Composition

Abstract

The present invention involves improving the expression of functional and stable recombinant proteins in mammalian expression systems using an oligosaccharide transferase (OST) enzyme obtained from Trypanosoma brucei. In particular, the present invention provides mammalian cells comprising at least one OST protein or at least one nucleic acid sequence encoding a functional fragment thereof, wherein the at least one OST protein is a Trypanosoma spp. OST protein, and preferably, the at least one OST protein is a Trypanosoma brucei OST protein.

Description

Technical Field

[0001] The present invention involves improving the expression of functional and stable recombinant proteins in mammalian expression systems by utilizing an oligosaccharyltransferase (OST) enzyme obtained from Trypanosoma brucei (T. brucei), which includes creating a novel engineered mammalian cell line and gene construct containing a nucleotide encoding the OST enzyme obtained from T. brucei.

Background Art

[0002] The addition of a glycan moiety to the surface of a protein, called glycosylation, is one of the most common post-translational modifications (PTMs) and is a natural process important for the function and stability of many proteins. This complex process begins in the endoplasmic reticulum where the glycan moiety is added to newly synthesized proteins. This in turn affects the protein's structure and biological activity. In fact, the unique structural variations of the glycan moiety can modulate the properties of the protein. Therefore, the production of therapeutic glycoproteins in the industry has attracted great interest. The production of proteins with added glycan moieties holds the potential for improving the yield and stability of therapeutic proteins, as well as modulating activity and new drug discovery. Protein glycosylation is a naturally occurring process, and approximately 50% of proteins in the human body are glycosylated and thus referred to as glycoproteins. In the pharmaceutical industry, two-thirds of the therapeutic proteins approved by regulatory authorities are glycoproteins (Delobel, Mass Spectrometry of Glycoproteins, pp 1 - 21, 2021), and they all have at least one glycan moiety (which is a specific sugar molecule) on their surface, and some have dozens of glycan moieties attached to them. These glycan moieties are fundamentally important for the functionality of the protein and affect the protein's activity, the period during which it is stable, and the state in which the protein is folded. Two types of glycosylation exist: N-glycosylation and O-glycosylation. Over 90% of glycoproteins are modified by N-glycosylation (Helenius, Mol Biol Cell, 5(3);253-65, 1994). In mammals, N-glycosylation is a covalent bond of Glc3MAN9GlcNAc2 to an asparagine (Asn or N) residue on the polypeptide chain and is located within the Asn-X-Ser / Thr motif (where X can be any amino acid other than proline) (Reilly et al., Nature Reviews Nephrology, 15(346-366), 2019). This can occur within the endoplasmic reticulum (ER) of cells, both during and after translation (Canada et al., Cell, 136(2):272-283, 2010) (Kleizen & Braakman, Curr Opin Cell Biol, 16(4):343-9, 2004).

[0003] The addition of N-glycans to proteins is catalyzed by multi-subunit enzymes known as oligosaccharide transferases (OSTs or OTases). Mammalian OSTs are multi-subunit membrane proteins containing two distinct catalytic subunits (STT3A and STT3B) along with at least six other non-catalyzable subunits, but their functions have not been fully studied (Mohanty et al., Biomolecules, 10(4):624, 2020), (Pfeffer et al., Nature Communications, 5(3072), 2014). The two catalytic subunits have distinct substrate specificities (Cherepanova & Gilmore, Scientific Reports, 6(20946), 2016), and the OST as a whole catalyzes the enblock transfer of pre-assembled high-mannose oligosaccharides to asparagine residues (Kheller & Gilmore, Glycobiology, 16(4):47-62, 2005).

[0004] However, in eukaryotes, OSTs that perform glycosylation are generally inefficient, with approximately 35% of N-glycan sequencers remaining unoccupied (Petrescu et al., Glycobiology, 14(2):103-14, 2004). This poses a significant limitation in industries where mammalian cells are commonly used for the bioproduction of glycosylated proteins, including therapeutic proteins. In fact, N-glycan site occupancy is one of the key quality attributes analyzed by the U.S. Food and Drug Administration (FDA) for regulatory approval of therapeutic proteins. This is because N-glycosylation plays a crucial role in many aspects of protein production, including protein yield, protein function, correct protein folding, and control of batch-to-batch variability. Therefore, in this field of technology, there is a need to improve the production of recombinantly produced proteins in mammalian cell systems. Specifically, in the drug production and drug discovery industries, there is a need to increase the N-glycan moiety occupancy of recombinant proteins to substantially improve the yield, activity, stability, and reproducibility of recombinant proteins. [Overview of the Initiative]

[0005] The inventors of this invention have surprisingly discovered that a specific OST enzyme derived from the parasite Trypanosoma bruseyi can be engineered in mammalian bioproduction cells to substantially improve the N-glycan profile of recombinant proteins produced in mammalian cells. This has the potential to improve the yield, stability, and activity of the recombinant proteins produced.

[0006] In a first aspect of the present invention, a mammalian cell is provided comprising at least one nucleic acid sequence encoding at least one oligosaccharide transferase (OST) protein or a functional fragment thereof, wherein the at least one OST protein is a Trypanosoma spp. OST protein, and preferably, the at least one OST protein is a Trypanosoma brusey OST protein. A second aspect of the present invention provides a mammalian cell comprising at least one nucleic acid sequence encoding at least one oligosaccharide transferase (OST) protein or a functional fragment thereof, wherein the at least one nucleic acid sequence encoding the at least one OST protein or functional fragment comprises the sequence according to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22, or a sequence having at least 70% sequence identity to those. In a preferred embodiment, the mammalian cell may comprise a combination of nucleic acids, such as the combinations described in the detailed description. A third aspect of the present invention provides a mammalian cell comprising at least one nucleic acid sequence encoding at least one oligosaccharide transferase (OST) protein or a functional fragment thereof, wherein the at least one OST protein comprises an amino acid sequence according to SEQ ID NOs. 14, 15, 16, 17, 18, 19, and / or 20, or a sequence having at least 70% sequence identity thereto. In a preferred embodiment, the mammalian cell may comprise a combination of nucleic acids, such as the combinations described in the detailed description.

[0007] In a fourth aspect of the present invention, an isolated nucleic acid molecule is provided which includes the sequence according to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22, or a sequence having at least 70% identity with them, preferably an isolated nucleic acid molecule which includes the sequence according to any of SEQ ID NOs: 3, 5, 7, 9, 11, and / or 13. A fifth aspect of the present invention provides a nucleic acid vector comprising (i) at least one nucleic acid sequence according to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21 and / or 22, or a sequence having at least 70% identity to them, or (ii) at least one isolated nucleic acid molecule according to a fourth aspect of the present invention, preferably a nucleic acid vector selected from the group including, but not limited to, plasmid-based expression vectors, bacterial artificial chromosome (BAC) vectors, and viral vectors such as adenovirus vectors, adeno-associated vectors (AAVs), retrovirus vectors, or lentivirus vectors. In a preferred embodiment, the vector may comprise a combination of nucleic acids, such as the combinations described in the detailed description.

[0008] A sixth aspect of the present invention provides a method for modifying the glycosylation profile of a recombinant protein, comprising contacting the recombinant protein with (i) a mammalian cell according to a first aspect of the present invention, or (ii) at least one OST protein or a functional fragment thereof, wherein the at least one OST protein is a trypanosoma OST protein, preferably a trypanosoma brusey OST protein, and more preferably the at least one OST protein comprises the amino acid sequence according to any of SEQ ID NOs. 14, 15, 16, 17, 18, 19, and / or 20, or a sequence having at least 70% sequence identity thereto, or producing the recombinant protein in a mammalian cell according to a first aspect of the present invention, wherein the mammalian cell is manipulated to express the recombinant protein. [Brief explanation of the drawing]

[0009] [Figure 1A]This figure shows the amino acid sequence alignment of residues 367-408 of OST proteins (TbSTT3A, B, and C) derived from T. brusey, including the predicted active region 371-408. Positively charged residues are shaded in gray, negatively charged residues are bold and underlined, and neutrally charged residues are bold. [Figure 1B] This figure shows the AlphaFold predicted structures of TbSTT3A, B, and C, with the predicted active region highlighted in dark gray. Important amino acid residues are shown and labeled in black. [Figure 2] This table shows the three OST enzymes isolated from T. brusey and their respective gene identification numbers. [Figure 3] This table shows the expression of T. brusey enzyme in mammalian cells, as verified by mass spectrometry. [Figure 4] This figure demonstrates that hormone production increases in the presence of kinetoplast-like OSTs. EPO hormone yield was detected 72 hours after transient transfection of cells. "Mock" refers to mock-transfected cells (control). [Figure 5] This figure demonstrates that N-glycosylation increases in the presence of T. brusey's OST. The data show the occupancy rate of N-glycan sites in the presence of OST (enzyme A) and mock-transfected cells (control). The percentage of N-glycosylated peptides was determined by mass spectrometry. [Figure 6] This figure demonstrates that transient expression of the T. brusey OST enzyme in Expi293 cells derived from HEK-293 cells does not affect the overall viability of the cell population. [Modes for carrying out the invention]

[0010] To make the present invention easier to understand, certain terms are defined first. Further definitions are provided throughout the detailed description.

[0011] As used herein, the term “mammalian cells” refers to eukaryotic cells derived from or isolated from mammalian tissue. In some embodiments, eukaryotic cells may be modified to grow immortalized cell lines indefinitely. In other embodiments, eukaryotic cells may not be immortalized. In the context of the present invention, mammalian cells are used as expression systems for recombinant protein expression. Therefore, any mammalian cells that achieve this function are within the scope of the present invention. For example, mammalian cells may be Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK21) cells, mouse myeloma cells (NS0 and Sp2 / 0), or human embryonic kidney (HEK) cells. Preferably, the mammalian cells of the present invention are Chinese hamster ovary (CHO) cells and / or immortalized human embryonic kidney (HEK) cells such as HEK293 cells.

[0012] As used herein, the term “exogenous” refers to a nucleic acid sequence (i.e., heterogeneous nucleic acid) that originates from outside a mammalian cell and is subsequently transformed into a mammalian cell, also referred to as a DNA sequence. As will be understood by those skilled in the art, the nucleic acid molecules of the present invention are exogenous to the mammalian cells of the present invention because they originate from the Trypanosoma bursey OST coding nucleic acid. The terms “nucleic acid sequence” and “gene” may be used interchangeably herein, but “gene” is generally understood to refer to a nucleic acid sequence or combination of nucleic acid sequences that are transcribed and translated into a single protein having a specified function, or into a complex of multiple proteins. In the context of the present invention, the term “exogenous gene” refers to a gene originating from a different species, specifically a parasite such as Trypanosoma bursey, and is therefore considered a heterogeneous gene. The exogenous nucleic acid sequences of the present invention may have the sequence of any one of SEQ ID NOs: 1 to 7.

[0013] As used herein, the terms “oligosaccharide transferase,” “OST,” and “OTase” are interchangeable and may refer to single and multi-subunit glycosyltransferase enzymes that catalyze the addition of N-glycans to proteins. In the context of the present invention, OSTs derived from or obtained from parasites are used. As used herein, the terms “sequence identity” and “sequence homology” are interchangeable and refer to the number of identical residues over a given length for a given alignment. To calculate the % sequence identity of any of the sequences disclosed herein, sequence comparison software may be used, for example, with default settings in the BLAST software package (V2.10.1).

[0014] As used herein, the term “recombinant protein” refers to a specific nucleic acid sequence cloned in an expression system that supports gene expression and messenger RNA (mRNA) translation, i.e., a protein encoded by a gene (recombinant DNA). The gene introduced into the expression system may be a heterologous gene (or exogenous gene), i.e., the gene is derived from or obtained from a cell type of an organism different from the recipient expression system. Methods for heterologous expression of recombinant proteins are well known to those skilled in the art. The expression system of the present invention is a mammalian expression system. Preferably, the mammalian expression system is a mammalian cell such as a CHO or HEK cell.

[0015] When used herein in connection with the methods of the present invention, the term “contact” is intended to include any means of bringing the recombinant protein and at least one OST protein into a state of contact sufficient to enable glycosylation of the recombinant protein by the at least one OST protein. This may involve chaperone proteins as needed. It is assumed that the recombinant protein can be contacted with at least one OST protein in mammalian cells or in cell-free systems, e.g., cell-free expression systems. The recombinant protein and at least one OST protein may be co-expressed in mammalian cells or cell-free systems, or they may be expressed at different times. For example, mammalian cells may constitutively express at least one OST protein before the expression of the recombinant protein by the mammalian cells. At least one OST protein may be expressed and effluxed from or extracted from mammalian cells before contact with the recombinant protein. For example, mammalian cells expressing at least one OST protein may be lysed to enable contact with the recombinant protein. As those skilled in the art will understand, there are many ways in which the method of the present invention can be carried out, but the central concept requires a method in which at least one OST protein is presented to the recombinant protein in such a way that glycosylation of the recombinant protein is possible. Ideally, the glycosylated recombinant protein is maintained in a conformation that preserves its biological activity, such that it is measured in vitro or in vivo by ligand affinity assays, activity assays, stability assays, half-life assays, pharmacokinetic assays, dose studies, cell uptake assays, and other assays for evaluating potency, efficacy and quality. Furthermore, those skilled in the art will know how to do this.

[0016] As used herein, the term “glycosylation” refers to the process by which oligosaccharides (glycan portions) are bound to target molecules such as proteins and lipids. It will be readily apparent to those skilled in the art that this process is essential for both the functionality and stability of proteins. Oligosaccharides are carbohydrates consisting of chains or polymers of monosaccharides (single sugar molecules). Glycosylation is a form of co-translational and post-translational modification of proteins in which carbohydrates are conjugated or covalently bound to proteins. In biology, the process of glycosylation is an enzyme-catalyzed reaction, and enzymes such as oligosaccharide transferases (OSTs) and glycosyltransferases may be involved. In mammalian cells, glycosylation mainly occurs in the rough endoplasmic reticulum, cytoplasm, and nucleus. There are two main types of glycosylation: N-glycosylation (also known as “N-linked glycosylation”) and O-glycosylation (also known as “O-linked glycosylation”), with the former being more widely recognized. Furthermore, C-glycosylation (also known as "C-linked glycosylation") exists, which occurs when glycans are linked to carbon atoms on the tryptophan side chain. In N-glycosylation, glycans are linked to the nitrogen atom of an amino acid (e.g., asparagine and arginine). In O-glycosylation, glycans are linked to the oxygen atom of the hydroxyl group on an amino acid (e.g., serine, threonine, and tyrosine).

[0017] As used herein, the term “active region” refers to at least one region of an OST protein that is proposed or predicted to interact with a target molecule, such as a polypeptide. Such interactions between the OST and the target molecule may be involved in glycosylation and / or binding. For example, such interactions may be involved in OST-mediated glycosylation of recombinant polypeptides or proteins. The term “active region” may also be used interchangeably with “active site,” “recognition site,” “consensus,” “conserved region,” and “binding site,” as used herein. Therefore, in the context of the present invention, the addition of "N-glycans" to the protein of interest is of particular interest. The addition of N-glycans refers to the addition of any of the three common types of N-glycans: oligomannoses, complexes, and hybrids, each containing a common core Man3GlcNAc2 having any number of branches and / or extensions.

[0018] As used herein, the terms “N-glycan occupancy,” “N-glycan site occupancy,” “N-glycosylation occupancy,” and “N-glycosylation site occupancy” may be used interchangeably and refer to the number of potential N-glycan (or N-glycosylation) sites on a protein that are occupied, i.e., the number of modified sequenceons. This is also known as the “macroheterogeneity” of N-glycosylation. For example, an N-glycan occupancy of 50% represents a protein in which half of its potential N-glycan sites are occupied. An N-glycan occupancy of 100% represents a protein in which all of its potential N-glycan sites are occupied. The present invention relates particularly to the N-glycan occupancy of an NXS / T sequenceon (i.e., the number of NXS / T consensus sites occupied by glycans).

[0019] As used herein, the term "N-glycosylation profile" refers to the sites of N-glycosylation on the surface of a fully folded protein and the types of N-glycans present at those sites. Thus, an N-glycosylation profile represents both the degree of N-glycosylation site occupancy and the types of individual N-glycans on a fully folded protein. As will be understood by those skilled in the art, when determining an N-glycosylation profile, the degree of N-glycosylation site occupancy and the types of N-glycans present represent the profile of all proteins in a given population of proteins (i.e., the proteins present in a sample). In particular, in the context of the present invention, the term "N-glycosylation profile" refers to the overall profile of the degree of N-glycosylation site occupancy and the types of N-glycans present in a given population of recombinant proteins. Preferably, in the context of the present invention, an improved glycosylation profile is constituted by an increase in the degree of N-glycosylation site occupancy on a recombinant protein having a reproducible and consistent N-glycan moiety between production batches of the recombinant protein. Mass spectrometry can be utilized to determine the percentage of N-glycosylation occupancy of a given protein. Preferably, the OST protein of the present invention is envisioned to increase the percentage of N-glycosylation occupancy of a given protein, such as a recombinant protein, to an occupancy of at least 90% of the sites, for example, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% of the N-glycan sites.

[0020] As used herein, the terms "expression vector" or "expression construct" refer to a recombinantly or synthetically produced nucleic acid construct having a series of specified nucleic acid elements that enable transcription of a particular nucleic acid sequence in a host cell. An expression vector can be a plasmid, a bacterial artificial chromosome, a virus, or a fragment of a nucleic acid sequence. Typically, an expression vector contains a nucleic acid sequence linked to a promoter sequence. An expression vector can encode at least one of any of the nucleic acid sequences of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22.

[0021] To produce T. brucei OST in mammalian cells, the above-described expression vector containing the nucleic acid sequence is used for stable transfection or transient transfection of mammalian cells. Stable transfection refers to any method by which the T. brucei OST gene is integrated into the host genome of mammalian cells that allows for stable expression of the OST gene under the control of a constitutive or inducible promoter. Stable transfection allows for integration into the host genome and thus the gene is replicated and the expression of the T. brucei OST gene persists for a long period. Transient transfection refers to any method by which the T. brucei OST gene is introduced and expressed under the control of a constitutive or inducible promoter. Transient transfection does not allow for integration into the host genome and thus the gene is not inherited during cell division, and as a result, the expression of the T. brucei OST gene is of a limited duration.

[0022] The use of alternatives (e.g., "or") should be understood to mean either, both, or any combination of the alternatives. As used herein, the indefinite articles "a" or "an" should be understood to refer to "one or more" of any recited or listed component.

[0023] As used herein, “approximately” means within the tolerance range of a particular value as determined by those skilled in the art, the tolerance range depending in part on the method by which the value is measured or determined, i.e., the limits of the measuring system. For example, “approximately” may mean within 1 or a standard deviation greater than 1, according to practice in the art. Alternatively, “approximately” may mean a range of up to 20%. Where a particular value is provided in this application and claims, unless otherwise stated, the meaning of “approximately” should be assumed to be within the tolerance range of that particular value. The expression systems and methods disclosed herein are expected to have numerous advantages compared to expression systems utilizing natural mammalian OSTs. For example, recombinant products produced in the expression systems specified herein are expected to have improved stability and functionality, enhanced efficacy, higher yields, and greater batch-to-batch uniformity, all of which are important and desirable characteristics in the bioproduction industry.

[0024] In a first aspect, the present invention provides mammalian cells comprising at least one oligosaccharide transferase (OST) protein or at least one nucleic acid sequence encoding a functional fragment thereof, wherein the at least one OST protein is a trypanosoma OST protein, and preferably the at least one OST protein is a trypanosoma brusey OST protein. In a second aspect, the present invention provides a mammalian cell comprising at least one nucleic acid sequence encoding at least one oligosaccharide transferase (OST) protein or a functional fragment thereof, wherein the at least one nucleic acid sequence encoding the at least one OST protein comprises the sequence according to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22, or a sequence having at least 70% identity to them.

[0025] Therefore, the present invention utilizes genetic engineering of highly efficient OST enzymes derived from single-celled parasites to mammalian cells. As a result, the inventors anticipate that improvements in N-glycan occupancy and sialylation of recombinant proteins can be obtained. The nucleic acid sequence according to the first embodiment may have the sequence of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22. The above sequence encodes an OST protein derived from Trypanosoma brucei (T. brucei), a known pathogen of human African trypanosomiasis. There are three subspecies that cause different types of trypanosomiasis: T. brucei brucei, T. brucei gambiense, and T. brucei rhodiense.

[0026] In all aspects or embodiments of the inventions disclosed herein, it is assumed that the cells or vectors may include any combination of one, two, three or more of the nucleic acid sequences disclosed herein, particularly those described in the detailed description. Sequence ID 1 corresponds to the consensus DNA sequence encoding a fragment of the TbSTT3A protein. Sequence ID 21 corresponds to the consensus DNA sequence encoding a fragment of the TbSTT3B protein. Sequence ID 22 corresponds to the consensus DNA sequence encoding a fragment of the TbSTT3C protein.

[0027] The highly conserved consensus sequences of SEQ ID NOs: 1, 21, and 22 may contain residues important for the activity of OST proteins. While not theoretically bound, it is assumed that SEQ ID NOs: 1, 21, and 22 may encode the active region of Trypanosoma OST proteins. This region, defined by SEQ ID NOs: 1, 21, and 22, may hereafter be referred to as the active region, active site, recognition site, binding site, consensus site, or highly conserved site. The residues highlighted in Figure 1A are considered to be particularly important. Therefore, the present invention also includes cells and vectors containing any sequence having at least 70% identity to any of SEQ ID NOs: 1, 21, and / or 22 listed herein, and further containing lysine-369, arginine-369, and / or lysine-381 when numbered according to their positions in SEQ ID NOs: 8, 10, and 12.

[0028] Sequence IDs 2, 4, and 6 correspond to DNA sequences encoding the active region sequences of the TbSTT3A, TbSTT3B, and TbSTT3C proteins, respectively. Sequence IDs 3, 5, and 7 correspond to DNA sequences encoding the active region sequences of the TbSTT3A, TbSTT3B, and TbSTT3C proteins, respectively, optimized for expression in CHO mammalian cells. Sequence IDs 8, 10, and 12 correspond to DNA sequences encoding the full-length sequences of the TbSTT3A, TbSTT3B, and TbSTT3C proteins, respectively. Sequence IDs 9, 11, and 13 correspond to DNA sequences encoding the full-length sequences of the TbSTT3A, TbSTT3B, and TbSTT3C proteins, respectively, optimized for expression in CHO mammalian cells. While Sequence IDs 1-13 and 21-22 disclosed herein represent DNA nucleic acid sequences, corresponding RNA nucleic acid sequences are also compatible with the disclosures of the present invention, and it will be understood by those skilled in the art that thymine residues may be replaced with uracil residues. The active region of the T. brusey OST enzyme is predicted to be located within the region spanning amino acid residue numbers 371-408 of TbSTT3A, B, and C. The inventors of this invention have identified potentially important residues within and around this highly conserved region that may affect peptide specificity. These residues include lysine-369 or arginine-369 or lysine-381, and arginine-397 or histidine-397. In particular, the important amino acid residues in TbSTT3A are lysine-369, lysine-381, and arginine-397. The important amino acid residues in TbSTT3B are arginine-369, lysine-381, and histidine-397. The important amino acid residues in TbSTT3C are arginine-369, lysine-381, and arginine-397.

[0029] A third aspect of the present invention provides a mammalian cell comprising at least one nucleic acid sequence encoding at least one oligosaccharide transferase (OST) protein or a functional fragment thereof, wherein the at least one OST protein comprises the amino acid sequence given by SEQ ID NOs: 14, 15, 16, 17, 18, 19, and / or 20, or a sequence having at least 70% sequence identity to those given thereto. The OST protein may comprise a full-length protein (i.e., including SEQ ID NOs: 17, 18, and / or 19), or the OST protein may comprise a highly conserved region (i.e., including SEQ ID NOs: 14, 15, 16, and / or 20). As will be readily apparent to those skilled in the art, amino acid sequences as defined in the third aspect of the present invention may derive from nucleic acid sequences as defined in the first and second aspects of the present invention. However, there are numerous rearrangements of nucleic acid codon combinations that can produce these amino acid sequences, which will also be apparent to those skilled in the art. Therefore, there may be other nucleic acid sequences from which the amino acid sequences of the third aspect of the present invention may derive.

[0030] Sequence IDs 14, 15, and 16 correspond to the amino acid sequences of the active regions of the TbSTT3A, TbSTT3B, and TbSTT3C proteins, respectively. Sequence IDs 17, 18, and 19 correspond to the amino acid sequences of the full-length TbSTT3A, TbSTT3B, and TbSTT3C proteins, respectively. Sequence ID 20 corresponds to an amino acid conserved across the active regions of TbSTT3A, TbSTT3B, and TbSTT3C. Sequence IDs 1, 21, and 22 are consensus DNA sequences encoding the amino acids according to Sequence ID 20. Jinnelov et al. (2017) have previously demonstrated that arginine-397 and histidine-397 in T. brusey OSTa (TbSTT3A) and T. brusey OSTb (TbSTT3B), respectively, are important for determining the peptide specificity between T. brusey OST enzymes. It is noteworthy that both of these residues are positively charged. We observed that residues that may be involved in peptide specificity in the active sites of L. major and T. cruzi OST enzymes are neutral or negatively charged amino acids (i.e., these important residues are not positively charged). Surprisingly, in contrast, important residues that are likely to be involved in peptide specificity in the active site of T. brusey OST enzymes are positively charged. This pattern is observed more than twice in the other residues mentioned above. Therefore, the inventors of the present invention hypothesized that the unique glycan specificity of the T. brusey OST protein may be influenced by, or a consequence of, the presence of positively charged residues at these positions.

[0031] The present invention also provides nucleic acids comprising or having sequences that have at least 70% identity to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22. For example, nucleic acid sequences have at least 75% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22; at least 80% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22; at least 85% identity to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22; SEQ ID NOs: 1, 2, 3 At least 90% identity with 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22; at least 91% identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22; at least 92% identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22; SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 1 At least 93% identity with 2, 13, 21, and / or 22; at least 94% identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22; at least 95% identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22 At least 96% identity; at least 97% identity with sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22; at least 98% identity with sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22; or may have at least 99% identity with sequence numbers 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22.

[0032] Mammalian cells may contain the sequence of SEQ ID NO: 1 or at least one exogenous nucleic acid having the same sequence. In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 1 or a sequence having at least 70% identity to the above sequence. For example, the exogenous nucleic acid may contain a sequence having at least 75% identity to SEQ ID NO: 1; at least 80% identity to SEQ ID NO: 1; at least 85% identity to SEQ ID NO: 1; at least 90% identity to SEQ ID NO: 1; at least 91% identity to SEQ ID NO: 1; at least 92% identity to SEQ ID NO: 1; at least 93% identity to SEQ ID NO: 1; at least 94% identity to SEQ ID NO: 1; at least 95% identity to SEQ ID NO: 1; at least 96% identity to SEQ ID NO: 1; at least 97% identity to SEQ ID NO: 1; at least 98% identity to SEQ ID NO: 1; or at least 99% identity to SEQ ID NO: 1. According to the sequence shown in Sequence ID No. 1, the amino acid residues at positions 369, 381, and 397 are positively charged amino acids, such as amino acids selected from lysine, arginine, and histidine, or their artificial analogues.

[0033] Mammalian cells may contain the sequence of SEQ ID NO: 21 or at least one exogenous nucleic acid having the same sequence. In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 21 or a sequence having at least 70% identity to the above sequence. For example, the exogenous nucleic acid may contain a sequence having at least 75% identity to SEQ ID NO: 21; at least 80% identity to SEQ ID NO: 21; at least 85% identity to SEQ ID NO: 21; at least 90% identity to SEQ ID NO: 21; at least 91% identity to SEQ ID NO: 21; at least 92% identity to SEQ ID NO: 21; at least 93% identity to SEQ ID NO: 21; at least 94% identity to SEQ ID NO: 21; at least 95% identity to SEQ ID NO: 21; at least 96% identity to SEQ ID NO: 21; at least 97% identity to SEQ ID NO: 21; at least 98% identity to SEQ ID NO: 21; or at least 99% identity to SEQ ID NO: 21. According to the sequence shown in Sequence ID No. 21, the amino acid residues at positions 369, 381, and 397 are positively charged amino acids, such as amino acids selected from lysine, arginine, and histidine, or their artificial analogues.

[0034] Mammalian cells may contain the sequence of SEQ ID NO: 22 or at least one exogenous nucleic acid having the same sequence. In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 22 or a sequence having at least 70% identity to the above sequence. For example, the exogenous nucleic acid may contain a sequence having at least 75% identity to SEQ ID NO: 22; at least 80% identity to SEQ ID NO: 22; at least 85% identity to SEQ ID NO: 22; at least 90% identity to SEQ ID NO: 22; at least 91% identity to SEQ ID NO: 22; at least 92% identity to SEQ ID NO: 22; at least 93% identity to SEQ ID NO: 22; at least 94% identity to SEQ ID NO: 22; at least 95% identity to SEQ ID NO: 22; at least 96% identity to SEQ ID NO: 22; at least 97% identity to SEQ ID NO: 22; at least 98% identity to SEQ ID NO: 21; or at least 99% identity to SEQ ID NO: 22. According to the sequence shown in Sequence ID No. 22, the amino acid residues at positions 369, 381, and 397 are positively charged amino acids, such as amino acids selected from lysine, arginine, and histidine, or their artificial analogues. In all embodiments of the present invention, Sequence IDs 1, 21, and 22 are interchangeable.

[0035] Mammalian cells may contain one or more exogenous nucleic acids encoding different OSTs, where the exogenous nucleic acids are selected from the group including any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22. Mammalian cells may contain a single exogenous nucleic acid sequence, two exogenous nucleic acid sequences, or three exogenous nucleic acid sequences. For example, a mammalian cell may contain any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22, or any combination thereof. If a mammalian cell contains two or more exogenous nucleic acids, the exogenous nucleic acids may be expressed separately, i.e., on separate vectors, or together, i.e., on the same vector. One or more nucleic acid molecules may be expressed as a fusion protein, or they may be expressed as separate proteins. One or more nucleic acid molecules may be expressed as a fusion protein containing a linker, such as a cleavable linker. The cleavable linker may be a protease-cleavable linker, as understood by those skilled in the art, or the cleavable linker may be a self-cleaving peptide, such as a viral self-cleaving peptide, e.g., P2A.

[0036] In a preferred embodiment, the mammalian cell may contain the exogenous nucleic acid sequence by SEQ ID NOs: 2, 4, or 6; 2 and 4; 2 and 6; 4 and 6; and / or 2, 4 and 6. In another preferred embodiment, the mammalian cell may contain the exogenous nucleic acid sequence by SEQ ID NOs: 8, 10, or 12; 8 and 10; 8 and 12; 10 and 12; and / or 8, 10, and 12. In yet another preferred embodiment, the mammalian cell may contain the exogenous nucleic acid sequence by SEQ ID NOs: 3, 5, or 7; 3 and 5; 3 and 7; 5 and 7; and / or 3, 5, and 7. In yet another preferred embodiment, the mammalian cell may contain the exogenous nucleic acid sequence by SEQ ID NOs: 9, 11, or 13; 9 and 11; 9 and 13; 11 and 13; and / or 9, 11, and 13. As will be understood by those skilled in the art, any combination of DNA sequences disclosed herein, including combinations of optimized and unoptimized nucleic acid sequences, may be used for the purposes of the present invention. Furthermore, it will be readily apparent to those skilled in the art that any nucleic acid sequence encoding an active region disclosed herein (whether optimized or unoptimized) can be combined with any nucleic acid sequence encoding a full-length protein disclosed herein (whether optimized or unoptimized).

[0037] In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 2, or a sequence that is at least 70% identical to the above sequence. For example, the exogenous nucleic acid may include sequences that are at least 75% identical to SEQ ID NO: 2; at least 80% identical to SEQ ID NO: 2; at least 85% identical to SEQ ID NO: 2; at least 90% identical to SEQ ID NO: 2; at least 91% identical to SEQ ID NO: 2; at least 92% identical to SEQ ID NO: 2; at least 93% identical to SEQ ID NO: 2; at least 94% identical to SEQ ID NO: 2; at least 95% identical to SEQ ID NO: 2; at least 96% identical to SEQ ID NO: 2; at least 97% identical to SEQ ID NO: 2; at least 98% identical to SEQ ID NO: 2; or at least 99% identical to SEQ ID NO: 2.

[0038] In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 3, or a sequence that is at least 70% identical to the above sequence. For example, the exogenous nucleic acid may include sequences that are at least 75% identical to SEQ ID NO: 3; at least 80% identical to SEQ ID NO: 3; at least 85% identical to SEQ ID NO: 3; at least 90% identical to SEQ ID NO: 3; at least 91% identical to SEQ ID NO: 3; at least 93% identical to SEQ ID NO: 3; at least 93% identical to SEQ ID NO: 3; at least 94% identical to SEQ ID NO: 3; at least 95% identical to SEQ ID NO: 3; at least 96% identical to SEQ ID NO: 3; at least 97% identical to SEQ ID NO: 3; at least 98% identical to SEQ ID NO: 3; or at least 99% identical to SEQ ID NO: 3.

[0039] In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 4, or a sequence that is at least 70% identical to the above sequence. For example, the exogenous nucleic acid may include sequences that are at least 75% identical to SEQ ID NO: 4; at least 80% identical to SEQ ID NO: 4; at least 85% identical to SEQ ID NO: 4; at least 90% identical to SEQ ID NO: 4; at least 91% identical to SEQ ID NO: 4; at least 94% identical to SEQ ID NO: 4; at least 94% identical to SEQ ID NO: 4; at least 94% identical to SEQ ID NO: 4; at least 95% identical to SEQ ID NO: 4; at least 96% identical to SEQ ID NO: 4; at least 97% identical to SEQ ID NO: 4; at least 98% identical to SEQ ID NO: 4; or at least 99% identical to SEQ ID NO: 4.

[0040] In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 5, or a sequence having at least 70% identity to the above sequence. For example, the exogenous nucleic acid may include sequences having at least 75% identity to SEQ ID NO: 5; at least 80% identity to SEQ ID NO: 5; at least 85% identity to SEQ ID NO: 5; at least 90% identity to SEQ ID NO: 5; at least 91% identity to SEQ ID NO: 5; at least 92% identity to SEQ ID NO: 5; at least 93% identity to SEQ ID NO: 5; at least 94% identity to SEQ ID NO: 5; at least 95% identity to SEQ ID NO: 5; at least 96% identity to SEQ ID NO: 5; at least 97% identity to SEQ ID NO: 5; at least 98% identity to SEQ ID NO: 5; or at least 99% identity to SEQ ID NO: 5. This particular exogenous nucleic acid is expected to be particularly successful in improving N-glycan occupancy due to its broad substrate specificity and expression.

[0041] In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 6, or a sequence that is at least 70% identical to the above sequence. For example, the exogenous nucleic acid may include sequences that are at least 75% identical to SEQ ID NO: 6; at least 80% identical to SEQ ID NO: 6; at least 85% identical to SEQ ID NO: 6; at least 90% identical to SEQ ID NO: 6; at least 91% identical to SEQ ID NO: 6; at least 92% identical to SEQ ID NO: 6; at least 93% identical to SEQ ID NO: 6; at least 94% identical to SEQ ID NO: 6; at least 95% identical to SEQ ID NO: 6; at least 96% identical to SEQ ID NO: 6; at least 97% identical to SEQ ID NO: 6; at least 98% identical to SEQ ID NO: 6; or at least 99% identical to SEQ ID NO: 6. In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 7, or a sequence that is at least 70% identical to the above sequence. For example, the exogenous nucleic acid may include sequences that are at least 75% identical to SEQ ID NO: 7; at least 80% identical to SEQ ID NO: 7; at least 85% identical to SEQ ID NO: 7; at least 90% identical to SEQ ID NO: 7; at least 91% identical to SEQ ID NO: 7; at least 92% identical to SEQ ID NO: 7; at least 93% identical to SEQ ID NO: 7; at least 94% identical to SEQ ID NO: 7; at least 95% identical to SEQ ID NO: 7; at least 96% identical to SEQ ID NO: 7; at least 97% identical to SEQ ID NO: 7; at least 98% identical to SEQ ID NO: 7; or at least 99% identical to SEQ ID NO: 7.

[0042] In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 8, or a sequence that is at least 70% identical to the above sequence. For example, the exogenous nucleic acid may include sequences that are at least 75% identical to SEQ ID NO: 8; at least 80% identical to SEQ ID NO: 8; at least 85% identical to SEQ ID NO: 8; at least 90% identical to SEQ ID NO: 8; at least 91% identical to SEQ ID NO: 8; at least 92% identical to SEQ ID NO: 8; at least 93% identical to SEQ ID NO: 8; at least 94% identical to SEQ ID NO: 8; at least 95% identical to SEQ ID NO: 8; at least 96% identical to SEQ ID NO: 8; at least 97% identical to SEQ ID NO: 8; at least 98% identical to SEQ ID NO: 8; or at least 99% identical to SEQ ID NO: 8. In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 9, or a sequence that is at least 70% identical to the above sequence. For example, the exogenous nucleic acid may include sequences that are at least 75% identical to SEQ ID NO: 9; at least 80% identical to SEQ ID NO: 9; at least 85% identical to SEQ ID NO: 9; at least 90% identical to SEQ ID NO: 9; at least 91% identical to SEQ ID NO: 9; at least 92% identical to SEQ ID NO: 9; at least 93% identical to SEQ ID NO: 9; at least 94% identical to SEQ ID NO: 9; at least 95% identical to SEQ ID NO: 9; at least 96% identical to SEQ ID NO: 9; at least 97% identical to SEQ ID NO: 9; at least 98% identical to SEQ ID NO: 9; or at least 99% identical to SEQ ID NO: 9.

[0043] In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 10, or a sequence that is at least 70% identical to the above sequence. For example, the exogenous nucleic acid may include sequences that are at least 75% identical to SEQ ID NO: 10; at least 80% identical to SEQ ID NO: 10; at least 85% identical to SEQ ID NO: 10; at least 90% identical to SEQ ID NO: 10; at least 91% identical to SEQ ID NO: 10; at least 92% identical to SEQ ID NO: 10; at least 93% identical to SEQ ID NO: 10; at least 94% identical to SEQ ID NO: 10; at least 95% identical to SEQ ID NO: 10; at least 96% identical to SEQ ID NO: 10; at least 97% identical to SEQ ID NO: 10; at least 98% identical to SEQ ID NO: 10; or at least 99% identical to SEQ ID NO: 10.

[0044] In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 11, or a sequence that is at least 70% identical to the above sequence. For example, the exogenous nucleic acid may include sequences that are at least 75% identical to SEQ ID NO: 11; at least 80% identical to SEQ ID NO: 11; at least 85% identical to SEQ ID NO: 11; at least 90% identical to SEQ ID NO: 11; at least 91% identical to SEQ ID NO: 11; at least 92% identical to SEQ ID NO: 11; at least 93% identical to SEQ ID NO: 11; at least 94% identical to SEQ ID NO: 11; at least 95% identical to SEQ ID NO: 11; at least 96% identical to SEQ ID NO: 11; at least 97% identical to SEQ ID NO: 11; at least 98% identical to SEQ ID NO: 11; or at least 99% identical to SEQ ID NO: 11.

[0045] In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 12, or a sequence that is at least 70% identical to the above sequence. For example, the exogenous nucleic acid may include a sequence that is at least 75% identical to SEQ ID NO: 12; at least 80% identical to SEQ ID NO: 12; at least 85% identical to SEQ ID NO: 12; at least 90% identical to SEQ ID NO: 12; at least 91% identical to SEQ ID NO: 12; at least 92% identical to SEQ ID NO: 12; at least 93% identical to SEQ ID NO: 12; at least 94% identical to SEQ ID NO: 12; at least 95% identical to SEQ ID NO: 12; at least 96% identical to SEQ ID NO: 12; at least 97% identical to SEQ ID NO: 12; at least 98% identical to SEQ ID NO: 12; or at least 99% identical to SEQ ID NO: 12.

[0046] In a preferred embodiment, the exogenous nucleic acid has the sequence of SEQ ID NO: 13, or a sequence that is at least 70% identical to the above sequence. For example, the exogenous nucleic acid may include sequences that are at least 75% identical to SEQ ID NO: 13; at least 80% identical to SEQ ID NO: 13; at least 85% identical to SEQ ID NO: 13; at least 90% identical to SEQ ID NO: 13; at least 91% identical to SEQ ID NO: 13; at least 92% identical to SEQ ID NO: 13; at least 93% identical to SEQ ID NO: 13; at least 94% identical to SEQ ID NO: 13; at least 95% identical to SEQ ID NO: 13; at least 96% identical to SEQ ID NO: 13; at least 97% identical to SEQ ID NO: 13; at least 98% identical to SEQ ID NO: 13; or at least 99% identical to SEQ ID NO: 13.

[0047] The three OST paralogs expressed in T. bursey possess distinct substrate specificities. Analysis of parasitic protein glycosylation revealed that TbSTT3A selectively transfers bibranched Man5GlcNAc2 glycan, while both TbSTT3B and TbSTT3C transfer ternary branched Man9GlcNAc2 glycan. This substrate specificity is suggested to stem from the presence of an ALG12-dependent c-branch of conventional ternary branched Man9GlcNAc2, which is required for TbSTT3B and TbSTT3C but not for TbSTT3A. However, studies have also reported that both TbSTT3A and TbSTT3B can transfer Man5GlcNAc2 and Man7GlcNAc2 glycan to the T. bursey VSG protein. The unique functionality of TbSTT3B, given its diverse substrate specificities, is therefore reasonable. Most OTases exhibit distinct preferences for oligosaccharides, thereby limiting the efficiency of N-glycosylation. In fact, N-glycosylation can be increased in mammalian cells by utilizing adaptable OTases that can transfer N-glycans to virtually any amino acid sequence without bias. Therefore, in alternative embodiments of the present invention, it is desirable that the T. brusey OST enzymes be provided in combinations that utilize their unique specificity and functionality.

[0048] Therefore, in an alternative preferred embodiment, the mammalian cell may contain two exogenous nucleic acids in between that encode at least the active regions of TbSTT3B and TbSTT3C. For example, the exogenous nucleic acids may include the sequence according to SEQ ID NO: 4, or a sequence having at least 70% identity to the above sequence, and a further sequence according to SEQ ID NO: 6, or a sequence having at least 70% identity to the above sequence. For example, an exogenous nucleic acid may contain sequences that are at least 75% identical to SEQ ID NO: 4 and / or 6; at least 80% identical to SEQ ID NO: 4 and / or 6; at least 85% identical to SEQ ID NO: 4 and / or 6; at least 90% identical to SEQ ID NO: 4 and / or 6; at least 91% identical to SEQ ID NO: 4 and / or 6; at least 92% identical to SEQ ID NO: 4 and / or 6; at least 93% identical to SEQ ID NO: 4 and / or 6; at least 94% identical to SEQ ID NO: 4 and / or 6; at least 95% identical to SEQ ID NO: 4 and / or 6; at least 96% identical to SEQ ID NO: 4 and / or 6; at least 97% identical to SEQ ID NO: 4 and / or 6; at least 98% identical to SEQ ID NO: 4 and / or 6; or at least 99% identical to SEQ ID NO: 4 and / or 6.

[0049] In an alternative, preferred embodiment, the mammalian cell may comprise two exogenous nucleic acids encoding full-length TbSTT3B and TbSTT3C. For example, the exogenous nucleic acids may comprise the sequence according to SEQ ID NO: 10, or a sequence having at least 70% identity to the above sequence, and a further sequence according to SEQ ID NO: 12, or a sequence having at least 70% identity to the above sequence. For example, an exogenous nucleic acid may contain sequences that are at least 75% identical to sequence numbers 10 and / or 12; at least 80% identical to sequence numbers 10 and / or 12; at least 85% identical to sequence numbers 10 and / or 12; at least 90% identical to sequence numbers 10 and / or 12; at least 91% identical to sequence numbers 10 and / or 12; at least 92% identical to sequence numbers 10 and / or 12; at least 93% identical to sequence numbers 10 and / or 12; at least 94% identical to sequence numbers 10 and / or 12; at least 95% identical to sequence numbers 10 and / or 12; at least 96% identical to sequence numbers 10 and / or 12; at least 97% identical to sequence numbers 10 and / or 12; at least 98% identical to sequence numbers 10 and / or 12; or at least 99% identical to sequence numbers 10 and / or 12.

[0050] In an alternative, preferred embodiment, the mammalian cell may comprise two exogenous nucleic acids encoding at least the active regions of TbSTT3A and TbSTT3B. For example, the exogenous nucleic acids may comprise the sequence according to Sequence ID No. 2, or a sequence having at least 70% identity to the above sequence, and a further sequence according to Sequence ID No. 4, or a sequence having at least 70% identity to the above sequence. For example, an exogenous nucleic acid may contain sequences that are at least 75% identical to sequence numbers 2 and / or 4; at least 80% identical to sequence numbers 2 and / or 4; at least 85% identical to sequence numbers 2 and / or 4; at least 90% identical to sequence numbers 2 and / or 4; at least 91% identical to sequence numbers 2 and / or 4; at least 92% identical to sequence numbers 2 and / or 4; at least 93% identical to sequence numbers 2 and / or 4; at least 94% identical to sequence numbers 2 and / or 4; at least 95% identical to sequence numbers 2 and / or 4; at least 96% identical to sequence numbers 2 and / or 4; at least 97% identical to sequence numbers 2 and / or 4; at least 98% identical to sequence numbers 2 and / or 4; or at least 99% identical to sequence numbers 2 and / or 4.

[0051] In an alternative, preferred embodiment, the mammalian cell may comprise two exogenous nucleic acids encoding full-length TbSTT3A and TbSTT3B. For example, the mammalian cell may comprise two exogenous nucleic acids, which may comprise the sequence according to Sequence ID No. 8, or a sequence having at least 70% identity to the above sequence, and a further sequence according to Sequence ID No. 10, or a sequence having at least 70% identity to the above sequence. For example, an exogenous nucleic acid may contain sequences that are at least 75% identical to sequence number 8 and / or 10; at least 80% identical to sequence number 8 and / or 10; at least 85% identical to sequence number 8 and / or 10; at least 90% identical to sequence number 8 and / or 10; at least 91% identical to sequence number 8 and / or 10; at least 92% identical to sequence number 8 and / or 10; at least 93% identical to sequence number 8 and / or 10; at least 94% identical to sequence number 8 and / or 10; at least 95% identical to sequence number 8 and / or 10; at least 96% identical to sequence number 8 and / or 10; at least 97% identical to sequence number 8 and / or 10; at least 98% identical to sequence number 8 and / or 10; or at least 99% identical to sequence number 8 and / or 10.

[0052] In an alternative, preferred embodiment, the mammalian cell may comprise two exogenous nucleic acids encoding at least the active regions of TbSTT3A and TbSTT3C. For example, the mammalian cell may comprise two exogenous nucleic acids, which may comprise the sequence according to Sequence ID No. 2, or a sequence having at least 70% identity to the above sequence, and a further sequence according to Sequence ID No. 6, or a sequence having at least 70% identity to the above sequence. For example, an exogenous nucleic acid may contain sequences that are at least 75% identical to sequence numbers 2 and / or 6; at least 80% identical to sequence numbers 2 and / or 6; at least 85% identical to sequence numbers 2 and / or 6; at least 90% identical to sequence numbers 2 and / or 6; at least 91% identical to sequence numbers 2 and / or 6; at least 92% identical to sequence numbers 2 and / or 6; at least 93% identical to sequence numbers 2 and / or 6; at least 94% identical to sequence numbers 2 and / or 6; at least 95% identical to sequence numbers 2 and / or 6; at least 96% identical to sequence numbers 2 and / or 6; at least 97% identical to sequence numbers 2 and / or 6; at least 98% identical to sequence numbers 2 and / or 6; or at least 99% identical to sequence numbers 2 and / or 6.

[0053] In an alternative, preferred embodiment, the mammalian cell may comprise two exogenous nucleic acids encoding full-length TbSTT3A and TbSTT3C. For example, the exogenous nucleic acids may comprise the sequence according to Sequence ID No. 8, or a sequence having at least 70% identity to the above sequence, and a further sequence according to Sequence ID No. 12, or a sequence having at least 70% identity to the above sequence. For example, an exogenous nucleic acid may contain sequences that are at least 75% identical to sequence number 8 and / or 12; at least 80% identical to sequence number 8 and / or 12; at least 85% identical to sequence number 8 and / or 12; at least 90% identical to sequence number 8 and / or 12; at least 91% identical to sequence number 8 and / or 12; at least 92% identical to sequence number 8 and / or 12; at least 93% identical to sequence number 8 and / or 12; at least 94% identical to sequence number 8 and / or 12; at least 95% identical to sequence number 8 and / or 12; at least 96% identical to sequence number 8 and / or 12; at least 97% identical to sequence number 8 and / or 12; at least 98% identical to sequence number 8 and / or 12; or at least 99% identical to sequence number 8 and / or 12.

[0054] In a more preferred embodiment, the mammalian cell may contain three exogenous nucleic acids encoding at least the active regions of TbSTT3A, TbSTT3B, and TbSTT3C. For example, the exogenous nucleic acids may include the sequence according to SEQ ID NO: 2, or a sequence having at least 70% identity to the above sequence, a further sequence according to SEQ ID NO: 4, or a sequence having at least 70% identity to the above sequence, and the sequence according to SEQ ID NO: 6, or a sequence having at least 70% identity to the above sequence. For example, an exogenous nucleic acid may contain sequences that are at least 75% identical to SEQ ID NOs: 2, 4 and / or 6; at least 80% identical to SEQ ID NOs: 2, 4 and / or 6; at least 85% identical to SEQ ID NOs: 2, 4 and / or 6; at least 90% identical to SEQ ID NOs: 2, 4 and / or 6; at least 91% identical to SEQ ID NOs: 2, 4 and / or 6; at least 92% identical to SEQ ID NOs: 2, 4 and / or 6; at least 93% identical to SEQ ID NOs: 2, 4 and / or 6; at least 94% identical to SEQ ID NOs: 2, 4 and / or 6; at least 95% identical to SEQ ID NOs: 2, 4 and / or 6; at least 96% identical to SEQ ID NOs: 2, 4 and / or 6; at least 97% identical to SEQ ID NOs: 2, 4 and / or 6; at least 98% identical to SEQ ID NOs: 2, 4 and / or 6, or at least 99% identical to SEQ ID NOs: 2, 4 and / or 6.

[0055] In an alternative, preferred embodiment, the mammalian cell may contain three exogenous nucleic acids encoding full-length TbSTT3A, TbSTT3B, and TbSTT3C. For example, the exogenous nucleic acids may include the sequence according to SEQ ID NO: 8, or a sequence that is at least 70% identical to the above sequence, a further sequence according to SEQ ID NO: 10, or a sequence that is at least 70% identical to the above sequence, and a further sequence according to SEQ ID NO: 12, or a sequence that is at least 70% identical to the above sequence. For example, an exogenous nucleic acid may contain sequences that are at least 75% identical to sequence numbers 8, 10 and / or 12; at least 80% identical to sequence numbers 8, 10 and / or 12; at least 85% identical to sequence numbers 8, 10 and / or 12; at least 90% identical to sequence numbers 8, 10 and / or 12; at least 91% identical to sequence numbers 5 and / or 3; at least 92% identical to sequence numbers 8, 10 and / or 12; at least 93% identical to sequence numbers 8, 10 and / or 12; at least 94% identical to sequence numbers 8, 10 and / or 12; at least 95% identical to sequence numbers 8, 10 and / or 12; at least 96% identical to sequence numbers 8, 10 and / or 12; at least 97% identical to sequence numbers 8, 10 and / or 12; at least 98% identical to sequence numbers 8, 10 and / or 12; or at least 99% identical to sequence numbers 8, 10 and / or 12.

[0056] It will be readily apparent to those skilled in the art that any of the above combinations can be used with optimized versions of the sequences disclosed herein. For example, a mammalian cell may contain two optimized exogenous nucleic acids encoding at least one active region of TbSTT3A, TbSTT3B, and / or TbSTT3C, and thus contain or have sequences such as SEQ ID NOs. 5 and / or 7; 3 and / or 5; 3 and / or 7; or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to them. Alternatively, mammalian cells may contain three optimized exogenous nucleic acids encoding any of the active regions of TbSTT3A, TbSTT3B, and TbSTT3C, and thus contain or have sequences with at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to them. Similarly, mammalian cells may contain or have two optimized exogenous nucleic acids encoding any of the full lengths of TbSTT3A, TbSTT3B, and / or TbSTT3C, and thus sequences according to SEQ ID NOs. 11 and / or 13; 9 and / or 11; 9 and / or 13; or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to them.Alternatively, mammalian cells may contain three optimized exogenous nucleic acids encoding the full lengths of any TbSTT3A, TbSTT3B, and TbSTT3C, and thus the sequences according to SEQ ID NOs. 9, 11, and / or 13; or contain or have sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to them. It will also be readily apparent to those skilled in the art that any combination of DNA sequences disclosed herein, including combinations of optimized and unoptimized nucleic acid sequences, can be used for the purposes of the present invention.

[0057] Furthermore, it will be readily apparent to those skilled in the art that any nucleic acid sequence encoding an active region disclosed herein (whether optimized or unoptimized) can be combined with any nucleic acid sequence encoding a full-length protein disclosed herein (whether optimized or unoptimized). For example, if two exogenous nucleic acids are present, the nucleic acid sequences may include any of the sequences according to SEQ ID NOs. 2, 3, 8 and / or 9, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with them, and may further include any of the sequences according to SEQ ID NOs. 4, 5, 10 and / or 11, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with them.

[0058] Furthermore, the nucleic acid sequence may include any sequence according to SEQ ID NOs. 2, 3, 8 and / or 9 or any sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to them, and may further include any sequence according to SEQ ID NOs. 6, 7, 12 and / or 13 or any sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to them.

[0059] Furthermore, the nucleic acid sequence may include any of the sequences according to SEQ ID NOs. 4, 5, 10 and / or 11, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to them, and may further include any of the sequences according to SEQ ID NOs. 6, 7, 12 and / or 13, or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to them.

[0060] If three exogenous nucleic acids are present, the nucleic acid sequences may include any of the sequences according to SEQ ID NOs. 2, 3, 8 and / or 9 or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to them, or sequences according to SEQ ID NOs. 4, 5, 10 and / or 11 or sequences having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 99% sequence identity to them. It may further include any sequence having 1%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity, and may further include any sequence according to sequence numbers 6, 7, 12 and / or 13, or any sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to them.

[0061] In a fourth aspect, the present invention provides an isolated nucleic acid molecule comprising the sequence of any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21 and / or 22, or a sequence having at least 70% identity to them, preferably an isolated nucleic acid molecule comprising the sequence of SEQ ID NOs: 3, 5, 7, 9, 11 and / or 13. Thus, in a preferred embodiment, the isolated nucleic acid molecule comprises the sequence of SEQ ID NOs: 3, 5, 7, 9, 11 and / or 13 (i.e., a sequence optimized for expression in mammalian cells such as CHO cells). It is assumed that the nucleic acid of the fourth aspect of the present invention can be successfully transfected into mammalian cells. In another embodiment, the nucleic acid comprises the sequence of SEQ ID NOs: 2, 4, 6, 8, 10 and / or 12 (i.e., a sequence not optimized for expression in mammalian cells such as CHO cells). Furthermore, it will be readily apparent to those skilled in the art that any combination of nucleic acid sequences disclosed herein, including combinations of optimized and unoptimized nucleic acid sequences, can be used for the purposes of the present invention. Furthermore, it will be readily apparent to those skilled in the art that any nucleic acid sequence encoding an active region disclosed herein (whether optimized or unoptimized) can be combined with any nucleic acid sequence encoding a full-length protein disclosed herein (whether optimized or unoptimized).

[0062] Accordingly, in a fifth aspect, the present invention provides a vector comprising at least one nucleic acid sequence according to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21 and / or 22, or an isolated nucleic acid molecule according to a fourth aspect of the present invention. A suitable vector is assumed to be designed to encode at least one nucleic acid comprising the sequences according to SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21 and / or 22, or having them, for transfection into mammalian cells. In one embodiment, the vector comprises at least one nucleic acid sequence according to SEQ ID NOs: 3, 5, 7, 9, 11 and / or 13 (i.e., a sequence optimized for expression in mammalian cells such as CHO cells). A vector according to a fourth aspect of the present invention is assumed to be successfully transfected into mammalian cells. In another embodiment, the vector comprises at least one nucleic acid sequence (i.e., a sequence not optimized for expression in mammalian cells such as CHO cells) according to SEQ ID NOs: 2, 4, 6, 8, 10, and / or 12. Furthermore, it will be readily apparent to those skilled in the art that any combination of DNA sequences disclosed herein, including combinations of optimized and unoptimized nucleic acid sequences, can be used for the purposes of the present invention. Suitable vectors may include, but are not limited to, plasmid-based expression vectors, bacterial artificial chromosome (BAC) vectors, or viral vectors (e.g., adenoviruses, retroviruses, lentiviruses). Methods for transfection into mammalian cells are common practice in the art and will be readily apparent to those skilled in the art. Suitable transfection methods may include, but are not limited to, electroporation, viral transfection, microinjection, and chemical transfection (e.g., reagent-based transfection).

[0063] The mammalian cells of the present invention may be any mammalian cells that can be successfully transfected with and express the parasite-derived OST disclosed herein. For example, the mammalian cells may be Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK21) cells, mouse myeloma cells (such as NS0 and Sp2 / 0), or human fetal kidney (such as HEK293) cells. In preferred embodiments, the mammalian cells are CHO cells or HEK293 cells. Both transient and stable mammalian cell lines can be used in the present invention. Preferably, the mammalian cells to be used are stable mammalian cells. More preferably, the mammalian cells to be used are stable CHO cells. Stable mammalian cells have advantages over transiently transfected mammalian cells in that they allow the genetic modification to be passed through subsequent generations of cells, enabling the genetically modified cells to maintain stable expression of the target gene over a long period of time.

[0064] The present invention discloses mammalian cells comprising at least one exogenous nucleic acid sequence encoding at least one OST derived from T. brusey. The exogenous nucleic acid sequence can be expressed by mammalian cells and may provide a functional protein. Expression of the nucleic acid sequence in mammalian cells can be achieved by inductive expression, constitutive expression, stable expression, and transient expression. In one embodiment, mammalian cells may express one or more recombinant proteins encoded by at least one T. brusey OST enzyme and further exogenous nucleic acid sequences. The recombinant protein is a protein of interest that is to be glycosylated by the T. brusey OST enzyme. As will be understood by those skilled in the art, the recombinant protein may be any protein or peptide containing amino acid residues suitable for N-glycosylation. For example, the recombinant protein may be a therapeutic protein or peptide, a diagnostic protein or peptide, a protein or peptide for research and development purposes and / or a protein or peptide for supplementing growth media, which requires or benefits from N-glycosylation in mammalian cells to improve its yield, activity, stability, and / or reproducibility.

[0065] Recombinant proteins can be any protein that can be produced using the mammalian expression systems disclosed herein. An important advantage of the present invention is that its application is not limited to a few recombinant proteins, but can be applied to any recombinant protein of interest. For example, recombinant proteins can be therapeutic proteins. Examples of therapeutic proteins include, but are not limited to, hormones, cytokines, antibodies, enzymes, complement proteins, blood coagulation factors, functional fragments thereof, or any combination thereof. Antibodies can be monoclonal or polyclonal antibodies. Antibodies can be derived from the following classes and subclasses: IgG including the IgG1, IgG2, IgG3, and IgG4 subclasses, IgA including the IgA1 and IgA2 subclasses, IgM, IgD, and IgE. Antibodies can be humanized monoclonal antibodies. Antibodies can be bispecific antibodies.

[0066] When the term “functional fragment” is used in reference to the OST protein in any embodiment of the present invention, or in reference to the recombinant protein or any other protein referred herein, it refers to a polypeptide derived from a longer polypeptide, e.g., a full-length polypeptide, which is cleaved at the N-terminal and / or C-terminal regions to produce a fragment of the full-length polypeptide. To be a functional fragment, the fragment must retain at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and most preferably at least 100% of the activity of the full-length / mature polypeptide.

[0067] In preferred embodiments, the recombinant protein may be a protein selected from the group including erythropoietin (EPO), retuximab, butyrylcholinesterase (BuChE), factor VIII, ENPP1-Fc, Olamkicept (sgp130-Fc), GM-CSF, FSH, eCG, alpha-1-antitrypsin, viral glycoproteins, such as the SARS-CoV-2 spike protein, or any combination thereof. In alternative embodiments, mammalian cells may express at least one T. brusey OST enzyme alone, without recombinant protein. Such cells are presumably further modified to express any recombinant protein(s) of interest that require N-glycosylation by the T. brusey OST enzyme. Furthermore, the mammalian cells may be further modified to become increasingly suitable for the introduction of additional exogenous nucleic acid sequences encoding recombinant proteins. In a preferred embodiment, mammalian cells express OST proteins and provide functional / active OST proteins. Specifically, mammalian cells can produce at least one OST protein containing the amino acid sequence according to any one of SEQ ID NOs: 14, 15, 16, 17, 18, 19 and / or 20, or a sequence having at least 70% identity to the above sequence.

[0068] Mammalian cells may produce at least one OST protein containing an amino acid sequence or functional fragment thereof according to any one of sequence numbers 14, 15, and / or 16, or a sequence having at least 70% identity to the above sequence or fragment. Alternatively, mammalian cells may produce at least one OST protein containing an amino acid sequence or functional fragment thereof according to any one of sequence numbers 17, 18, and / or 19, or a sequence having at least 70% identity to the above sequence or fragment. Mammalian cells may produce at least one OST protein containing, or having, any combination of, the amino acid sequence or functional fragment thereof according to any one of sequence numbers 14, 15, 16, 17, 18, 19, and / or 20. Mammalian cells may contain a single OST protein, two OST proteins, or three OST proteins.

[0069] Therefore, in one embodiment, a mammalian cell may contain one OST protein. In a preferred embodiment, the OST protein has the sequence of SEQ ID NO: 14, or a sequence that is at least 70% identical to the above sequence. For example, the OST protein may have a sequence that is at least 75% identical to SEQ ID NO: 14; at least 80% identical to SEQ ID NO: 14; at least 85% identical to SEQ ID NO: 14; at least 90% identical to SEQ ID NO: 14; at least 91% identical to SEQ ID NO: 14; at least 92% identical to SEQ ID NO: 14; at least 93% identical to SEQ ID NO: 14; at least 94% identical to SEQ ID NO: 14; at least 95% identical to SEQ ID NO: 14; at least 96% identical to SEQ ID NO: 14; at least 97% identical to SEQ ID NO: 14; at least 98% identical to SEQ ID NO: 14; or at least 99% identical to SEQ ID NO: 14.

[0070] In a preferred embodiment, the OST protein has the sequence of SEQ ID NO: 15, or a sequence that is at least 70% identical to the above sequence. For example, the OST protein may have a sequence that is at least 75% identical to SEQ ID NO: 15; at least 80% identical to SEQ ID NO: 15; at least 85% identical to SEQ ID NO: 15; at least 90% identical to SEQ ID NO: 15; at least 91% identical to SEQ ID NO: 15; at least 92% identical to SEQ ID NO: 15; at least 93% identical to SEQ ID NO: 15; at least 94% identical to SEQ ID NO: 15; at least 95% identical to SEQ ID NO: 15; at least 96% identical to SEQ ID NO: 15; at least 97% identical to SEQ ID NO: 15; at least 98% identical to SEQ ID NO: 15; or at least 99% identical to SEQ ID NO: 15. In a preferred embodiment, the OST protein has the sequence of SEQ ID NO: 16, or a sequence that is at least 70% identical to the above sequence. For example, the OST protein may have a sequence that is at least 75% identical to SEQ ID NO: 16; at least 80% identical to SEQ ID NO: 16; at least 85% identical to SEQ ID NO: 16; at least 90% identical to SEQ ID NO: 16; at least 91% identical to SEQ ID NO: 16; at least 92% identical to SEQ ID NO: 16; at least 93% identical to SEQ ID NO: 16; at least 94% identical to SEQ ID NO: 16; at least 95% identical to SEQ ID NO: 16; at least 96% identical to SEQ ID NO: 16; at least 97% identical to SEQ ID NO: 16; at least 98% identical to SEQ ID NO: 16; or at least 99% identical to SEQ ID NO: 16.

[0071] In a preferred embodiment, the OST protein has the sequence of SEQ ID NO: 17, or a sequence that is at least 70% identical to the above sequence. For example, the OST protein may have a sequence that is at least 75% identical to SEQ ID NO: 17; at least 80% identical to SEQ ID NO: 17; at least 85% identical to SEQ ID NO: 17; at least 90% identical to SEQ ID NO: 17; at least 91% identical to SEQ ID NO: 17; at least 92% identical to SEQ ID NO: 17; at least 93% identical to SEQ ID NO: 17; at least 94% identical to SEQ ID NO: 17; at least 95% identical to SEQ ID NO: 17; at least 96% identical to SEQ ID NO: 17; at least 97% identical to SEQ ID NO: 17; at least 98% identical to SEQ ID NO: 17; or at least 99% identical to SEQ ID NO: 17. In a preferred embodiment, the OST protein has the sequence of SEQ ID NO: 18, or a sequence that is at least 70% identical to the above sequence. For example, the OST protein may have a sequence that is at least 75% identical to SEQ ID NO: 18; at least 80% identical to SEQ ID NO: 18; at least 85% identical to SEQ ID NO: 18; at least 90% identical to SEQ ID NO: 18; at least 91% identical to SEQ ID NO: 18; at least 92% identical to SEQ ID NO: 18; at least 93% identical to SEQ ID NO: 18; at least 94% identical to SEQ ID NO: 18; at least 95% identical to SEQ ID NO: 18; at least 96% identical to SEQ ID NO: 18; at least 97% identical to SEQ ID NO: 18; at least 98% identical to SEQ ID NO: 18; or at least 99% identical to SEQ ID NO: 18.

[0072] In a preferred embodiment, the OST protein has the sequence of SEQ ID NO: 19, or a sequence that is at least 70% identical to the above sequence. For example, the OST protein may have a sequence that is at least 75% identical to SEQ ID NO: 19; at least 80% identical to SEQ ID NO: 19; at least 85% identical to SEQ ID NO: 19; at least 90% identical to SEQ ID NO: 19; at least 91% identical to SEQ ID NO: 19; at least 92% identical to SEQ ID NO: 19; at least 93% identical to SEQ ID NO: 19; at least 94% identical to SEQ ID NO: 19; at least 95% identical to SEQ ID NO: 19; at least 96% identical to SEQ ID NO: 19; at least 97% identical to SEQ ID NO: 19; at least 98% identical to SEQ ID NO: 19; or at least 99% identical to SEQ ID NO: 19.

[0073] In a preferred embodiment, the OST protein has the sequence of SEQ ID NO: 20, or a sequence that is at least 70% identical to the above sequence. For example, the OST protein may have a sequence that is at least 75% identical to SEQ ID NO: 20; at least 80% identical to SEQ ID NO: 20; at least 85% identical to SEQ ID NO: 20; at least 90% identical to SEQ ID NO: 20; at least 91% identical to SEQ ID NO: 20; at least 92% identical to SEQ ID NO: 20; at least 93% identical to SEQ ID NO: 20; at least 94% identical to SEQ ID NO: 20; at least 95% identical to SEQ ID NO: 20; at least 96% identical to SEQ ID NO: 20; at least 97% identical to SEQ ID NO: 20; at least 98% identical to SEQ ID NO: 20; or at least 99% identical to SEQ ID NO: 20.

[0074] Mammalian cells may produce at least one OST protein containing or having any combination of amino acid sequences according to any one of sequence numbers 14, 15, 16, 17, 18, 19, and / or 20. Mammalian cells may contain a single OST protein, two OST proteins, or three OST proteins.

[0075] Therefore, in another embodiment, a mammalian cell may contain or express two separate OST proteins. For example, the OST protein may have sequences containing the active regions of TbSTT3B and TbSTT3C, and thus contain or have the sequence according to SEQ ID NO: 15, or a sequence having at least 70% identity to the above sequence, and a further sequence according to SEQ ID NO: 16, or a sequence having at least 70% identity to the above sequence. For example, an OST protein may have a sequence that is at least 75% identical to sequence number 15 and / or 16; at least 80% identical to sequence number 15 and / or 16; at least 85% identical to sequence number 15 and / or 16; at least 90% identical to sequence number 15 and / or 16; at least 91% identical to sequence number 15 and / or 16; at least 92% identical to sequence number 15 and / or 16; at least 93% identical to sequence number 15 and / or 16; at least 94% identical to sequence number 15 and / or 16; at least 95% identical to sequence number 15 and / or 16; at least 96% identical to sequence number 15 and / or 16; at least 97% identical to sequence number 15 and / or 16; at least 98% identical to sequence number 15 and / or 16; or at least 99% identical to sequence number 15 and / or 16.

[0076] In another embodiment, the OST protein may have sequences comprising full-length TbSTT3B and TbSTT3C, and thus comprises or has the sequence according to SEQ ID NO: 18, or a sequence having at least 70% identity to the above sequence, and a further sequence according to SEQ ID NO: 19, or a sequence having at least 70% identity to the above sequence. For example, an OST protein may have a sequence that is at least 75% identical to SEQ ID NO: 18 and / or 19; at least 80% identical to SEQ ID NO: 18 and / or 19; at least 85% identical to SEQ ID NO: 18 and / or 19; at least 90% identical to SEQ ID NO: 18 and / or 19; at least 91% identical to SEQ ID NO: 18 and / or 19; at least 92% identical to SEQ ID NO: 18 and / or 19; at least 93% identical to SEQ ID NO: 18 and / or 19; at least 94% identical to SEQ ID NO: 18 and / or 19; at least 95% identical to SEQ ID NO: 18 and / or 19; at least 96% identical to SEQ ID NO: 18 and / or 19; at least 97% identical to SEQ ID NO: 18 and / or 19; at least 98% identical to SEQ ID NO: 18 and / or 19; or at least 99% identical to SEQ ID NO: 18 and / or 19.

[0077] In another embodiment, the OST protein may have sequences containing the active regions of TbSTT3A and TbSTT3B, and therefore include or have the sequence according to SEQ ID NO: 14, or a sequence having at least 70% identity to the above sequence, and a further sequence according to SEQ ID NO: 15, or a sequence having at least 70% identity to the above sequence. For example, an OST protein may have a sequence that is at least 75% identical to SEQ ID NO: 14 and / or 15; at least 80% identical to SEQ ID NO: 14 and / or 15; at least 85% identical to SEQ ID NO: 14 and / or 15; at least 90% identical to SEQ ID NO: 14 and / or 15; at least 91% identical to SEQ ID NO: 14 and / or 15; at least 92% identical to SEQ ID NO: 14 and / or 15; at least 93% identical to SEQ ID NO: 14 and / or 15; at least 94% identical to SEQ ID NO: 14 and / or 15; at least 95% identical to SEQ ID NO: 14 and / or 15; at least 96% identical to SEQ ID NO: 14 and / or 15; at least 97% identical to SEQ ID NO: 14 and / or 15; at least 98% identical to SEQ ID NO: 14 and / or 15; or at least 99% identical to SEQ ID NO: 14 and / or 15.

[0078] In another embodiment, the OST protein may have sequences including full-length TbSTT3A and TbSTT3B, and thus include or have the sequence according to SEQ ID NO: 17, or a sequence having at least 70% identity to the above sequence, and a further sequence according to SEQ ID NO: 18, or a sequence having at least 70% identity to the above sequence. For example, an OST protein may have a sequence that is at least 75% identical to SEQ ID NO: 17 and / or 18; at least 80% identical to SEQ ID NO: 17 and / or 18; at least 85% identical to SEQ ID NO: 17 and / or 18; at least 90% identical to SEQ ID NO: 17 and / or 18; at least 91% identical to SEQ ID NO: 17 and / or 18; at least 92% identical to SEQ ID NO: 17 and / or 18; at least 93% identical to SEQ ID NO: 17 and / or 18; at least 94% identical to SEQ ID NO: 17 and / or 18; at least 95% identical to SEQ ID NO: 17 and / or 18; at least 96% identical to SEQ ID NO: 17 and / or 18; at least 97% identical to SEQ ID NO: 17 and / or 18; at least 98% identical to SEQ ID NO: 17 and / or 18; or at least 99% identical to SEQ ID NO: 17 and / or 18.

[0079] In another embodiment, the OST protein may have a sequence containing the active regions of TbSTT3A and TbSTT3C, and thus include or have the sequence according to SEQ ID NO: 14, or a sequence having at least 70% identity to the above sequence, and a further sequence according to SEQ ID NO: 16, or a sequence having at least 70% identity to the above sequence. For example, an OST protein may have a sequence that is at least 75% identical to SEQ ID NO: 14 and / or 16; at least 80% identical to SEQ ID NO: 14 and / or 16; at least 85% identical to SEQ ID NO: 14 and / or 16; at least 90% identical to SEQ ID NO: 14 and / or 16; at least 91% identical to SEQ ID NO: 14 and / or 16; at least 92% identical to SEQ ID NO: 14 and / or 16; at least 93% identical to SEQ ID NO: 14 and / or 16; at least 94% identical to SEQ ID NO: 14 and / or 16; at least 95% identical to SEQ ID NO: 14 and / or 16; at least 96% identical to SEQ ID NO: 14 and / or 16; at least 97% identical to SEQ ID NO: 14 and / or 16; at least 98% identical to SEQ ID NO: 14 and / or 16; or at least 99% identical to SEQ ID NO: 14 and / or 16.

[0080] In another embodiment, the OST protein may have sequences including full-length TbSTT3A and TbSTT3C, and thus include or have the sequence according to SEQ ID NO: 17, or a sequence having at least 70% identity to the above sequence, and a further sequence according to SEQ ID NO: 19, or a sequence having at least 70% identity to the above sequence. For example, an OST protein may have a sequence that is at least 75% identical to sequence number 17 and / or 19; at least 80% identical to sequence number 17 and / or 19; at least 85% identical to sequence number 17 and / or 19; at least 90% identical to sequence number 17 and / or 19; at least 91% identical to sequence number 17 and / or 19; at least 92% identical to sequence number 17 and / or 19; at least 93% identical to sequence number 17 and / or 19; at least 94% identical to sequence number 17 and / or 19; at least 95% identical to sequence number 17 and / or 19; at least 96% identical to sequence number 17 and / or 19; at least 97% identical to sequence number 17 and / or 19; at least 98% identical to sequence number 17 and / or 19; or at least 99% identical to sequence number 17 and / or 19.

[0081] To avoid any doubt, if a cell contains nucleic acids encoding more than one OST protein as described herein, these sequences are not necessarily located on the same nucleic acid molecule or construct. Currently, nucleic acids encoding OST proteins reside on separate nucleic acid molecules or gene constructs used for transient and stable expression of OST proteins. Nevertheless, the possibility of having more than one OST protein encoding nucleic acids on the same nucleic acid molecule is not excluded by the disclosure herein, and the disclosure herein also includes the co-expression of multiple OST molecules or multiple expression cassettes as fusion proteins within the same vector, or as proteins separated by enzymatically cleavable sequences such as self-cleaving peptide sequences (e.g., viral P2A sequences) or sequences susceptible to protease-mediated degradation. It will be readily apparent to those skilled in the art that any of the amino acid sequences containing the active region can be combined with any of the amino acid sequences corresponding to a full-length protein.

[0082] Mammalian cells may produce OST proteins containing or having any one of sequence numbers 14, 15, 16, 17, 18, 19, and / or 20, or any combination thereof. Mammalian cells may contain a single OST protein, two OST proteins, or three OST proteins.

[0083] Therefore, in one embodiment, a mammalian cell may contain three distinct OST proteins. For example, the OST protein may have sequences containing the proposed polypeptide binding sites TbSTT3A, TbSTT3B, and TbSTT3C, and therefore contain or have the sequence according to SEQ ID NO: 14, or a sequence having at least 70% identity to the above sequence, and a further sequence according to SEQ ID NO: 15, or a sequence having at least 70% identity to the above sequence, and a further sequence according to SEQ ID NO: 16, or a sequence having at least 70% identity to the above sequence. For example, the OST protein is at least 75% identical to SEQ ID NOs: 14, 15 and / or 16; at least 80% identical to SEQ ID NOs: 14, 15 and / or 16; at least 85% identical to SEQ ID NOs: 14, 15 and / or 16; at least 90% identical to SEQ ID NOs: 14, 15 and / or 16; at least 91% identical to SEQ ID NOs: 14, 15 and / or 16; at least 92% identical to SEQ ID NOs: 14, 15 and / or 16 It may have sequences that are at least 93% identical to; at least 94% identical to sequence numbers 14, 15 and / or 16; at least 95% identical to sequence numbers 14, 15 and / or 16; at least 96% identical to sequence numbers 14, 15 and / or 16; at least 97% identical to sequence numbers 14, 15 and / or 16; at least 98% identical to sequence numbers 14, 15 and / or 16; or sequences that are at least 99% identical to sequence numbers 14, 15 and / or 16.

[0084] In another embodiment, the OST protein may have sequences including full-length TbSTT3A, TbSTT3B and TbSTT3C, and thus include or have sequences according to SEQ ID NO: 17, or sequences having at least 70% identity to the above sequence, and further sequences according to SEQ ID NO: 18, or sequences having at least 70% identity to the above sequence, and further sequences according to SEQ ID NO: 19, or sequences having at least 70% identity to the above sequence. For example, the OST protein may have at least 75% identity to SEQ ID NOs: 17, 18 and / or 19; at least 80% identity to SEQ ID NOs: 17, 18 and / or 19; at least 85% identity to SEQ ID NOs: 17, 18 and / or 19; at least 90% identity to SEQ ID NOs: 17, 18 and / or 19; at least 91% identity to SEQ ID NOs: 17, 18 and / or 19; at least 92% identity to SEQ ID NOs: 17, 18 and / or 19 It may have sequences that are at least 93% identical to sequence 17, 18 and / or 19; at least 94% identical to sequence numbers 17, 18 and / or 19; at least 95% identical to sequence numbers 17, 18 and / or 19; at least 96% identical to sequence numbers 17, 18 and / or 19; at least 97% identical to sequence numbers 17, 18 and / or 19; at least 98% identical to sequence numbers 17, 18 and / or 19; or sequences that are at least 99% identical to sequence numbers 17, 18 and / or 19. It will be readily apparent to those skilled in the art that any of the amino acid sequences containing the proposed polypeptide binding site (a highly conserved region) can be combined with any of the amino acid sequences corresponding to a full-length protein.

[0085] The native gene sequence from T. brusey that encodes the OST enzyme has been codon-optimized for recombinant protein production in CHO cells. As will be readily apparent to those skilled in the art, codon optimization is a genetic engineering tool performed to improve gene expression and protein production. Codon optimization attempts to address the problem of codon bias, which refers to synonymous codons that are used more frequently than other synonymous codons within a region of a transcribed gene. The frequencies of these synonymous codons vary within and between species. Codon optimization modifies the gene sequence to adapt to the codon bias of the host organism without altering the amino acid sequence in order to increase protein production. The GenScript codon optimization tool was used. Thus, while this invention discloses a codon-optimized sequence for recombinant protein production in CHO cells, those skilled in the art will understand that the same can be done for recombinant protein production in other mammalian cells, including HEK cells (such as HEK293 cells).

[0086] By SEQ ID NOs. 3, 5, 7, 9, 11, and / or 13 disclosed herein, the inventors of the present invention have developed nucleic acid sequences (by SEQ ID NOs. 14, 15, 16, 17, 8, and / or 19) that encode the T. brusey OST protein, with codons optimized for production in mammalian cells.

[0087] In a sixth aspect, the present invention provides a method for modifying the glycosylation profile of a recombinant protein, comprising contacting the recombinant protein with (i) a mammalian cell according to a first aspect of the present invention, or (ii) an OST protein or a functional fragment thereof, wherein the OST protein is a trypanosoma OST protein, preferably a trypanosoma brusey OST protein, and more preferably the OST protein comprises the amino acid sequence of any of SEQ ID NOs: 14, 15, 16, 17, 18, 19 and / or 20, or producing the recombinant protein in a mammalian cell according to a first aspect of the present invention, wherein the mammalian cell is manipulated to express the recombinant protein.

[0088] The TbSTT3 protein targets certain proteins through the recognition of a consensus region on the target by the TbSTT3 active domain. The TbSTT3 protein exhibits polypeptide specificity for certain acceptor substrates, and the active domain involved in peptide acceptor specificity by TbSTT3 is predicted to be located within a region spanning amino acid positions 371-408 for TbSTT3A, TbSTT3B, and TbSTT3C, respectively (Figure 1). In particular, arginine-397 in TbSTT3A and TbSTT3C, and histidine-397 in TbSTT3B are known to interact with NX(S / T) adjacent amino acids, influencing the interaction between the acceptor peptide and the enzyme surface (Jinnelov et al., 2017). These interactions can enhance the efficiency of substrate recognition, and therefore, this region is important for the specific function of the TbSTT3 protein. The residues arginine-397 and histidine-397 in TbSTT3 are both positively charged amino acids. While we do not wish to be constrained by theory, potentially, the positively charged active region helps define TbSTT3 function by promoting specific sequence preference or polypeptide acceptor specificity. Therefore, this unique property of the TbSTT3 protein may be particularly useful for biotechnology platforms to enhance the efficient N-glycosylation of recombinant glycoproteins in eukaryotic expression systems.

[0089] The three STT3 paralogs in T. brusey exhibit distinct substrate specificities. For example, TbSTT3A has been found to selectively transfer the bibranched Man5GlcNAc2 glycan, while both TbSTT3B and TbSTT3C transfer the tribranched Man9GlcNAc2 glycan (Jinnelov et al., 2017). The ability of TbSTT3 proteins to transfer the Man9GlnNAc2 moiety, which is a preferred substrate for mammalian OST proteins, is particularly important.

[0090] This invention discloses the genetic engineering of a highly efficient enzyme derived from a unicellular parasite (T. bruseyi) into a mammalian cell line to enhance the N-glycan occupancy of recombinant proteins. Accordingly, the methods disclosed herein provide a method in which the frequency of N-glycan occupancy on recombinant proteins can be altered. In preferred embodiments, the methods disclosed herein provide a method in which the frequency of N-glycan on recombinant proteins can be increased. However, increased sialylation and density of glycans on the surface of recombinant proteins may also be observed. In the context of this invention, the term "increased" refers to a comparison between the number of potential N-glycosylation sites (i.e., N-glycan occupancy) on recombinant proteins produced in a mammalian cell line having OST in a natural / unmodified form and the N-glycan occupancy on recombinant proteins produced in a mammalian cell line having OST obtained from the parasite disclosed herein.

[0091] As disclosed above, recombinant proteins involved in the method of modifying the glycosylation profile of recombinant proteins can be any protein that can be produced using the mammalian expression systems disclosed herein. For example, recombinant proteins can be therapeutic proteins. Examples of therapeutic proteins include, but are not limited to, hormones, cytokines, antibodies, enzymes, complement proteins, blood coagulation factors, functional fragments thereof, or any combination thereof. Antibodies can be monoclonal or polyclonal antibodies. Antibodies can be humanized antibodies. Antibodies can be bispecific antibodies. The term “functional fragment thereof” refers to a polypeptide derived from a longer polypeptide, e.g., a full-length polypeptide, which is cleaved at the N-terminal and / or C-terminal regions to produce a fragment of the full-length polypeptide. To be a functional fragment, the fragment must retain at least 40%, preferably at least 50%, more preferably at least 60%, more preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, most preferably at least 95%, and most most preferably at least 100% of the activity of the full-length / mature polypeptide.

[0092] In preferred embodiments, the recombinant protein may be a protein selected from the group including erythropoietin (EPO), retuximab, butyrylcholinesterase (BuChE), factor VIII, ENPP1-Fc, Olamkicept (sgp130-Fc), GM-CSF, FSH, eCG, alpha-1-antitrypsin, viral glycoproteins, such as the SARS-CoV-2 spike protein, or any combination thereof. The present invention is further described with reference to the following non-limiting embodiments: [Examples]

[0093] Expression of trypanosome OST in mammalian cells leads to improved yield of therapeutic targets and N-glycan moiety occupancy. ( Example 1): Results The inventors sought to study the effects of expressing OST derived from T. brusey in mammalian cells used for therapeutic bioproduction (Figure 2). Therefore, the study was conducted using a pre-validated suspension-adapted CHO cell line (CHO-EPO) engineered to stably express the recombinant hormone erythropoietin. Four OST enzymes derived from T. brusey were selected. Each OST was cloned into a mammalian expression vector for transient expression. The OST vectors were transfected into the CHO-EPO cell line, and the expression of OST and EPO was monitored. As a control, mock transfection using an empty mammalian expression vector was performed in the CHO-EPO cell line.

[0094] T. bruseyOST is expressed in mammalian cells. After transfection, cell number, viability, aggregation, and cell diameter were evaluated. To confirm the expression of related OSTs, cell pellets from each transiently transfected cell line were collected 72 hours after transfection and sent for proteomic analysis by mass spectrometry (MS) to identify the presence of OSTs. Related OSTs, as well as stably expressed EPO, were detected by MS in all transiently transfected cell lines (Figure 3). T. bruseyOST increases the yield of therapeutic proteins secreted by CHO cells. To investigate the effect of OST expression on the yield of therapeutic proteins produced by cells, total protein yield in the supernatant was measured after 3 days. This showed an increase in EPO production compared to mock-transfected cell lines upon introduction of each OST (Figure 4). For example, in the case of enzyme A (TbSTT3A), we observed a 2.75-fold increase in recombinant EPO production, demonstrating the predicted and intended effects of heterologous OST enzymes on improving protein secretion. T. bruseyOST increases the occupancy rate of the N-glycan site of EPO produced in CHO cells. Next, the inventors determined whether heterologous OST expression alters the N-glycosylation level of the secreted therapeutic protein. To this end, EPO was purified from OST-transfected cells and digested with PNGaseF for analysis of N-glycan moiety occupancy. The number of N-glycan moieties occupied on the EPO-derived peptide increased from 80% to 89% (Figure 5), demonstrating that OST expression can modify the N-glycosylation state of the therapeutic protein even when expressed transiently for a short period.

[0095] conclusion This study demonstrates that OST derived from T. brusey can be successfully expressed in mammalian cells, and that its presence increases both the yield and modification of therapeutic proteins produced in the cells. While this study focuses exclusively on EPO hormone production, similar findings are expected for the production of other therapeutic proteins in mammalian cells.

[0096] (Example 2): Method Transfection Cells were transfected according to a standard protocol. Briefly, the cells were washed in electroporation buffer, and 160 μg of total plasmid DNA was mixed with 40,000,000 CHO cells to a final volume of 400 μL in electroporation buffer. Electroporation was performed according to the manufacturer's instructions, and immediately after transfection, 400 μL of cells was transferred to a 125 mL Erlenmeyer flask and placed in a 37°C CO2 incubator. After 40 minutes, 30 mL of growth medium was added to the Erlenmeyer flask. Subsequently, the protein concentration in the supernatant was measured.

[0097] purification The cell pellet was collected for total proteome analysis. EPO was purified using an EPO purification kit. mass spectrometry Total proteome S-Trap treatment of the sample Cell pellets were dissolved in lysis buffer (100 mM TEAB, 5% SDS), sonicated three times to shear the DNA, and protein concentrations were estimated using a micro-BCA assay. 350 μg aliquots of protein were treated using the S-Trap miniprotocol. Protein disulfide bonds were reduced in the presence of 20 mM DTT, followed by alkylation in 40 mM IAA. After applying the sample to the S-Trap minispin column, five washes were performed with S-Trap binding buffer. Peptides were digested overnight with trypsin (1:40) at 37°C, the trypsin peptides were pooled, dried, and quantified. mass spectrometry A peptide (equivalent to 1.5 μg) was injected into a nanoscale C18 reversed-phase chromatography system and electrosprayed onto an Orbitrap mass spectrometer. The peptide was eluted from the column at a constant flow rate, and two blanks were performed between each sample to reduce carryover. The column was maintained at a constant temperature of 50°C. The MS was operated in DIA mode.

[0098] Study on the occupancy rate of N-glycan moieties on purified EPO PNGase treatment The purified EPO samples were mixed with 4 μl of PNGase buffer and dried in a speed vac. Then, 20 μl of heavy water was added to each sample, and the mixture was dried again. The dried PNGase F was resuspended in 10 μl of heavy water, vortexed for 3 seconds, and the contents were added to the sample. Another 10 μl of heavy water was added to the PNGase vial, vortexed for 2 seconds, and the contents were added to the sample. Subsequently, the mixture (20 μl) was incubated at 50°C for 20 minutes. Peptide digestion The sample was reduced using DTT and alkylated by adding 5 μl of iodoacetamide (final concentration 300 mM). Next, the sample and marker were flowed onto a Bis-Tris 4-12% gradient gel, washed, stained with Coomassie stain, the protein bands were excised, and the gel fragments were washed. Intragellation was performed using trypsin at a final concentration of 12 μg / mL (in 20 mM ammonium bicarbonate) and incubated on a shaker at 30°C for 16 hours. The peptide mixture was extracted by adding acetonitrile (an equal amount used to cover the gel piece) and shaking at 30°C for 15 minutes. The resulting supernatant was transferred to a fresh microcentrifuge tube. Peptides in the gel piece were further extracted by adding 5% formic acid, followed by 100% acetonitrile. The supernatant was collected and transferred to the first fraction. The gel piece was finally washed with acetonitrile for 10 minutes, and the three pooled fractions were dried in a speed-vac. LC-MS / MS analysis of N-glycan moiety occupancy Peptide readings were analyzed using a Q-exactive HF mass spectrometer connected to a Dionex Ultimate 3000 RS. Samples were reconstituted in 1% formic acid, and aliquots of each sample were loaded at 10 μL / min onto a trap column equilibrated in 0.1% TFA. Peptides were eluted from the column at a constant flow rate of 300 nl / min, and the column was maintained at a constant temperature of 50°C. The Q-exactive HF was operated in data-dependent positive ionization mode. Three blanks were run between each sample to reduce carryover and confirm mass accuracy before sample analysis began.

[0099] Database search, protein and peptide identification Raw MS data was searched against the human uniport proteome using the MASCOT search engine (Matrix Science, Version 2.6). Protein identifications and peptides were exported to Microsoft Excel.

[0100] (Example 3): Stable expression of OST enzyme in CHO-S cells Using bacterial artificial chromosomes (BACs) containing the T. brusey OST nucleic acid sequence, the OST gene was randomly incorporated into the CHO-S genome, resulting in stable expression of the T. brusey OST enzyme. Briefly, BACs were linearized and transfected into CHO-S cells using an Amaxa nucleofector. After 7 days of antibiotic selection, viable and dead cells were separated using fluorescence-assisted cell sorting (FACS). Single viable cells were isolated, and then single cell clones were recovered and propagated. The clones were monitored for cell viability, gene copy number, titer, and glycosylation pattern. Stable cell lines currently in production can be found in Table 1 below.

[0101] [Table 1]

[0102] Transient expression of OST enzyme in Expi293 cells We transiently expressed the OST enzyme in Expi293 cells derived from the HEK293 cell line using a mammalian expression plasmid containing the T. brusey OST nucleic acid sequence. In short, the plasmid was transfected into Expi293 cells using an Expifectamine reagent derived from the Expi293 expression line. Constitutive expression of the OST enzyme was performed for 6 days, during which time the viability of Expi293 cells was monitored (Figure 6). The results demonstrate that the expression of the OST cell line in the mammalian-derived Expi293 cell line does not adversely affect cell viability.

[0103] A sequence that forms part of the explanation Sequence ID 1 - Consensus DNA sequence of the active region of TbSTT3A CGGCGTCCGTGCGTTGTTCGTGAAACATACGCGTACCGGAAATCCCTCGTGGATTCTGTGGCTGAGCAT Sequence ID 2 - DNA sequence of the active region of TbSTT3A AAACCTACAGCGTACCGCGTCCGTGCGTTGTTCGTGAAACATACGCGTACCGGAAATCCCTCGTGGATTCTGTGGCTGAGCATCGGCCGACGACTGCCGGGGCGTATCTGCGCTACTTT Sequence ID 3 - DNA sequence of the active region of TbSTT3A (optimized CHO) AAGCCTACCGCCTACAGAGTGCGGGCCCTGTTTGTGAAGCACACCAGAACCGGCAATCCTCTGGTGGACTCTGTGGCCGAGCATAGACCTACAACCGCTGGCGCCTACCTGCGGTACTTT Sequence ID 4 - DNA sequence of the active region of TbSTT3B AGGCCGTTTTCTTCTCGTGTCCGTGCGTTGTTCGTGAAACATACGCGTACCGGAAATCCCTCGTGGATTCTGTGGCTGAGCACCATCCGGCGTCGAATGATGATTTCTTTGGTTACCTT

[0104] Sequence ID 5 - DNA sequence of the active region of TbSTT3B (optimized CHO) CGGCCTTTCAGCTCTAGAGTGCGGGCCCTGTTTGTGAAGCACACCAGAACCGGCAATCCTCTGGTGGACTCTGTGGCCGAGCACCATCCTGCCTCTAACGACGACTTCTTCGGCTACCTG Sequence ID 6 - DNA sequence of the active region of TbSTT3C AGGTCGTTTTCTTCTCGTGTCCGTGCGTTGTTCGTGAAACATACGCGTACCGGAAATCCCTCGTGGATTCTGTGGCTGAGCATCGGCCGACGACTGCCGGGGCCTTCCTACGTCATCTT Sequence ID 7 - DNA sequence of the active region of TbSTT3C (optimized CHO) CGGTCCTTCAGCTCTAGAGTGCGGGCCCTGTTTGTGAAGCACACCAGAACCGGCAATCCTCTGGTGGACTCTGTGGCCGAGCATCGGCCTACAACAGCTGGCGCCTTTCTGAGACATCTG Sequence ID 8 - DNA sequence of full-length TbSTT3A

[0105] Sequence ID 9 - Full-length DNA sequence of TbSTT3A (optimized CHO)

[0106] Sequence ID 10 - Full-length DNA sequence of TbSTT3B

[0107] Sequence ID 11 - Full-length TbSTT3B DNA sequence (optimized CHO)

[0108] Sequence ID 12 - DNA sequence of full-length TbSTT3C

[0109] Sequence ID 13 - Full-length TbSTT3C DNA sequence (optimized CHO)

[0110] Sequence ID 14 - Amino acid sequence of the active region of TbSTT3A FFKPTAYRVRALFVKHTRTGNPLVDSVAEHRPTTAGAYLRYF Sequence ID 15 - Amino acid sequence of the active region of TbSTT3B YFRPFSSRVRALFVKHTRTGNPLVDSVAEHHPASNDDFFGYL Sequence ID 16 - Amino acid sequence of the active region of TbSTT3C YFRPFSSRVRALFVKHTRTGNPLVDSVAEHRPTTAGAFLRHL

[0111] Sequence ID 17 - Amino acid sequence of full-length TbSTT3A MTKGGKVAVTKGSAQSDGAGEGGMSKAKSSTTFVATGGGSLPAWALKAVSTIVSAVILIYSVHRAYDIRLTSVRLYGELIHEFDPWFNYRATQYLSDNGWRAFFQWYDYMSWYPLGRPVGTTIFPGMQLTGVAIHRVLEMLGRGMSINNICVYIPAWFGSIATVLAALIAYESSNSLSVMAFTAYFFSIVPAHLMRSMAG EFDNECVAMAAMLLTFYMWVRSLRSSSSWPIGALAGVAYGYMVSTWGGYIFVLNMVAFHASVCVLLDWARGIYSVSLLRAYSLFFVIGTALAICVPPVEWTPFRSLEQLTALFVFVFMWALHYSEYLRERARAPIHSSKALQIRARIFMGTLSLLLIVASLLAPFGFFKPTAYRVRALFVKHTRTGNPLVDSVAEHRPTT AGAYLRYFHVCYPLWGCGGLSMLVFMKKDRWRAIVFLASLSTVTMYFSARMSRLLLLAGPAATACAGMFIGGLFDLALSQFGDLHSPKDASGDSDPAGGSKRAKGKVVNEPSKRAIFSHRWFQRLVQSLPVPLRRGIAVVVLVCLFANPMRHSFEKSCEKMAHALSSPRIIAVTDLPNGERVLADDYYVSYLWLRNNTPE DARILSWWDYGYQITGIGNRTTLADGNTWSHKHIATIGKMLTSPVKESHALIRHLADYVLIWAGEDRGDLLKSPHMARIGNSVYRDMCSEDDPRCRQFGFEGGDLNKPTPMQRSLLYNLHRFGTDGGKTQLDKNMFQLAYVSKYGLVKIYKVVNVSEESKAWVADPKNRVCDPPGSWICAGQYPPAKEIQDMLAKRFHYE

[0112] SEQ ID NO: 18 - Amino acid sequence of full-length TbSTT3B MTKGGKVAVTKGSAQSDGAGEGGMSKAKSSTTFVATGGGSLPAWALKAVSTVVSAVILIYSVHRAYDIRLTSVRLYGELIHEFDPWFNYRATQYLSDNGWRAFFQWYDYMSWYPLGRPVGTTIFPGMQLTGVAIHRVLEMLGRGMSINNICVYIPAWFGSIATVLAALIAYESSNSLSVMAFTAYFFSIVPAHLMRSMAGEFDNE CVAMAAMLLTFYMWVRSLRSSSSWPIGALAGVAYGYMVSTWGGYIFVLNMVAFHASVCVLLDWARGTYSVSLLRAYSLFFVIGTALAICVPPVEWTPFRSLEQLTALFVFVFMWALHYSEYLRERARAPIHSSKALQIRARIFMGTLSLLLIVAIYLFSTGYFRPFSSRVRALFVKHTRTGNPLVDSVAEHHPASNDDFFGYLHV CYNGWIIGFFFMSVSCFFHCTPGMSFLLLYSILAYYFSLKMSRLLLLSAPVASILTGYVVGSIVDLAADCFAASGTEHADSKEHQGKARGKGQKEQITVECG CHNPFYKLWCNSFSSRLVVGKFFVVVVLSICGPTFLGSNFRIYSEQFADSMSSPQIIMRATVGGRRVILDDYYVSYLWLRNNTPEDARILSWWDYGYQITGIG NRTTLADGNTWNHEHIATIGKMLTSPVKESHALIRHLADYVLIWAGYDGSDLLKSPHMARIGNSVYRDICSEDDPLCTQFGFYSGDFSKPTPMMQRSLLYNLHRFGTDGGKTQLDKNMFQLAYVSKYGLVKIYKVMNVSEESKAWVADPKNRKCDAPGSWICTGQYPPAKEIQDMLAKRIDYEQLEDFNRRNRSDAYYRAYMRQMG

[0113] SEQ ID NO: 19 - Amino acid sequence of the full-length TbSTT3C MTKGGKVAVTKGSAQSDGAGEGGMSKAKSSTTFVATGGGSLPAWALKAVSTVVSAVILIYSVHRAYDIRLTSVRLYGELIHEFDPWFNYRATQYLSDNGWRAFFQWYDYMSWYPLGRPVGTTIFPGMQLTGVAIHRVLEMLGRGMSINNICVYIPAWFGSIATVLAALIAYESSNSLSVMAFTAYFFSIVPAHLMRSMAGEFDNE CVAMAAMLLTFYMWVRSLRSSSSWPIGALAGVAYGYMVSTWGGYIFVLNMVAFHASVCVLLDWARGTYSVSLLRAYSLFFVIGTALAICVPPVEWTPFRSLEQLTALFVFVFMWALHYSEYLRERARAPIHSSKALQIRARIFMGTLSLLLIVAIYLFSTGYFRSFSSRVRALFVKHTRTGNPLVDSVAEHRPTTAGAFLRHLHV CYNGWIIGFFFMSVSCFFHCTPGMSFLLLYSILAYYFSLKMSRLLLLSAPVASILTGYVVGSIVDLAADCFAASGTEHADSKEHQGKARGKGQKRQITVECG CHNPFYKLWCNSFSSRLVVGKFFVVVVLSICGPTFLGSEFRAHCERFSVSVANPRIISSIRHSGKLVLADDYYVSYLWLRNNTPEDARILSWWDYGYQITGIG NRTTLADGNTWNHEHIATIGKMLTSPVKESHALIRHLADYVLIWAGEDRGDLRKSRHMARIGNSVYRDMCSEDDPLCTQFGFYSGDFNKPTPMQRSLLYNLHRFGTDGGKTQLDKNMFQLAYVSKYGLVKIYKVMNVSEESKAWVADPKNRKCDAPGSWICAGQYPPAKEIQDMLAKRIDYEQLEDFNRRNRSDAYYRAYMRQMG

[0114] Sequence ID No. 20 - Conserved amino acid residues in the active regions of TbSTT3A, B, and C RVRALFVKHTRTGNPLVDSVAEH Sequence ID 21 - Consensus DNA sequence of the active region of TbSTT3B CGTGTCCGTGCGTTGTTCGTGAAACATACGCGTACCGGAAATCCCTCGTGGATTCTGTGGCTGAGCAC Sequence ID 22 - Consensus DNA sequence of the active region of TbSTT3C CGTGTCCGTGCGTTGTTCGTGAAACATACGCGTACCGGAAATCCCTCGTGGATTCTGTGGCTGAGCAT

[0115] [Table 2] References

[0116] JPEG2026523077000003.jpg145123

Claims

1. A mammalian cell comprising at least one nucleic acid sequence encoding at least one oligosaccharide transferase (OST) protein or a functional fragment thereof, wherein the at least one OST protein is a Trypanosoma spp. OST protein, and preferably the at least one OST protein is a Trypanosoma brucei OST protein.

2. The mammalian cell according to claim 1, wherein the at least one nucleic acid sequence encoding the at least one OST protein or a functional fragment thereof includes the sequence according to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22, or a sequence having at least 70% sequence identity with respect to them.

3. A mammalian cell comprising at least one nucleic acid sequence encoding at least one oligosaccharide transferase (OST) protein or a functional fragment thereof, wherein the at least one nucleic acid sequence encoding the at least one OST protein or functional fragment comprises the sequence according to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22, or a sequence having at least 70% sequence identity with respect to those.

4. A mammalian cell comprising at least one oligosaccharide transferase (OST) protein or at least one nucleic acid sequence encoding a functional fragment thereof, wherein the at least one OST protein comprises an amino acid sequence according to any of SEQ ID NOs: 14, 15, 16, 17, 18, 19 and / or 20, or a sequence having at least 70% sequence identity thereto.

5. A mammalian cell according to any one of claims 1 to 4, wherein the at least one nucleic acid sequence encoding the at least one OST protein or a functional fragment thereof comprises the sequence according to SEQ ID NOs: 2, 3, 8 and / or 9, or a sequence having at least 70% identity thereto, and the cell further comprises a nucleic acid sequence encoding a further OST protein or a functional fragment thereof, the further nucleic acid sequence comprising the sequence according to SEQ ID NOs: 4, 5, 10 and / or 11, or a sequence having at least 70% identity thereto, and further comprising the sequence according to SEQ ID NOs: 6, 7, 12 and / or 13, or a sequence having at least 70% identity thereto.

6. A mammalian cell according to any one of claims 1 to 5, wherein the at least one nucleic acid sequence encoding the at least one OST protein or a functional fragment thereof comprises the sequence according to SEQ ID NOs: 4, 5, 10 and / or 11, or a sequence having at least 70% identity thereto, and the cell further comprises a nucleic acid sequence encoding a further OST protein or a functional fragment thereof, the further nucleic acid comprising the sequence according to SEQ ID NOs: 6, 7, 12 and / or 13, or a sequence having at least 70% identity thereto.

7. The mammalian cell according to any one of claims 1 to 4, wherein the at least one nucleic acid sequence encoding the at least one OST protein or a functional fragment thereof comprises the sequence according to SEQ ID NOs: 2, 3, 8 and / or 9, or a sequence having at least 70% identity thereto, and the cell further comprises a nucleic acid sequence encoding a further OST protein or a functional fragment thereof, wherein the further nucleic acid sequence comprises the sequence according to SEQ ID NOs: 4, 5, 10 and / or 11, or a sequence having at least 70% identity thereto.

8. A mammalian cell according to any one of claims 1 to 4, wherein the at least one nucleic acid sequence encoding the at least one OST protein or a functional fragment thereof comprises the sequence according to SEQ ID NOs: 2, 3, 8 and / or 9, or a sequence having at least 70% identity thereto, and the cell further comprises a nucleic acid sequence encoding a further OST protein or a functional fragment thereof, wherein the further nucleic acid sequence comprises the sequence according to SEQ ID NOs: 6, 7, 12 and / or 13, or a sequence having at least 70% identity thereto.

9. An isolated nucleic acid molecule comprising a sequence according to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22, or a sequence having at least 70% identity with them, preferably an isolated nucleic acid molecule comprising a sequence according to any of SEQ ID NOs: 3, 5, 7, 9, 11, and / or 13.

10. A nucleic acid vector comprising at least one nucleic acid sequence according to any of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 21, and / or 22, or at least one nucleic acid as described in claim 9, preferably selected from the group including, but not limited to, plasmid-based expression vectors, bacterial artificial chromosome (BAC) vectors, and viral vectors such as adenovirus vectors, adeno-associated vectors (AAVs), retrovirus vectors, or lentiviral vectors.

11. The mammalian cell according to any one of claims 1 to 8, wherein the mammalian cell is a Chinese hamster ovary (CHO) cell, a baby hamster kidney (BHK21) cell, a mouse myeloma cell, or a human fetal kidney (HEK293) cell, preferably the mammalian cell is a Chinese hamster ovary (CHO) cell, and more preferably the CHO mammalian cell is a stable CHO (CHO-S) mammalian cell.

12. The OST protein or functional fragment thereof has an amino acid sequence according to SEQ ID NOs: 14, 15, 16, 17, 18, 19 and / or 20, or a sequence having at least 70% identity thereto, and preferably the OST protein is expressed by the mammalian cell according to any one of claims 1 to 3, 5 to 8 and 11, the mammalian cell according to any one of claims 1 to 3, 5 to 8 and 11.

13. The mammalian cell according to claim 11 or 12, wherein the mammalian cell is manipulated to express a recombinant protein, preferably the recombinant protein is a therapeutic protein.

14. A method for modifying the glycosylation profile of a recombinant protein, comprising: (i) contacting a recombinant protein with a mammalian cell according to any one of claims 1 to 8 and 11 to 13; or (ii) contacting an OST protein or a functional fragment thereof, wherein the OST protein is a trypanosoma OST protein, preferably a trypanosoma brusey OST protein, and more preferably the OST protein comprises the amino acid sequence according to any one of SEQ ID NOs: 14, 15, 16, 17, 18, 19 and / or 20; or producing the recombinant protein in a mammalian cell according to any one of claims 1 to 8 and 11 to 13, preferably wherein the mammalian cell is manipulated to express the recombinant protein.

15. The method according to claim 14, wherein the frequency of N-glycans on the recombinant protein is changed, preferably, increased, and / or the recombinant protein is a protein selected from the group including hormones, cytokines, antibodies, enzymes, complement proteins, blood coagulation factors, functional fragments thereof, or any combination thereof.

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