Plasmids from E. coli cells were isolated using a GeneJET Plasmid Miniprep Kit (ThermoFisher®, Cat. No. K502). Proper assembly was confirmed by Sanger DNA sequencing.

[0157] Primers PMR17 (SEQ ID NO:32) and PMR18 (S Using the plasmid P1, P2, P3, P4 or P5 as a template, and the Q5 high-fidelity DNA polymerase, linear dsDNA fragments for integration into yeast were obtained. Electrocompetent Pichia pastoris cells were incubated with 100 ng / mL PBS as described by Madden, Tolstorukov, & Cregg (2014) Fungi, Volume 1, Fungal Biology. The cells were transformed as described above. The cells were plated on MD plates (1.34% yeast nitrogen base, 4 × 10 -5 % biotin, 2% glucose, 20% agar) and that + Cells were allowed to select and incubated at 30°C for 72 hours. Individual yeast colonies (~10 The cells (~20 cells) were restreaked on MD plates and grown at 30°C for 24 hours. The transformed cells were analyzed for the expression of SP1 (SEQ ID NO: 1) in the secretion of the protein of interest (POI). SP2 (SEQ ID NO:1), SP2 (SEQ ID NO:2), SP3 (SEQ ID NO:3) and SP4 (SEQ ID NO:5) were used as controls to evaluate the higher efficiency of SP2 (SEQ ID NO:1), SP3 (SEQ ID NO:2), SP4 (SEQ ID NO:3) and SP5 (SEQ ID NO:5).

[0158]

[0141] Individual colonies from the restreaked plates were cultured in 600 μl of 2% YPD (2% Inoculate 96 deep-well plates using 1% ethanol (1% glycerol, 2% kistulose, 2% peptone, 1% yeast extract). Grow cells at 1,000 rpm and 30°C for 48 hours. A quart of the resulting cell suspension was diluted with 550 μl of BMG (100 mM potassium phosphate buffer (pH = 6.0), 1.34% yeast nitrogen base, 4 × 10 -5 The cells were transferred to a 100% PBS containing 1% biotin and 1% glycerol and incubated at 1,000 rpm and 30°C for 48 hours. The cells were then centrifuged at 4,500 x g for 5 minutes. and incubated at 1,000 rpm and 20°C for 72 h induction. in 1% BMM (100 mM potassium phosphate buffer (pH = 6.0), 1.34% yeast nitrogen base, 4 × 10 -5 % biotin, 1% methanol) and the proteins secreted into the extracellular medium were then analyzed by SDS-PAGE, ELISA and Western blot.

[0159] All of the methods disclosed and claimed herein are herein believed to be of a similar nature and are not to be construed as limiting the scope of the invention in light of the present disclosure. The compositions and methods disclosed herein have been described with respect to specific embodiments, but it will be apparent to those skilled in the art that variations may be applied to the methods described herein, and in the steps, or in the sequence of steps of the methods, without departing from the concept, spirit and scope of the disclosed embodiments. More specifically, it will be understood that certain agents that are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the embodiments disclosed herein, as defined by the appended claims.

[0160] This specification includes the disclosure of the following inventions: [Item 1] An isolated nucleic acid encoding a polypeptide comprising a sequence having at least 90% sequence identity to SEQ ID NO:1, 2, 3 or 4. [Item 2] An isolated nucleic acid according to item 1, the sequence of which comprises SEQ ID NO:1, 2, 3, or 4. [Item 3] An isolated nucleic acid according to item 1 or 2, wherein the polypeptide further comprises a sequence of a mammalian protein. [Item 4] An isolated nucleic acid according to item 3, wherein the mammalian protein is a human milk protein. [Item 5] The isolated nucleic acid according to Item 4, wherein the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme or α-lactalbumin. [Item 6] The isolated nucleic acid according to Item 5, wherein the human milk protein is human lactoferrin. [Item 7] The isolated nucleic acid according to any one of items 1 to 6, wherein the sequence is SEQ ID NO: An isolated nucleic acid having at least 90% sequence identity to ID NO:1. [Item 8] An isolated nucleic acid according to any one of items 1 to 6, wherein the sequence comprises SEQ ID NO:1. [Item 9] The isolated nucleic acid according to item 8, wherein the isolated nucleic acid has SEQ ID An isolated nucleic acid comprising a nucleic acid sequence having at least 80% identity to NO:41. [Item 10] An isolated nucleic acid according to Item 9, wherein the nucleic acid sequence comprises SEQ ID NO:41. [Item 11] The isolated nucleic acid according to item 8, wherein the polypeptide is selected from the group consisting of SEQ ID NO: An isolated nucleic acid comprising NO:5. [Item 12] An isolated nucleic acid according to item 11, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:46. [Item 13] An isolated nucleic acid according to Item 12, wherein the nucleic acid sequence comprises SEQ ID NO:46. [Item 14] The isolated nucleic acid according to any one of items 1 to 6, wherein the sequence is SEQ ID NO: An isolated nucleic acid having at least 90% sequence identity to ID NO:2. [Item 15] An isolated nucleic acid according to any one of items 1 to 6, wherein the sequence comprises SEQ ID NO:2. [Item 16] An isolated nucleic acid according to Item 15, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:42. [Item 17] An isolated nucleic acid according to Item 16, wherein the nucleic acid sequence comprises SEQ ID NO:42. [Item 18] An isolated nucleic acid according to Item 15, wherein the polypeptide comprises SEQ ID NO:6. [Item 19] An isolated nucleic acid according to item 18, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:47. [Item 20] An isolated nucleic acid according to Item 19, wherein the nucleic acid sequence comprises SEQ ID NO:47. [Item 21] An isolated nucleic acid according to any one of items 1 to 6, wherein the sequence has at least 90% sequence identity to SEQ ID NO:3. [Item 22] An isolated nucleic acid according to any one of items 1 to 6, wherein the sequence comprises SEQ ID NO:3. [Item 23] An isolated nucleic acid according to Item 22, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:43. [Item 24] An isolated nucleic acid according to Item 23, wherein the nucleic acid sequence comprises SEQ ID NO:43. [Item 25] An isolated nucleic acid according to Item 22, wherein the polypeptide comprises SEQ ID NO:7. [Item 26] An isolated nucleic acid according to item 25, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:48. [Item 27] ​​An isolated nucleic acid according to Item 26, wherein the nucleic acid sequence comprises SEQ ID NO:48. [Item 28] An isolated nucleic acid according to any one of Items 1 to 6, wherein the sequence has at least 90% sequence identity to SEQ ID NO:4. [Item 29] An isolated nucleic acid according to any one of items 1 to 6, wherein the sequence comprises SEQ ID NO:4. [Item 30] An isolated nucleic acid according to Item 29, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:44. [Item 31] An isolated nucleic acid according to Item 30, wherein the nucleic acid sequence comprises SEQ ID NO:44. [Item 32] An isolated nucleic acid according to Item 32, wherein the polypeptide comprises SEQ ID NO:8. [Item 33] An isolated nucleic acid according to Item 32, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:49. [Item 34] An isolated nucleic acid according to Item 33, wherein the nucleic acid sequence comprises SEQ ID NO:49. [Item 35] A vector comprising the nucleic acid according to any one of Items 1 to 34. [Item 36] An engineered eukaryotic cell comprising the nucleic acid according to any one of items 1 to 34 or the vector according to item 35. [Item 37] The engineered eukaryotic cell according to Item 36, wherein the cell is a fungal cell. [Item 38] The engineered eukaryotic cell according to Item 37, wherein the fungal cell is selected from the group consisting of the genus Arcthura, Aspergillus, Aurantiochytrium, Candida, Cryptococcus, Csudaema, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidia, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Tribolium, and Trichoderma. The engineered eukaryotic cell is a cell of the genus Cosporon, Wickerhamomyces or Yarrowia. [Item 39] The engineered eukaryotic cell according to Item 38, wherein the cell is a yeast cell. [Item 40] The engineered eukaryotic cell according to Item 39, wherein the yeast cell is a cell of the genus Komagataella. [Item 41] The engineered eukaryotic cell according to Item 40, wherein the yeast cell is a Komagataella puffii, Komagataella pastoris or Komagataella pseudopastoris cell. [Item 42] An engineered eukaryotic cell according to any one of items 36 to 41, wherein the nucleic acid is integrated into the genome of the cell. [Item 43] An engineered eukaryotic cell according to any one of items 36 to 41, wherein the nucleic acid is not integrated into the genome of the cell. [Item 44] A method for producing a secreted protein, the method comprising growing a cell according to any one of Items 36 to 43 under conditions sufficient to secrete the polypeptide from the cell. [Item 45] The method described in Item 44, further comprising collecting the secreted protein. [Item 46] The method according to item 44 or 45, wherein the secreted protein is a human milk protein. [Item 47] The method according to Item 46, wherein the human milk protein is a secretory IgA ( sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, The method is butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin. [Item 48] The method according to any one of Items 44 to 47, wherein the human milk protein contains one or more human-like N-glycans. [Item 49] The method according to any one of Items 44 to 48, further comprising producing a mixture comprising the human milk protein and one or more components of infant formula. [Item 50] An engineered yeast cell comprising a nucleic acid encoding a polypeptide comprising a sequence having at least 90% sequence identity to SEQ ID NO:1, 2, 3 or 4. [Item 51] An engineered yeast cell according to Item 50, wherein the sequence comprises SEQ ID NO:1, 2, 3 or 4. [Item 52] An engineered yeast cell according to Item 51, wherein the sequence comprises SEQ ID NO:3. [Item 53] The engineered yeast cell according to any one of Items 50 to 52, wherein the polypeptide further comprises a sequence of a mammalian protein. [Item 54] The engineered yeast cell according to Item 53, wherein the mammalian protein is a human milk protein. [Item 55] The engineered yeast cell according to Item 54, wherein the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme or α-lactalbumin. [Item 56] The engineered yeast cell according to Item 55, wherein the human milk protein is human lactoferrin. [Item 57] An engineered yeast cell comprising: (a) a first nucleic acid encoding a polypeptide comprising: (i) at least 90% sequence identity to SEQ ID NO:1, 2, 3, or 4 Sequence identity and (ii) a human milk protein sequence; and (b) an engineered yeast cell that contains a second nucleic acid encoding an α-1,2-mannosidase (Man-I) protein, wherein the cell does not express a functional OCH1 protein. [Item 58] The engineered yeast cell according to Item 57, wherein the sequence of (i) is SEQ ID NO: An engineered yeast cell comprising ID NO:1, 2, 3 or 4. [Item 59] The engineered yeast cell according to Item 57 or 58, wherein the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin. [Item 60] The engineered yeast cell according to Item 57, wherein the human milk protein is human lactoferrin. [Item 61] The engineered yeast cell according to Item 57, wherein the human milk protein is human alpha-lactalbumin. [Item 62] The engineered yeast cell according to any one of Items 57 to 61, wherein the Man-I protein is fused to an HDEL C-terminal tag. [Item 63] The engineered yeast cell according to any one of Items 57 to 62, further comprising: (a) N-acetylglucosaminyltransferase I (GnT-I) protein; (b) α-1,3 / 6-mannosidase (Man-II) protein; (c) β-1,2-acetylglucosaminyltransferase (GnT-II) protein; and (d) β-1,4-galactosyltransferase (GalT) protein; The engineered yeast cell further comprising a third nucleic acid encoding one or more of the following: [Item 64] An infant formula containing human glycoproteins with human-like N-linked glycosylation. [Item 65] The infant formula according to Item 64, wherein the human glycoprotein is a human milk protein. [Item 66] The infant formula according to Item 65, wherein the human milk protein is a secretory IgA ( sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, An infant formula that is butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin. [Item 67] The infant formula according to Item 66, wherein the human milk protein is human lactoferrin. [Item 68] An infant formula according to any one of Items 64 to 67, wherein the lactoferrin has a glycan pattern that is different from the glycan pattern of any human lactoferrin naturally present in human breast milk. [Item 69] The infant formula according to any one of Items 64 to 68, wherein the human glycoprotein is produced by the method according to any one of Items 44 to 49. References The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specifically incorporated herein by reference.

[0161] [ka] U.S. Patent No. 4,977,137 (Nichols et al.) U.S. Patent No. 5,571,691 (Conneely et al.) U.S. Patent No. 7,335,512 (Callewaert et al.) U.S. Patent No. 7,344,867 (Connolly) U.S. Patent No. 7,749,960 (Vidal et al.) U.S. Patent No. 7,524,815 (Vidal et al.) U.S. Patent No. 7,914,822 (Medo) U.S. Patent No. 8,440,456 (Callewaert et al.) U.S. Patent No. 8,871,445 (Cong et al.) U.S. Patent No. 8,802,650 (Buck et al.) U.S. Patent No. 8,821,878 (Medo et al.) U.S. Patent No. 8,927,027 (Fournell et al.) U.S. Patent No. 7,449,308 (Gerngross et al.) U.S. Patent Application Publication No. 2012 / 0142580 (Nutten et al.)

Claims

1. An isolated nucleic acid encoding a polypeptide comprising a sequence having at least 90% sequence identity to SEQ ID NO:1, 2, 3 or 4.

2. 2. The isolated nucleic acid of claim 1, wherein the sequence comprises SEQ ID NO: 1, 2, 3, or 4.

3. 3. The isolated nucleic acid of claim 1 or 2, wherein the polypeptide further comprises a sequence of a mammalian protein.

4. 4. The isolated nucleic acid of claim 3, wherein the mammalian protein is a human milk protein.

5. 5. The isolated nucleic acid of claim 4, wherein the human milk protein is secretory IgA(s). IgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, The isolated nucleic acid is tyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme or α-lactalbumin.

6. 6. The isolated nucleic acid of claim 5, wherein the human milk protein is human lactoferrin.

7. 7. The isolated nucleic acid of any one of claims 1 to 6, wherein said sequence has at least 90% sequence identity to SEQ ID NO:

1.

8. 7. The isolated nucleic acid according to any one of claims 1 to 6, wherein the sequence is An isolated nucleic acid comprising NO:

1.

9. 9. The isolated nucleic acid of claim 8, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:

41.

10. 10. The isolated nucleic acid of claim 9, wherein the nucleic acid sequence comprises SEQ ID NO:

41.

11. 9. The isolated nucleic acid of claim 8, wherein the polypeptide comprises SEQ ID NO:

5.

12. 12. The isolated nucleic acid of claim 11, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:

46.

13. 13. The isolated nucleic acid of claim 12, wherein the nucleic acid sequence comprises SEQ ID NO:

46.

14. 7. The isolated nucleic acid of any one of claims 1 to 6, wherein said sequence has at least 90% sequence identity to SEQ ID NO:

2.

15. 7. The isolated nucleic acid according to any one of claims 1 to 6, wherein the sequence is An isolated nucleic acid comprising NO:

2.

16. 16. The isolated nucleic acid of claim 15, wherein the isolated nucleic acid has SEQ ID NO:

5. An isolated nucleic acid comprising a nucleic acid sequence having at least 80% identity to SEQ ID NO:

42.

17. 17. The isolated nucleic acid of claim 16, wherein the nucleic acid sequence comprises SEQ ID NO:

42.

18. 16. The isolated nucleic acid of claim 15, wherein the polypeptide comprises SEQ ID NO:

6.

19. 20. The isolated nucleic acid of claim 18, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:

47.

20. 20. The isolated nucleic acid of claim 19, wherein the nucleic acid sequence comprises SEQ ID NO:

47.

21. 7. The isolated nucleic acid according to any one of claims 1 to 6, wherein said sequence has at least 90% sequence identity to SEQ ID NO:

3.

22. 7. The isolated nucleic acid according to any one of claims 1 to 6, wherein the sequence is An isolated nucleic acid comprising NO:

3.

23. 23. The isolated nucleic acid of claim 22, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:

43.

24. 24. The isolated nucleic acid of claim 23, wherein the nucleic acid sequence comprises SEQ ID NO:

43.

25. 23. The isolated nucleic acid of claim 22, wherein the polypeptide comprises SEQ ID NO:

7.

26. 26. The isolated nucleic acid of claim 25, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:

48.

27. 27. The isolated nucleic acid of claim 26, wherein the nucleic acid sequence comprises SEQ ID NO:

48.

28. 7. The isolated nucleic acid according to any one of claims 1 to 6, wherein said sequence has at least 90% sequence identity to SEQ ID NO:

4.

29. 7. The isolated nucleic acid according to any one of claims 1 to 6, wherein the sequence is An isolated nucleic acid comprising NO:

4.

30. 30. The isolated nucleic acid of claim 29, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:

44.

31. 31. The isolated nucleic acid of claim 30, wherein the nucleic acid sequence comprises SEQ ID NO:

44.

32. 33. The isolated nucleic acid of claim 32, wherein the polypeptide comprises SEQ ID NO:

8.

33. 33. The isolated nucleic acid of claim 32, wherein the isolated nucleic acid comprises a nucleic acid sequence having at least 80% identity to SEQ ID NO:

49.

34. 34. The isolated nucleic acid of claim 33, wherein the nucleic acid sequence comprises SEQ ID NO:

49.

35. A vector comprising the nucleic acid according to any one of claims 1 to 34.

36. 36. An engineered eukaryotic cell comprising a nucleic acid according to any one of claims 1 to 34 or a vector according to claim 35.

37. 37. The engineered eukaryotic cell of claim 36, wherein the cell is a fungal cell.

38. 38. The engineered eukaryotic cell of claim 37, wherein the fungal cell is a cell of the genus Arcthura, Aspergillus, Aurantiochytrium, Candida, Cryptococcus, Csudaema, Geotrichum, Hansenula, Kluyveromyces, Kodamaea, Komagataella, Leucosporidia, Lipomyces, Mortierella, Ogataea, Pichia, Prototheca, Rhizopus, Rhodosporidium, Rhodotorula, Saccharomyces, Schizosaccharomyces, Tremella, Trichosporon, Wickerhamomyces, or Yarrowia.

39. 40. The engineered eukaryotic cell of claim 38, wherein the cell is a yeast cell.

40. 40. The engineered eukaryotic cell of claim 39, wherein the yeast cell is a Komagataella cell.

41. 41. The engineered eukaryotic cell of claim 40, wherein the yeast cell is a Komagataella puffii, Komagataella pastoris or Komagataella pseudopastoris cell.

42. 42. The engineered eukaryotic cell of any one of claims 36 to 41, wherein said nucleic acid is integrated into the genome of said cell.

43. 42. The engineered eukaryotic cell of any one of claims 36 to 41, wherein said nucleic acid is not integrated into the genome of said cell.

44. 44. A method for producing a secreted protein, the method comprising growing a cell according to any one of claims 36 to 43 under conditions sufficient to secrete the polypeptide from the cell.

45. 45. The method of claim 44, further comprising collecting the secreted protein.

46. 46. ​​The method of claim 44 or 45, wherein the secreted protein is a human milk protein.

47. 47. The method of claim 46, wherein the human milk protein is selected from the group consisting of secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, and lipase. zozyme, or α-lactalbumin.

48. 48. The method of any one of claims 44 to 47, wherein the human milk protein comprises one or more human-like N-glycans.

49. 49. The method of any one of claims 44-48, further comprising producing a mixture comprising the human milk protein and one or more components of an infant formula.

50. An engineered yeast cell comprising a nucleic acid encoding a polypeptide comprising a sequence having at least 90% sequence identity to SEQ ID NO:1, 2, 3 or 4.

51. 51. The engineered yeast cell of claim 50, wherein the sequence comprises SEQ ID NO: 1, 2, 3 or 4.

52. 52. The engineered yeast cell of claim 51 , wherein the sequence comprises SEQ ID NO:

3.

53. 53. The engineered yeast cell of any one of claims 50 to 52, wherein the polypeptide further comprises a sequence of a mammalian protein.

54. 54. The engineered yeast cell of claim 53, wherein the mammalian protein is a human milk protein.

55. 55. The engineered yeast cell of claim 54, wherein the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin.

56. 56. The engineered yeast cell of claim 55, wherein the human milk protein is human lactoferrin.

57. 1. An engineered yeast cell comprising: (a) a first nucleic acid encoding a polypeptide comprising: (i) a sequence having at least 90% sequence identity to SEQ ID NO: 1, 2, 3 or 4; and (ii) a human milk protein sequence; and (b) an engineered yeast cell that contains a second nucleic acid encoding an α-1,2-mannosidase (Man-I) protein, wherein the cell does not express a functional OCH1 protein.

58. 58. The engineered yeast cell of claim 57, wherein the sequence of (i) comprises SEQ ID NO: 1, 2, 3 or 4.

59. 59. The engineered yeast cell of claim 57 or 58, wherein the human milk protein is secretory IgA (sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin.

60. 58. The engineered yeast cell of claim 57, wherein the human milk protein is human lactoferrin.

61. 58. The engineered yeast cell of claim 57, wherein the human milk protein is human alpha-lactalbumin.

62. 62. The engineered yeast cell of any one of claims 57 to 61, wherein the Man-I protein is fused to a HDEL C-terminal tag.

63. 63. The engineered yeast cell of any one of claims 57 to 62, further comprising: (a) N-acetylglucosaminyltransferase I (GnT-I) protein; (b) α-1,3 / 6-mannosidase (Man-II) protein; (c) β-1,2-acetylglucosaminyltransferase (GnT-II) protein; and (d) β-1,4-galactosyltransferase (GalT) protein; The engineered yeast cell further comprising a third nucleic acid encoding one or more of the following:

64. An infant formula comprising a human glycoprotein having human-like N-linked glycosylation.

65. 65. The infant formula of claim 64, wherein the human glycoprotein is a human milk protein.

66. 66. The infant formula of claim 65, wherein the human milk protein is secretory IgA ( sIgA), xanthine dehydrogenase, lactoferrin, lactoperoxidase, An infant formula that is butyrophilin, lactadherin, adiponectin, β-casein, κ-casein, leptin, lysozyme, or α-lactalbumin.

67. 67. The infant formula of claim 66, wherein the human milk protein is human lactoferrin.

68. 68. An infant formula according to any one of claims 64 to 67, wherein the lactoferrin has a glycan pattern which differs from the glycan pattern of any human lactoferrin naturally occurring in human breast milk.

69. 69. An infant formula according to any one of claims 64 to 68, wherein the human glycoprotein is produced by the method according to any one of claims 44 to 49.

Images