Proteins with minimal N-terminal initiator methionine

Abstract

The present invention relates to recombinant plasmids, methods, and compositions for the expression of recombinant proteins with minimal N-terminal initiator methionines. The present invention also provides strategies for the efficient removal of N-terminal initiator methionines in recombinant proteins expressed on an industrial scale, and provides methods for producing proteins with minimal N-terminal initiator methionines.

Description

[Technical Field]

[0001] The present disclosure relates generally to the field of biotechnology. In particular, the present invention relates to recombinant plasmids, methods, and compositions for the expression and purification of recombinant proteins with a minimal N-terminal initiator methionine. [Background technology]

[0002] A revolution in industrial biotechnology was sparked by the discovery of DNA structure and the development of recombinant DNA technology. Traditional microbiological techniques were integrated with molecular biology to lead to improved recombinant processes for the industrial production of primary and secondary metabolites, protein biopharmaceuticals, and industrial enzymes. Recombinant DNA technology demonstrated that it was possible to improve the desirable properties of industrially synthesized proteins by controlling the expression of target genes.

[0003] Due to the unique biosynthetic production processes and molecular properties of biotechnology and biological products, drug substances contain multiple molecular entities or variants, and thus the desired product may be a mixture of expected post-translationally modified forms (e.g., glycoforms). Heterogeneity may also be introduced during the manufacturing and / or storage of a drug substance or drug product. Such forms have the potential to be active / inactive, and their presence may have adverse effects on the safety and efficacy of the biological product.

[0004] Protein synthesis is initiated by formylmethionine (N-terminal methionine) in prokaryotic cells and methionine in eukaryotic cells. During recombinant protein expression, the N-terminal methionine is co-translationally cleaved by endogenous methionine aminopeptidase (MAP). The cleavage process is inefficient because the amount of expressed recombinant protein exceeds the capacity of the limited amount of MAP to cleave N-terminal methionine, and therefore a significant amount of the expressed recombinant protein contains methionine as the first amino acid (N-terminal initiator methionine) that is not required for the mature protein.

[0005] Furthermore, recombinant proteins with an N-terminal initiator methionine are prone to oxidation during production and storage, and this oxidation must be carefully monitored and protected against. Methionine sulfoxide or methionine sulfone can be generated when methionine residues within proteins are oxidized. Increased immunogenicity, inactivity, and aggregation can result from methionine oxidation. Methionine oxidation can limit the clinical efficacy, stability, and regulatory approval of therapeutics.

[0006] Biological products with N-terminal methionine impurities may not have the same structure, activity, and stability as the native protein, and therefore may not exert an effective therapeutic function when administered to human subjects due to an unexpected immune response from the subject. Therefore, it is important that methionine impurities be removed or minimized from biological products before formulation.

[0007] Various attempts have been made to remove N-terminal methionine in the preparation of recombinant proteins with a natural N-terminus. Mostly, cyanogen bromide is used to cleave the N-terminal methionine under extremely acidic conditions, but this method is limited to proteins that do not have any internal methionine residues. Furthermore, the use of extremely acidic conditions can have a detrimental effect on the target protein. Therefore, it is necessary to explore alternative methods for N-terminal methionine removal.

[0008] Other strategies used by researchers in the past include changing the second amino acid (the position adjacent to the methionine) to allow for efficient cleavage of the methionine, but these strategies result in structural and functional changes in the molecular structure that can lead to undesirable effects along with the alteration of the molecule.

[0009] Researchers have also experimented with in vitro degradation with purified methionine aminopeptidase to remove the N-terminal methionine (Miller et al., 1987), but this method requires additional purification steps and enzyme costs and is therefore economically unfeasible.

[0010] N-terminal methionine can be cotranslationally cleaved by methionine aminopeptidase (MAP) enzymes. In eukaryotic cells, methionine is removed either by cleavage of the N-terminal signal peptide used for secretion or by MAP. In prokaryotic cells, formylmethionine is first removed by formylmethionine deformylase to generate N-terminal methionine, which is then processed by MAP. In Escherichia coli (E. coli), there is only one copy of the MAP gene, which is responsible for the removal of N-terminal methionine from 70% of proteins expressed in E. coli. Especially in E. coli, N-terminal methionine is retained in approximately 30% of expressed proteins during large-scale recombinant protein expression, likely due to saturation of MAP (see Paul Wingfield et al., 2018, "N-Terminal Methionine Processing"). Therefore, researchers have been interested in coexpressing MAP by genetically engineering E. coli. Various combinations of promoters were used to co-express MAP and the protein of interest to remove the methionine impurity.

[0011] Neupogen® (filgrastim, r-met-huG-CSF), a recombinant protein expressed in Escherichia coli, has been approved by the USFDA for use during or after chemotherapy in patients suffering from neutropenia. Neupogen® consists of a polypeptide chain 175 amino acids long (Souza et al., 1986; Lu et al., 1989b). When this protein is directly expressed in Escherichia coli, large amounts of the expression product aggregate into inclusion bodies. It has been reported that Neupogen® production lots typically contain very low levels (approximately 1%) of formylmethionine-G-CSF (f-metG-CSF). Similarly, this oxidized form represents less than 2% of the total Neupogen® [see: Alan C. Herman et al., study entitled "Characterization, Formulation, and Stability of Neupogen® (Filgrastim), a Recombinant Human Granulocyte Colony Stimulating Factor," published in Formulation, Characterization, and Stability of Protein Drugs, Rodney Pearlman and Y. John Wang, eds., Plenum Press, New York, 1996].

[0012] U.S. Patent No. 6,071,718A disclosed the use of a TAC promoter for MAP expression and a T7 promoter for the expressed gene of interest. This patent also disclosed the use of methionine aminopeptidase to cleave the N-terminal amino acid of an expressed protein (beta-casein). This patent further discloses the requirements for expression of iminopeptidase and aminopeptidase, as well as the use of two proline amino acids for efficient cleavage of the methionine initiator. The addition of the two prolines may add unwanted impurities or amino acids adjacent to the N-terminus of the expressed protein. Adding amino acids to a recombinant protein may further alter the structure and function of the expressed molecule.

[0013] U.S. Patent No. 1,106,0123 B2 discloses the use of a TAC promoter for MAP expression and a T7 promoter for the expressed gene of interest. In this patent, the MAP gene is inserted into the E. coli genome to restrict expression of the protein of interest to the modified host, more specifically, to the use of only E. coli (the B121 Gormet strain). Furthermore, the only copy of MAP is integrated as a substitution into a single locus (gor) that also carries an additional chloramphenicol gene for selection, limiting the effectiveness of MAP in cleaving and removing the N-terminal methionine in the expressed protein. Because this strategy involves re-modifying the E. coli genome, its applicability to specific E. coli strains is limited, and therefore its use for protein expression in other E. coli strains may be impossible.

[0014] European Patent No. 2430041B1 discloses a modified lectin protein with 141 amino acids that is expressed in Escherichia coli as a stable and soluble protein using recombinant DNA technology. The modified lectin resembles natural lectins in terms of carbohydrate-binding specificity and apoptotic properties, enabling its use as a drug delivery agent for cancer therapy and as a diagnostic tool.

[0015] A significant amount of purified recombinant proteins contain methionine as the first amino acid, which is not required in the mature protein sequence and is therefore an undesirable moiety in most protein formulations. Currently, there is no widely accepted strategy for efficiently and completely removing N-terminal methionine in recombinant proteins expressed on an industrial scale. Therefore, there is a need for a protein expression strategy that has high expression / yields, minimal impurities, and a simple purification process. [Prior art documents] [Patent documents]

[0016] [Patent Document 1] U.S. Patent No. 6,071,718A [Patent Document 2] U.S. Patent No. 11060123B2 [Patent Document 3] European Patent No. 2430041B1 [Non-patent literature]

[0017] [Non-Patent Document 1] Miller et al., 1987 [Non-patent document 2] Paul Wingfield et al. 2018 "N-Terminal Methionine Processing" [Non-patent document 3] Souza et al., 1986 [Non-patent document 4] Lu et al., 1989b [Non-Patent Document 5] A study by Alan C. Herman et al. entitled "Characterization, Formulation, and Stability of Neupogen® (Filgrastim), a Recombinant Human Granulocyte Colony Stimulating Factor," published in Formulation, Characterization, and Stability of Protein Drugs, edited by Rodney Pearlman and Y. John Wang, Plenum Press, New York, 1996. Summary of the Invention [Means for solving the problem]

[0018] The present disclosure relates to recombinant plasmids, methods, and compositions for the expression and purification of recombinant proteins with minimal N-terminal initiator methionine. Furthermore, the present invention provides strategies for the efficient or complete removal of N-terminal initiator methionine in recombinant proteins expressed on an industrial scale. The present invention also provides recombinant plasmids containing DNA constructs for the expression of methionine aminopeptidase (MAP), which enables the efficient removal of N-terminal initiator methionine in expressed proteins. The present invention also provides methods for producing proteins with minimal N-terminal initiator methionine.

[0019] In one aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a nucleotide sequence encoding a methionine aminopeptidase (MAP) protein or a variant thereof operably linked to a promoter sequence of SEQ ID NO: 5; a nucleotide sequence encoding a protein of interest operably linked to the promoter sequence of SEQ ID NO: 7; wherein the recombinant plasmid results in a protein of interest consisting of less than 2% N-terminal initiator methionines.

[0020] In another embodiment of the present invention, the recombinant plasmid is a modified pET27b plasmid.

[0021] In another embodiment of the present invention, the MAP protein or variant thereof is further operably linked to a terminator.

[0022] In another embodiment of the invention, the protein of interest is further operably linked to a terminator.

[0023] In another embodiment of the present invention, the nucleotide sequence encoding the MAP protein operably linked to a promoter sequence is represented as SEQ ID NO:2.

[0024] In another embodiment of the invention, the protein of interest is the protein of SEQ ID NO:10.

[0025] In another embodiment of the invention, the protein of interest is the protein of SEQ ID NO:13 or SEQ ID NO:16.

[0026] In another aspect of the invention, the MAP protein is the protein of SEQ ID NO: 4 or a variant thereof.

[0027] In another aspect of the invention, the protein of interest can be used in cancer diagnosis or cancer treatment.

[0028] In another embodiment, the present invention relates to the use of recombinant plasmids to produce proteins of interest with less than 2% N-terminal initiator methionines.

[0029] In another embodiment of the present invention, the nucleotide sequence encoding a protein of interest operably linked to a promoter is the sequence depicted as FIG.

[0030] In another aspect, the present invention relates to a recombinant plasmid of SEQ ID NO: 18, further comprising a nucleotide sequence encoding a protein of interest operably linked to a promoter sequence of SEQ ID NO: 7.

[0031] In yet another aspect, the present invention relates to a host cell comprising a recombinant plasmid according to claim 1, wherein the host cell is a prokaryotic cell.

[0032] In another embodiment of the invention, the prokaryotic cell is E. coli strain DE3.

[0033] In yet another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) providing a host cell harboring the recombinant plasmid of claim 1; b) culturing the host cells in a culture medium; c) purifying the protein of interest from the cultured host cells or the culture medium; 1. A method for producing a protein of interest, comprising: The purified protein comprises less than 2% N-terminal initiator methionine.

[0034] In another embodiment, the present invention provides a nucleotide sequence of SEQ ID NO: 3 operably linked to a promoter sequence and a terminator sequence of SEQ ID NO: 5; a nucleotide sequence encoding a protein of interest operably linked to the promoter and terminator sequences of SEQ ID NO: 7; wherein the recombinant plasmid results in a protein of interest consisting of less than 2% N-terminal initiator methionines. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 is a diagram depicting a DNA construct containing a TAC promoter, a methionine aminopeptidase gene, and a T3te terminator nucleotide sequence. [Figure 2] FIG. 1 is a diagram representing the pET27b plasmid. [Figure 3] FIG. 1 is a diagram depicting the pET27b plasmid, containing the TAC promoter, the methionine aminopeptidase gene, and the T3te terminator. [Figure 4] FIG. 1 is a diagram depicting a DNA construct carrying the methionine aminopeptidase gene together with a gene expressing a protein of interest. [Figure 5] FIG. 1 shows the pET27b plasmid construct carrying the methionine aminopeptidase gene along with a gene expressing a protein of interest. [Figure 6] FIG. 1 shows Western blot analysis of the reduction in add-Met content of protein variant 1 and protein variant 2 co-expressed with methionine aminopeptidase, along with their respective controls.

[0036] Brief description of the sequence listing: SEQ ID NO: 1 represents the DNA construct of the pET27b plasmid.

[0037] SEQ ID NO: 2 represents a DNA construct having a TAC promoter, a methionine aminopeptidase (MAP) gene, and a T3te terminator. ttgacaattaatcatcggctcgtataatgtgtggaattgtgagcggataacaatttcacacaggaaacagccagtccgtttaggtgttttcacgagcacttcaccaacaaggaccatagactagtatggctatctcaatcaagaccccagaagatatcgaaaaaatgcgcgtcgctggccgactggctgccgaagtgctggagatgatcgaaccgtatgttaaaccgggcgtcagcaccggcgagctggatcgcatctgtaatgattacattgttaatgaacaacacgcggtttctgcctgcctcggctatcacggctatccgaaatccgtttgcatctctattaatgaagtggtgtgccacggtatcccggacgatgctaagctgctgaaagatggcgatatcgttaacattgatgtcaccgtaatcaaagatggtttccacggcgatacctcgaaaatgtttatcgtcggtaagccgaccatcatgggcgaacgtctgtgccgcatcacgcaagaaagcctgtacctggcgctacgcatggtaaaaccaggcattaatctgcgcgaaatcggtgcggcgattcagaaatttgtcgaagcagaaggcttctccgtcgttcgtgaatattgcggacacggtattggtcgcggcttccatgaagaaccgcaggtgctgcactatgactcccgtgaaaccaacgtcgtactgaaacctgggatgacgttcaccatcgagccaatggtcaacgcgggtaaaaaagagatccgcaccatgaaagatggctggacggtaaaaaccaaagatcgcagcttgtctgcacaatatgagcatactattgtggtgactgataacggctgcgaaattctgacgctacgcaaggatgacaccatcccggcgataatctcgcacgacgaataataggctcaccttcacgggtgggcctttcttcg

[0038] SEQ ID NO: 3 represents the nucleotide sequence of methionine aminopeptidase (MAP). atggctatctcaatcaagaccccagaagatatcgaaaaaatgcgcgtcgctggccgactggctgccgaagtgctggagatgatcgaaccgtatgttaaa cggggcgtcagcaccggcgagctggatcgcatctgtaatgattacattgttaatgaacaacacgcggtttctgcctgcctcggctatcacggctatccga aatccgtttgcatctctattaatgaagtggtgtgccacggtatcccggacgatgctaagctgctgaaagatggcgatatcgttaacattgatgtcaccg taatcaaagatggtttccacggcgatacctcgaaaatgtttatcgtcggtaagccgaccatcatgggcgaacgtctgtgccgcatcacgcaagaaagcct gtacctggcgctacgcatggtaaaaccaggcattaatctgcgcgaaatcggtgcggcgattcagaaatttgtcgaagcagaaggcttctccgtcgttcg tgaatattgcggacacggtattggtcgcggcttccatgaagaaccgcaggtgctgcactatgactcccgtgaaaccaacgtcgtactgaaacctgggatg acgttcaccatcgagccaatggtcaacgcgggtaaaaaagagatccgcaccatgaaagatggctggacggtaaaaaccaaagatcgcagcttgtctgcac aatatgagcatactattgtggtgactgataacggctgcgaaattctgacgctacgcaaggatgacaccatcccggcgataatctcgcacgacgaataata

[0039] SEQ ID NO: 4 represents the amino acid sequence of methionine aminopeptidase (MAP).

[0040] MAISIKTPEDIEKMRVAGRLAAEVLEMIEPYVKPGVSTGELDRICNDYIVNEQHAVSACLGYHGYPKSVCISINEVVCHGIPDDAKLLKDGDIVNIDVTVIKDGFHGDTSKMFIVGKPTIMGERLCRITQES LYLALRMVKPGINLREIGAAIQKFVEAEGFSVVREYCGGHGIGRGFHEEPQVLHYDSRETNVVLKPGMTFTIEPMVNAGKKEIRTMKDGWTVKTKDRSLSAQYEHTIVVTDNGCEILTLRKDDTIPAIISHDE

[0041] SEQ ID NO: 5 represents the nucleotide sequence of the TAC promoter. ttgacaattaatcatcggctcgtataatg

[0042] SEQ ID NO: 6 represents the nucleotide sequence of the T3te terminator. ggctcaccttcacgggtgggcctttcttcg

[0043] SEQ ID NO: 7 represents the nucleotide sequence of the T7 promoter. taatacgactcactatagg

[0044] SEQ ID NO: 8 represents the nucleotide sequence of the T7 terminator. ctagcataaccccttggggcctctaaacgggtcttgaggggttttttg

[0045] SEQ ID NO: 9 represents the nucleotide sequence of modified Sclerotium rolfsii lectin (protein variant 1). acctataaaattaccgtgcgcgtgtatcagaccaacccggatgcctttttccatccggtggaaaaaaccgtgtggaaatatgcgaatggcggtacctggacgatt acggatgatcagcatgtgctgacgatgggtggtagcggtaccagcggcaccctgcgttttcacgcagataatggcgaaagcttcaccgccacctttggtgtgcata attataaacgctggtgtgatattgtgaccaacctggcagcggatgaaaccggcatggttattaatcagcagtattatagtcagaaaaaccgcgaagaagcgcgtga acgccagctgagtaactatcaggtgaaaaatgcgaaaggccgtaacttccagattgtttataccgaagcggaaggcaatgatctgcatgcgaacctgattatcggc

[0046] SEQ ID NO: 10 represents the amino acid sequence of modified Sclerotium rolfsii lectin (protein variant 1). TYKITVRVYQTNPDAFFHPVEKTVWKYANGGTWTITDDQHVLTMGGSGTSGTLRFHADNGESFTATFGVHNYKRWCDIVTNLAADETGMVINQQYYSQKNREEARERQLSNYQVKNAKGRNFQIVYTEAEGNDLHANLIIG

[0047] SEQ ID NO: 11 represents the nucleotide sequence of a DNA construct for expression of modified Sclerotium rolfsii lectin (protein variant 1). taatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacatatgacctataaaattaccgtgcgtgtatcagaccaacccggatgcctttttccatccggtggaaaaaaccgtgtggaaatatgcgaatggcggtacct ggacgattacggatgatcagcatgtgctgacgatgggtggtagcggtaccagcggcaccctgcgttttcacgcagataatggcgaaagcttcaccgccacctttggtgtgcataattataaacgctggtgtgatattgtgaccaacctggcagcggatgaaaccggcatggttattaatcagcagtat tatagtcagaaaaaccgcgaagaagcgcgtgaacgccagctgagtaactatcaggtgaaaaatgcgaaaaggccgtaacttccagattgtttataccgaagcggaaggcaatgatctgcatgcgaacctgattatcggctaatgaggatccgaattcgagctccgtcgacaagcttgcggccgcactcg agatcaaacgggctagccagccagaactcgccccggaagaccccgaggatgtcgagcaccaccaccaccaccactgagatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttg

[0048] SEQ ID NO: 12 represents the nucleotide sequence of modified Sclerotium rolfsii lectin (protein variant 2). agctataaaattaccgtgcgcgtgtatcagaccaacccggatgcctttttccatccggtggaaaaaaccgtgtggaaatatgcgaatggcggtacctggacgatta cggatgatcagcatgtgctgacgatgggtggtagcggtaccagcggcaccctgcgttttcacgcagataatggcgaaagcttcaccgccacctttggtgtgcataat tataaacgctggggcgatattgtgaccaacctggcagcggatgaaaccggcatggttattaatcagcagtattatagtcagaaaaaccgcgaagaagcgcgtgaac gccagctgagtaactatcaggtgaaaaatgcgaaaggccgtaacttccagattgtttataccgaagcggaaggcaatgatctgcatgcgaacctgattatcggctgc

[0049] SEQ ID NO: 13 represents the amino acid sequence of modified Sclerotium rolfsii lectin (protein variant 2). SYKITVRVYQTNPDAFFHPVEKTVWKYANGGTWTITDDQHVLTMGGSGTSGTLRFHADNGESFTATFGVHNYKRWGDIVTNLAADETGMVINQQYYSQKNREEARERQLSNYQVKNAKGRNFQIVYTEAEGNDLHANLIIGC

[0050] SEQ ID NO: 14 represents the nucleotide sequence of a DNA construct for expression of modified Sclerotium rolfsii lectin (protein variant 2). taatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacatatgagctataaaattaccgtgcgcgtgtatcagaccaacccggatgcctttttccatccggtggaaaaaaccgtgtggaaatatgcgaatggcggtacct ggacgattacggatgatcagcatgtgctgacgatgggtggtagcggtaccagcggcaccctgcgttttcacgcagataatggcgaaagcttcaccgccacctttggtgtgcataattataaacgctggggcgatattgtgaccaacctggcagcggatgaaaccggcatggttattaatcagcagtatt atagtcagaaaaaccgcgaagaagcgcgtgaacgccagctgagtaactatcaggtgaaaaatgcgaaaaggccgtaacttccagattgtttataccgaagcggaaggcaatgatctgcatgcgaacctgattatcggctgctaatgaggatccgaattcgagctccgtcgacaagcttgcggccgcactc gagatcaaacgggctagccagccagaactcgccccggaagaccccgaggatgtcgagcaccaccaccaccaccactgagatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttg

[0051] SEQ ID NO: 15 represents the nucleotide sequence of modified Sclerotium rolfsii lectin (protein variant 3). agctataaaattaccgtgcgcgtgtatcagaccaacccggatgcctttttccatccggtggaaaaaaccgtgtggaaatatgcgaatggcggtacctggacgattac ggatgatcagcatgtgctgacgatgggtggtagcggtaccagcggcaccctgcgttttcacgcagataatggcgaaagcttcaccgccacctttggtgtgcataatt ataaacgctggggcgatattgtgaccaacctggcagcggatgaaaccggcatggttattaatcagcagtattatagtcagaaaaaccgcgaagaagcgcgtgaacgc cagctgagtaactatcaggtgaaaaatgcgaaaggccgtaacttccagattgtttataccgaagcggaaggcaatgatctgcatgcgaacctgattatcggcagctgc

[0052] SEQ ID NO: 16 represents the amino acid sequence of modified Sclerotium rolfsii lectin (protein variant 3). SYKITVRVYQTNPDAFFHPVEKTVWKYANGGTWTITDDQHVLTMGGSGTSGTLRFHADNGESFTATFGVHNYKRWGDIVTNLAADETGMVINQQYYSQKNREEARERQLSNYQVKNAKGRNFQIVYTEAEGNDLHANLIIGSC

[0053] SEQ ID NO: 17 represents the nucleotide sequence of a DNA construct for expression of modified Sclerotium rolfsii lectin (protein variant 3). taatacgactcactataggggaattgtgagcggataacaattcccctctagaaataattttgtttaactttaagaaggagatatacatatgagctataaaattaccgtgcgcgtgtatcagaccaacccggatgcctttttccatccggtggaaaaaaccgtgtggaaatatgcgaatggcggtacctg gacgattacggatgatcagcatgtgctgacgatgggtggtagcggtaccagcggcaccctgcgttttcacgcagataatggcgaaagcttcaccgccacctttggtgtgcataattataaacgctggggcgatattgtgaccaacctggcagcggatgaaaccggcatggttattaatcagcagtattat agtcagaaaaaccgcgaagaagcgcgtgaacgccagctgagtaactatcaggtgaaaaatgcgaaaaggccgtaacttccagattgtttataccgaagcggaaggcaatgatctgcatgcgaacctgattatcggcagctgctaatgaggatccgaattcgagctccgtcgacaagcttgcggccgcact cgagatcaaacgggctagccagccagaactcgccccggaagaccccgaggatgtcgagcaccaccaccaccaccactgagatccggctgctaacaaagcccgaaaggaagctgagttggctgctgccaccgctgagcaataactagcataaccccttggggcctctaaacgggtcttgaggggttttttg

[0054] SEQ ID NO: 18 represents the nucleotide sequence of the modified plasmid, having the TAC promoter, the methionine aminopeptidase (MAP) gene, and the T3te terminator.

[0055] SEQ ID NO: 19 represents the nucleotide sequence of the plasmid carrying the modified Sclerotium rolfsii lectin (protein variant 1) gene.

[0056] SEQ ID NO: 20 represents the nucleotide sequence of the plasmid carrying the modified Sclerotium rolfsii lectin (protein variant 2) gene.

[0057] SEQ ID NO: 21 represents the nucleotide sequence of the plasmid carrying the modified Sclerotium rolfsii lectin (protein variant 3) gene. DETAILED DESCRIPTION OF THE INVENTION

[0058] Definition: As used herein, the term "comprises" refers to an open definition and should be understood to allow for additional members of similar or other characteristics.

[0059] As used herein, the term "consists of" or "consisting of" should be understood as a restrictive definition relating to a limited range of features.

[0060] As used herein, the term "methionine aminopeptidase" or MAP refers to a metalloenzyme that cleaves N-terminal methionine from newly synthesized peptides and proteins. MAP initiates co- and post-translational modifications essential for protein translocation, activation, regulation, and degradation. MAP, as used herein, can be an enzyme expressed in its native form or can be genetically modified to suit particular needs.

[0061] As used herein, the terms "N-terminal initiator methionine" or "N-terminal methionine" refer to a methionine amino acid present at the amino terminus of an expressed protein that is later co-translationally cleaved by a methionine aminopeptidase (MAP) enzyme to form the mature protein.

[0062] As used herein, the term "DNA construct" or "gene construct" refers to an artificially designed DNA segment present on a vector that can be used to integrate genetic material into target tissues or cells. The terms "DNA construct" and "gene construct" may be used interchangeably and should be interpreted as meaning the same thing. A "DNA construct" or "gene construct" includes a promoter sequence, an open reading frame sequence, and a terminator sequence. The construct may further include special sequences, such as enhancers, silencers, or reporter sequences, depending on the nature of the protein to be expressed.

[0063] As used herein, a "host cell" is a cell used to express a protein of interest. Host cells can be modified by transformation with a vector that allows for the expression of the protein of interest. Non-limiting examples of host cells include plant, animal, human, bacterial, yeast, or filamentous fungal cells.

[0064] As used herein, the phrase "protein with minimal N-terminal initiator methionines" refers to proteins that contain less than or equal to 2% N-terminal initiator methionines.

[0065] As used herein, the term "promoter" refers to a region of DNA at which RNA polymerase initiates transcription of a gene. Promoter sequences are typically located immediately upstream or 5' of the transcription initiation site. Promoters also function as regulatory regions for gene expression.

[0066] As used herein, the term "terminator" refers to a DNA region at the end of a gene or operon that terminates transcription. Terminator sequences follow the promoter and coding region and are typically located downstream or 3' of the gene.

[0067] As used herein, the term "pET plasmid" refers to a low copy number plasmid, which reduces expression leakage before induction. Target genes are cloned into pET plasmids under the control of the strong bacteriophage T7 transcriptional and (optionally) translational signals, and their expression is induced by providing the host cell with a source of T7 RNA polymerase.

[0068] As used herein, the term "open reading frame (ORF)" refers to the portion of a DNA sequence that spans between a start codon and a stop codon. In other words, an ORF is a nucleotide sequence that can be translated into a polypeptide.

[0069] As used herein, the term "operably linked to" refers to a sequence that has an activity of the present disclosure in functional association with other nucleotide components of the nucleic acid molecule.

[0070] As used herein, the term "lectin" refers to a carbohydrate-binding protein that is highly specific for sugars that are part of other molecules, resulting in the agglutination of specific cells or the precipitation of glycoconjugates and polysaccharides.

[0071] As used herein, the term "Sclerotium rolfsii" is used to refer to a soil-borne plant pathogen capable of purifying a lectin from its sclerotial body.

[0072] As used herein, the phrase "the recombinant plasmid results in a protein of interest" refers to the use of a recombinant plasmid of the invention in a method for preparing a protein of interest that results in a protein of interest that consists of less than 2% N-terminal initiator methionines.

[0073] Source of biological material: The biological material used in this disclosure is Escherichia coli strain BL21 DE3, commercially available from Stratagene, USA (a division of Agilent Technologies).

[0074] The embodiments of the present disclosure are not intended to limit the scope of the disclosure to the embodiments illustrated and disclosed herein.

[0075] The embodiments are provided to thoroughly and fully convey the scope of the present disclosure to those skilled in the art. Numerous details regarding specific components and processes are set forth to provide a thorough understanding of the embodiments of the present disclosure. It will be apparent to those skilled in the art that the details provided in the embodiments should not be construed as limiting the scope of the present disclosure. In some embodiments, well-known compositions, well-known processes, and well-known techniques are not described in detail.

[0076] It is understood that each feature or embodiment or combination described herein is non-limiting, and thus, any illustrative example of an aspect of the present disclosure is intended to be combinable with any other feature or embodiment or combination described herein. For example, when a feature is described with terms such as "an embodiment," "one of the embodiments," "another embodiment," "a further embodiment," "a specific example embodiment," and / or "another embodiment," each such type of embodiment is a non-limiting example of a feature that is intended to be combined with any other feature or combination of features described herein without enumerating all possible combinations. Such features or combinations of features apply to all aspects of the present disclosure.

[0077] Removal of the N-terminal initiator methionine is a critical step in the maturation of expressed proteins. Protein synthesis is initiated by either methionine in eukaryotic cells or formylmethionine in prokaryotic cells. Methionine aminopeptidase (MAP) is a metalloenzyme that cleaves N-terminal methionine from newly synthesized peptides and proteins. MAP initiates co- and post-translational modifications essential for protein translocation, activation, regulation, and degradation. Such MAP enzymes exist in prokaryotes, yeast, and eukaryotes. Methionine aminopeptidase isolated from prokaryotic cells is MAP1, while methionine aminopeptidase isolated from eukaryotic cells is MAP2. Host cells without exogenous or additional copies of the MAP gene reportedly express recombinant proteins with N-terminal methionine concentrations ranging from 10% to 90% of the total expressed protein.

[0078] In one embodiment of the present disclosure, a plasmid is designed and constructed for the expression of a protein with a minimal N-terminal initiator methionine.

[0079] In one embodiment of the present disclosure, a DNA construct is designed containing a promoter region, a gene / ORF, and a terminator / termination region. The construct may further contain special sequences, such as enhancer, silencer, or reporter sequences, depending on the nature of the protein to be expressed.

[0080] In one embodiment, the present invention provides a nucleotide sequence encoding a methionine aminopeptidase (MAP) protein or a variant thereof operably linked to a promoter sequence of SEQ ID NO: 5; a nucleotide sequence encoding a protein of interest operably linked to the promoter sequence of SEQ ID NO: 7; wherein the recombinant plasmid results in a protein of interest consisting of less than 2% N-terminal initiator methionines.

[0081] In another embodiment, the present invention provides a nucleotide sequence of SEQ ID NO: 3 operably linked to a promoter sequence and a terminator sequence of SEQ ID NO: 5; a nucleotide sequence encoding a protein of interest operably linked to the promoter and terminator sequences of SEQ ID NO: 7; wherein the recombinant plasmid results in a protein of interest consisting of less than 2% N-terminal initiator methionines.

[0082] In one embodiment of the present disclosure, a DNA construct is designed that includes a gene that expresses methionine aminopeptidase (MAP). The gene is flanked by a promoter sequence 5' to the open reading frame (ORF) of the methionine aminopeptidase gene and a terminator sequence 3' to the ORF. In another embodiment of the present disclosure, the designed DNA construct, including the promoter, the methionine aminopeptidase (MAP) gene, and the terminator sequence, is as shown in Figure 1. It is further contemplated that the sequence used to express methionine aminopeptidase (MAP) may include a natural (wild) sequence or a modified sequence. It is also contemplated that the MAP may be replaced with an enzyme having an activity similar to that of MAP.

[0083] In one embodiment of the present disclosure, a promoter operably linked to the MAP gene is selected to provide optimal expression of the MAP and, upon further MAP activity, to produce a protein with a minimal N-terminal initiator methionine. It will be very well understood by those skilled in the art that the promoter can be selected so as not to affect the yield of the expressed protein of interest.

[0084] In one embodiment of the present disclosure, a promoter selected from araE, UV5, and / or TAC (pTac) is evaluated by the promoter operably linked to the MAP gene for expression of a protein with a minimal N-terminal initiator methionine. In another embodiment of the present disclosure, the promoter sequence operably linked to the MAP gene is the TAC promoter, specifically having the nucleic acid sequence set forth in SEQ ID NO:5.

[0085] In one embodiment of the present disclosure, the terminator sequence operably linked to the MAP gene is selected to provide optimal expression of MAP and to further result in the expression of a protein with a minimal N-terminal initiator methionine due to MAP activity. It will be well understood by those skilled in the art that the terminator sequence can be selected so as not to affect the yield of the expressed protein. In another embodiment of the present disclosure, the terminator sequence operably linked to the MAP gene is a T3te terminator, particularly having the nucleic acid sequence set forth in SEQ ID NO:6.

[0086] In one of the embodiments of the present disclosure, a DNA construct comprising a gene expressing methionine aminopeptidase (MAP) comprises a TAC promoter sequence specified in SEQ ID NO: 5, a methionine aminopeptidase (MAP) gene specified in SEQ ID NO: 3, and a T3te terminator sequence specified in SEQ ID NO: 6. In another embodiment, the prepared DNA construct is represented as SEQ ID NO: 2.

[0087] In one embodiment of the present disclosure, a DNA construct containing a gene expressing methionine aminopeptidase (MAP) is cloned into a suitable vector or plasmid. A suitable vector or plasmid for expression of a recombinant protein in a host cell can be selected based on its compatibility with the expression of the recombinant protein in the host cell. A suitable vector can also be selected based on the copy number of the vector or plasmid, which allows for efficient expression of the protein of interest and the MAP protein. The expression vector can be selected from the pGEX and pET series of vectors. The pET series of plasmids are low-copy-number plasmids, which reduce expression leakage before induction. The target gene is cloned into the pET plasmid under the control of the strong bacteriophage T7 transcriptional and (optionally) translational signals, and its expression is induced by providing the host cell with a source of T7 RNA polymerase.

[0088] Plasmids of the pET family are envisioned to contain one or more of the key components mentioned below. - T7 promoter: drives high levels of transcription of the gene of interest in the presence of T7 RNA polymerase. When placed immediately upstream of the LacO element, the entire cassette is known as the T7lac promoter. - LacO:Lacl binding site. This element inhibits the activity of the T7 promoter in the presence of the Lacl protein, preventing leaky expression of the gene of interest. - RBS: ribosome binding site and translation initiation element from T7 bacteriophage, which allows efficient production of the protein of interest. - ORF: Open reading frame. Place your gene of interest here. - T7 terminator: a signal sequence that terminates the transcript produced from the gene of interest, preventing unterminated transcription. - Ampicillin: Ampicillin resistance gene. This gene allows the plasmid to be maintained in E. coli by ampicillin selection. - pBR322 ori: pBR322 replication origin. The plasmid carrying this origin and the Rop gene exists in E. coli at low copy number. - Rop: primer repressor. Encodes a small protein that regulates the copy number of the plasmid. The presence of the Rop protein in conjunction with the pBR322 origin of replication on the plasmid results in a low copy number of the plasmid. - LacI: the native promoter of E. coli and the coding sequence for the lac repressor. In the absence of induction of the system (i.e., in the absence of IPTG), the LacI protein represses transcription of the gene of interest from the T7lac promoter and transcription of T7 RNA polymerase from the LacUV5 promoter in host strains used for recombinant protein production.

[0089] In one embodiment of the present disclosure, a DNA construct containing a gene expressing methionine aminopeptidase (MAP) is cloned into a pET plasmid. In another embodiment, the modified pET plasmid contains the DNA construct set forth in SEQ ID NO:2.

[0090] In one embodiment of the present disclosure, a DNA construct containing a gene expressing methionine aminopeptidase (MAP) is cloned into a pET plasmid. In one embodiment, the DNA construct is cloned between the BglII and SphI restriction sites in a pET27b plasmid. In another embodiment, the modified pET27b plasmid containing the DNA construct (SEQ ID NO: 2) is represented as SEQ ID NO: 18.

[0091] In one embodiment of the present disclosure, a DNA construct is designed that includes a gene that expresses a protein of interest (hereinafter referred to as the protein). The gene is flanked by a promoter sequence 5' to the open reading frame (ORF) of the gene encoding the protein and a terminator sequence 3' to the open reading frame (ORF) of the gene encoding the protein. In another embodiment of the present disclosure, a designed DNA construct including a promoter, a gene encoding the protein, and a terminator sequence is as shown in Figure 4. It is further contemplated that the gene sequence used to express the protein can include a gene sequence encoding a protein, depending on the desires of those skilled in the art. It is further contemplated that the expressed protein can be selected from the group consisting of an antibody, a contractile protein, an enzyme, a hormone protein, a structural protein, a storage protein, and a transport protein.

[0092] In one embodiment of the present disclosure, a promoter operably linked to a gene encoding a protein is selected to provide optimal expression of the protein with a minimal N-terminal initiator methionine. It will be well understood by those skilled in the art that the promoter selection can be made so as not to affect the yield of the expressed protein.

[0093] In another embodiment of the present disclosure, the promoter sequence operably linked to the protein-encoding gene is a T7 promoter, particularly having the nucleic acid sequence set forth in SEQ ID NO:7.

[0094] In another embodiment of the present disclosure, the terminator sequence operably linked to the protein-encoding gene is a T7 terminator, particularly having the nucleic acid sequence set forth in SEQ ID NO:8.

[0095] In one embodiment of the present disclosure, the DNA construct comprising a gene encoding a protein comprises a T7 promoter sequence identified in SEQ ID NO: 7, a gene encoding the protein of interest, and a T7 terminator sequence identified by SEQ ID NO: 8. In another embodiment, the DNA construct prepared is the construct depicted in Figure 4. In another embodiment of the present disclosure, the gene encoding the protein of interest is selected from SEQ ID NO: 9, SEQ ID NO: 12, and SEQ ID NO: 15.

[0096] In one of the embodiments of the present disclosure, the DNA construct comprising the gene encoding the protein, represented as FIG. 4, is one or more selected from SEQ ID NO:11, SEQ ID NO:14, and SEQ ID NO:17.

[0097] In one embodiment of the present disclosure, a nucleotide sequence encoding a protein was cloned into a modified pET27b plasmid shown in Figure 5. It is contemplated that the nucleotide sequence may be any sequence designed by one skilled in the art based on the protein to be expressed. In another embodiment of the present disclosure, the nucleotide sequence expressing the protein is cloned between the NdeI and BamHI restriction sites in the MCS region on the antisense strand of the modified plasmid.

[0098] In one embodiment of the present disclosure, the nucleotide sequence (SEQ ID NO: 9) encoding the protein of SEQ ID NO: 10 (protein variant 1) was cloned between the NdeI and BamHI restriction sites in the MCS region on the antisense strand of a modified plasmid (SEQ ID NO: 18). In another embodiment of the present disclosure, the modified plasmid having the nucleotide sequence encoding the protein (protein variant 1) is SEQ ID NO: 19.

[0099] In one embodiment of the present disclosure, the nucleotide sequence (SEQ ID NO: 12) encoding the protein of SEQ ID NO: 13 (protein variant 2) was cloned between the NdeI and BamHI restriction sites in the MCS region on the antisense strand of a modified plasmid (SEQ ID NO: 18). In another embodiment of the present disclosure, the modified plasmid having the nucleotide sequence encoding the protein (protein variant 2) is SEQ ID NO: 20.

[0100] In another embodiment of the present disclosure, the nucleotide sequence (SEQ ID NO: 15) encoding the protein of SEQ ID NO: 16 (protein variant 3) was cloned between the NdeI and BamHI restriction sites in the MCS region on the antisense strand of a modified plasmid (SEQ ID NO: 18). In another embodiment of the present disclosure, the modified plasmid having the nucleotide sequence encoding the protein (protein variant 3) is SEQ ID NO: 21.

[0101] Another aspect of the disclosure provides a method for expressing a protein of interest with minimal or reduced N-terminal initiator methionine.

[0102] In yet another aspect, the present invention provides a method for producing a pharmaceutical composition comprising: a) providing a host cell harboring the recombinant plasmid of claim 1; b) culturing the host cells in a culture medium; c) purifying the protein of interest from the cultured host cells or the culture medium; 1. A method for producing a protein of interest, comprising: The purified protein comprises less than 2% N-terminal initiator methionine.

[0103] In one embodiment of the present disclosure, a method for expressing a protein with minimal or reduced N-terminal initiator methionine comprises constructing a vector containing a nucleic acid sequence encoding the protein and a nucleic acid sequence encoding a MAP (methionine aminopeptidase), introducing the vector into a host cell, allowing the host cell to co-express the gene of interest and the MAP, and allowing the expressed MAP to optimally cleave the N-terminal methionine of the protein. The expressed protein is further subjected to downstream purification and evaluation of purity and quality.

[0104] In one embodiment of the present disclosure, a modified plasmid containing a nucleotide sequence encoding a protein is transformed into a suitable host cell by any method known in the art. In another embodiment, the modified plasmid containing a nucleotide sequence encoding a protein is transformed into the host cell by heat shock. Selection of transformed cells from a non-transformed population can be achieved by using a selectable marker gene that confers resistance to antibiotics, and additional clones with the desired outcome are selected for protein expression and analysis. The host cell can be selected from prokaryotic and eukaryotic cells and contain a copy of the T7 RNA polymerase gene driven by the LacUV5 promoter. Expression of the T7 polymerase is induced by adding the lactose analog IPTG to the bacterial culture. The host cell may be selected from the group of prokaryotic cells including Corynebacterium, Bacillus, Caulobacteria, phototrophic bacteria, psychrophilic bacteria, halophilic bacteria, Streptomycete, Nocardia, Mycobacteria, lactic acid bacteria, and Escherichia coli. In another embodiment of the present disclosure, the host cell is a prokaryotic cell, more particularly, Escherichia coli. In a preferred embodiment, an E. coli strain having an inducible T7lac promoter (DE3) system can be used for protein production using the constructs or plasmids of the present disclosure. The E. coli strain may be selected from BL21(DE3) and its derivatives Rosetta2(DE3), BL21(DE3) CodonPlus RP / RIL, Tuner(DE3), BL21(DE3)Star, BL21-AI, BL21-SI, C41(DE3), C43(DE3); redox-modified strains such as AD494(DE3), BL21(DE3)trxB, Origami(DE3), OrigamiB(DE3); and rec strains such as BLR(DE3), JM109(DE3), HMS174(DE3).

[0105] In one embodiment of the present disclosure, the transformed host cells expressing the protein are subjected to a fermentation process. The fermentation process may include a suitable fermentation medium known to those skilled in the art. The medium may be prepared in-house or a commercially available medium may be utilized. It is further contemplated that the fermentation culture may include induction of protein expression by the introduction of the inducer isopropyl β-D thiogalactopyranoside (IPTG). The fermentation broth is harvested at the onset of the decay phase of the fermentation growth curve and subjected to a downstream purification process. It is contemplated that the downstream purification process may be designed by those skilled in the art based on the properties of the protein to be purified. The downstream process may include centrifugation, chromatography, filtration, dialysis, and combinations thereof.

[0106] In certain embodiments of the present disclosure, the expressed protein upon purification may be subjected to qualitative and quantitative analysis. It is contemplated that qualitative and quantitative analysis may be performed by those skilled in the art using any suitable method reported in the art. Proteins may be qualitatively and quantitatively analyzed for estimation of protein yield, protein purity, and analysis of N-terminal initiator methionine. Protein analysis may include, but is not limited to, the use of reverse-phase liquid chromatography (RP-HPLC), liquid chromatography mass spectrometry (LC-MS), high-resolution mass spectrometry (HR-MS), isoelectric focusing, capillary isoelectric focusing (cIEF), SDS-polyacrylamide gel electrophoresis, immunoblotting, etc.

[0107] Another aspect of the disclosure provides proteins having an N-terminal initiator methionine content of 2% or less.

[0108] In one embodiment of the present disclosure, the protein is contemplated to be a recombinant protein of therapeutic, industrial, and / or economic value. In another embodiment of the present disclosure, the protein having an N-terminal initiator methionine content of 2% or less is a lectin protein. Lectins are a unique group of proteins (or glycoproteins) with potent biological activity. They are an important group of bioactive proteins found in most living organisms and have long attracted widespread attention in the pharmaceutical field as diagnostic and therapeutic agents. To date, lectins from various sources, including plants, algae, fungi, and cyanobacteria, have been isolated and characterized and found to differ in physicochemical properties, such as molecular size and sugar specificity. Plant lectins are found in wheat, corn, tomato, peanut, kidney bean, banana, snow pea, lentil, soybean, mushroom, tuber, seed, mistletoe, potato, and many others. Similarly, fungal lectins have been isolated from mycelia, conidia, and Sclerotium rolfsii, among others.

[0109] In another embodiment of the present disclosure, the protein having an N-terminal initiator methionine content of 2% or less is a wild-type or modified lectin of Sclerotium rolfsii. Sclerotium rolfsii lectin (SRL) is a lectin isolated from the fungus Sclerotium rolfsii and has high binding specificity for the oncofetal Thomsen-Friedenreich saccharide antigen (Galβ1-3GalNAc-α-O-Ser / Thr, T or TF), which is expressed in over 90% of human cancers. This specificity for the TF antigen has been highly explored for its potential in cancer diagnostics and therapeutics. As disclosed in EP 2430041, a modified lectin protein with 141 amino acids was expressed in Escherichia coli as a stable and soluble protein using recombinant DNA technology. The modified lectin is similar to the native lectin in terms of its carbohydrate-binding specificity and apoptotic properties, enabling its use as a drug delivery agent for cancer therapy and as a diagnostic tool.

[0110] In another embodiment of the present disclosure, the protein having an N-terminal initiator methionine content of 2% or less is a wild-type or modified lectin of Sclerotium rolfsii. The lectin, expressed and purified as in the above-described embodiment of the present disclosure, comprises a protein selected from SEQ ID NO: 10, SEQ ID NO: 13, and SEQ ID NO: 16, wherein the protein upon purification comprises 2% or less N-terminal initiator methionines.

[0111] While considerable emphasis has been placed herein on particular features of the preferred embodiment, it will be understood that many additional features may be added to the preferred embodiment and that many modifications may be made thereto without departing from the principles of the present disclosure. These and other modifications in the preferred embodiment of the present disclosure will be apparent to those skilled in the art from the disclosure herein, and it is therefore clearly understood that the foregoing description is to be interpreted merely as an illustration of the present disclosure, and not as a limitation thereof. [Example]

[0112] The following examples are included to demonstrate preferred embodiments of the present disclosure. Those of skill in the art should understand that the compositions and techniques disclosed in the examples below represent techniques discovered by the inventors to function well in the practice of the present disclosure, and therefore can be considered to constitute preferred methods for doing so. However, those of skill in the art should, in light of the present disclosure, understand that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the present disclosure.

[0113] Example 1 DNA construct carrying the MAP gene A DNA construct containing a promoter, a methionine aminopeptidase (MAP) gene, and a terminator sequence was designed as shown in FIG.

[0114] The construct comprises the TAC promoter sequence specified in SEQ ID NO:5, the methionine aminopeptidase (MAP) gene specified in SEQ ID NO:3, and the T3te terminator sequence specified by SEQ ID NO:6.

[0115] The DNA construct prepared is represented as SEQ ID NO:2.

[0116] Example 2 Cloning of the DNA construct of SEQ ID NO:2 into the pET27b plasmid Cloning of the DNA construct of SEQ ID NO: 2 into the pET27b plasmid was carried out as shown in FIG.

[0117] The DNA construct of SEQ ID NO: 2 was cloned into the pET27b plasmid between the restriction sites BglII and SphI.

[0118] The modified pET27b plasmid containing the DNA construct (SEQ ID NO:2) is represented as SEQ ID NO:18.

[0119] Example 3 A DNA construct containing the nucleotide sequence of the protein to be expressed A DNA construct containing a promoter, the nucleotide sequence of the protein to be expressed, and a terminator was designed as shown in FIG.

[0120] The nucleotide sequence (SEQ ID NO: 9) expressing the recombinant protein of SEQ ID NO: 10 was used in the DNA construct.

[0121] SEQ ID NO:9 is amplified using gene-specific forward and reverse primers containing NdeI and BamHI sites, respectively, which are digested with restriction enzymes and used for cloning into the respective sites in the multiple cloning site of the plasmid of SEQ ID NO:18.

[0122] The DNA construct comprised the T7 promoter sequence specified in SEQ ID NO: 7, the nucleic acid sequence of SEQ ID NO: 9, and the T7 terminator sequence specified in SEQ ID NO: 8. The DNA construct is represented as SEQ ID NO: 11.

[0123] Similarly, DNA constructs containing the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 15 were prepared, respectively, and are represented as SEQ ID NO: 14 and SEQ ID NO: 17.

[0124] Example 4 Cloning of SEQ ID NO:9 into modified plasmid of SEQ ID NO:18 Cloning of the nucleotide sequence expressing the recombinant protein into the modified pET27b plasmid was performed as shown in FIG.

[0125] The nucleotide sequence (SEQ ID NO: 9) expressing the recombinant protein of SEQ ID NO: 10 was cloned between the restriction sites NdeI and BamHI in the MCS region on the antisense strand of the modified plasmid (SEQ ID NO: 18).

[0126] Similarly, modified plasmids containing the nucleotide sequences of SEQ ID NO: 12 and SEQ ID NO: 15 were prepared, respectively.

[0127] Example 5 Transformation of E. coli with vectors Each of the modified plasmids prepared in Example 4 was transformed into E. coli cells by the heat shock method as follows.

[0128] 100 ng of plasmid DNA was added to 100 μl of competent E. coli BL21 DE3 cells and incubated on ice for 30 minutes. The transformation mixture was then subjected to a 90-second heat shock at 42°C in a dry bath / water bath. 1 ml of Luria broth was gently added to the transformation mixture and incubated at 37°C ± 2°C for 60 minutes. 100 μl of the transformation mixture was plated onto Luria agar plates containing the appropriate antibiotic and incubated overnight at 37°C ± 2°C. Selected colonies were then subcultured into production medium to assess protein expression. Clones with the desired results were selected for further analysis.

[0129] Example 6 Expression and purification of expressed proteins Transformed E. coli BL21 DE3 cells were used to express the proteins specified in SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 16 using the process mentioned below.

[0130] The inoculation medium used contained 1.5% yeast extract (w / v), 0.75% Na2HPO42H2O (w / v), 0.5% dextrose (w / v), 0.1% MgSO4.7H2O (w / v), 0.5% NaCl (w / v), kanamycin at a final concentration of 20 μg / ml, and 0.1% (v / v) trace metal solution.

[0131] The fermentation medium contained 1% yeast extract (w / v), 0.3% KH2PO4 (w / v), 1.25% K2HPO4 (w / v), 0.5% (NH4)2SO4 (w / v), 1.2% dextrose (w / v), 0.1% MgSO4.7H2O (w / v), kanamycin at a final concentration of 20 μg / ml, and 0.1% (v / v) trace metal solution.

[0132] The transformed E. coli BL21 DE3 cells were inoculated into the inoculation medium under continuous shaking at 110 rpm at 30°C for 15 hours. Then, 600 ml of the inoculum was transferred to 6 L of fermentation medium at 14.7 OD at 600 nm. The fermentation process was carried out at 18°C ​​with an air flow rate of 0.5 to 2.0 VVM throughout the process.

[0133] Dissolved oxygen was maintained at approximately 60% by stirring and aeration with oxygen input. Foaming during the fermentation process was controlled with an antifoaming agent. Agitation was maintained at 400-950 RPM. Fermentation pH was maintained at 6.8 ± 0.6 using 10 N sodium hydroxide solution. At 5 hours, feeding with glycerol as a carbon source and yeast extract as a nitrogen source was initiated. At 10 hours, the fermentation culture was induced with 0.25 mM isopropyl β-D-thiogalactopyranoside (IPTG) to allow protein expression. Harvesting was performed at 48 hours / OD 105 at 600 nm.

[0134] The collected broth was centrifuged to obtain a pellet. The pellet was subjected to chromatography techniques. The purification involved passing through four columns. Column 1 was a Cellufine Max Qr ion exchange chromatography column. The eluate was further passed through a hydrophobic interaction chromatography column. The resulting eluate was passed through an SP Sepharose ion exchange chromatography column. The final column was a Source 30Q ion exchange chromatography column.

[0135] The purified proteins were further analyzed for quantitative and qualitative analysis.

[0136] Example 7 Analysis of expressed proteins The purified protein obtained in Example 6 was analyzed for the yield, purity, and content of the expressed protein.

[0137] The protein of SEQ ID NO: 10 expressed in E. coli without an exogenous MAP gene expression system was used as a control for analytical purposes.

[0138] The results obtained are tabulated in TABLE NO: 1.

[0139] Western blot analysis of the protein of SEQ ID NO: 10 co-expressed with methionine aminopeptidase and the protein of SEQ ID NO: 16 co-expressed with methionine aminopeptidase was performed to confirm expression of methionine aminopeptidase by the pET27b plasmid, with the respective control being the pET27b plasmid without the additional methionine aminopeptidase gene.

[0140] ULLB-0005(-) MAP uninduced - SEQ ID NO: 10 (control) without MAP gene ULLB-0005(-)MAP induced - SEQ ID NO: 10 without MAP gene (induced control) ULLB-1411-0011(-) MAP induction-free - SEQ ID NO: 16 (control) without MAP gene ULLB-1411-0011(-) MAP induction - SEQ ID NO: 16 without MAP gene (induction control) ULLB-0005 (+) MAP induction-free MAP gene SEQ ID NO: 10 ULLB-0005 (+) MAP induction - SEQ ID NO: 10 with MAP gene induction ULLB-1411-0011 (+) MAP induction-free MAP gene SEQ ID NO: 16 ULLB-1411-0011 (+) MAP induced - SEQ ID NO: 16 with MAP gene induced

[0141] The results can be seen in Figure 6.

[0142] [Table 1]

[0143] Conclusion: The resulting expressed protein of SEQ ID NO: 10 was observed to have less than 2% N-terminal methionine. Similar findings were found for the expressed proteins of SEQ ID NO: 13 and SEQ ID NO: 16. Western blot analysis revealed that upon induction of the expression vector, the bands were intense in the induced plasmids carrying the expressed proteins of SEQ ID NO: 13 and SEQ ID NO: 16 along with the methionine aminopeptidase gene.

[0144] Example 8 Analysis of various promoters for MAP expression The N-terminal initiator methionine content of the expressed protein in which the MAP gene was operably linked to one of the promoters selected from the araE promoter, the UV5 promoter, and the TAC promoter was evaluated.

[0145] The purified protein of SEQ ID NO: 10 (protein variant 1) obtained using three different promoters carrying the MAP gene was analyzed for yield as well as purity and content of the expressed protein.

[0146] The expressed protein of SEQ ID NO: 10 in E. coli without an exogenous MAP gene expression system (protein variant 1) was used as a control for analytical purposes.

[0147] [Table 2]

[0148] Conclusion: Upon evaluation, it was observed that expression of the MAP gene under the control of the TAC promoter resulted in a protein with an N-terminal methionine content of 2% or less.

Claims

1. a nucleotide sequence encoding a methionine aminopeptidase (MAP) protein or a variant thereof operably linked to a promoter sequence of SEQ ID NO: 5; a nucleotide sequence encoding a protein of interest operably linked to the promoter sequence of SEQ ID NO: 7; A recombinant plasmid comprising: A recombinant plasmid resulting in a protein of interest consisting of less than 2% N-terminal initiator methionines.

2. 2. The recombinant plasmid of claim 1, which is a modified pET27b plasmid.

3. The recombinant plasmid of claim 1 , wherein the MAP protein or variant thereof is further operably linked to a terminator.

4. The recombinant plasmid of claim 1, wherein the protein of interest is further operably linked to a terminator.

5. 2. The recombinant plasmid of claim 1, wherein the nucleotide sequence encoding a MAP protein is operably linked to a promoter sequence represented as SEQ ID NO:

2.

6. 2. The recombinant plasmid of claim 1, wherein the protein of interest is the protein of SEQ ID NO:

10.

7. 2. The recombinant plasmid of claim 1, wherein the target protein is the protein of SEQ ID NO: 13 or SEQ ID NO:

16.

8. 2. The recombinant plasmid of claim 1, wherein the MAP protein is the protein of SEQ ID NO: 4 or a variant thereof.

9. 6. Use of the recombinant plasmid according to claim 4 or 5, wherein the target protein can be used in cancer diagnosis or cancer treatment.

10. 10. Use of the recombinant plasmid of claim 1, which can be used to produce a protein of interest having less than 2% N-terminal initiator methionines.

11. 2. The recombinant plasmid of claim 1, wherein the nucleotide sequence encoding a protein of interest operably linked to a promoter is the sequence depicted in Figure 4.

12. A recombinant plasmid of SEQ ID NO: 18, further comprising a nucleotide sequence encoding a protein of interest operably linked to the promoter sequence of SEQ ID NO:

7.

13. A host cell comprising the recombinant plasmid of claim 1, which is a prokaryotic cell.

14. The host cell of claim 13, wherein the prokaryotic cell is Escherichia coli strain DE3.

15. a) providing a host cell harboring the recombinant plasmid of claim 1; b) culturing the host cells in a culture medium; c) purifying the protein of interest from the cultured host cells or the culture medium; 1. A method for producing a protein of interest, comprising: The method, wherein the purified protein consists of less than 2% N-terminal initiator methionines.

16. a nucleotide sequence of SEQ ID NO: 3 operably linked to a promoter sequence and a terminator sequence of SEQ ID NO: 5; a nucleotide sequence encoding a protein of interest operably linked to the promoter and terminator sequences of SEQ ID NO: 7; A recombinant plasmid comprising:

2. The recombinant plasmid of claim 1, which results in a protein of interest consisting of less than 2% N-terminal initiator methionines.

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