CN115867661A

CN115867661A - Production of soluble recombinant proteins

Info

Publication number: CN115867661A
Application number: CN202180021748.8A
Authority: CN
Inventors: M-J·张; N·奥加内森; A·李斯
Original assignee: Fina BioSolutions LLC
Current assignee: Fina BioSolutions LLC
Priority date: 2020-03-16
Filing date: 2021-03-12
Publication date: 2023-03-28
Also published as: KR20220154221A; EP4121541A2; JP2023517708A; CA3168571A1; WO2021188379A3; AU2021239914A1; WO2021188379A2; JP7449000B2

Abstract

The present invention relates to methods and compositions for expressing and purifying products, such as peptides and proteins, in microorganisms. In particular, the preproduct is expressed recombinantly, wherein the cytoplasm of the microorganism alters the expressed preproduct to produce the active/final or other desired form of the product. Alterations associated with expression of the desired recombinant product include altering the redox state of the cytoplasm to allow proper protein folding, site-directed cleavage of the preprotein to activate the protein, site-directed cleavage of the unwanted methionine from the N-terminus of the protein, and/or one or more linkages to form the desired protein configuration, all within the same cell.

Description

Production of soluble recombinant proteins

In the invention

The invention was made with U.S. government support under grant number 1R43AI148018-01A1 fain, R43ai148018, awarded by the national institutes of health, and the U.S. government has certain rights in the invention.

Reference to related applications

This application claims U.S. provisional application No. 63/152,954, filed 24/2/2021; U.S. provisional application No. 62/990,083, filed 3, 16, 2020; and U.S. application No. 16/819,775, filed 3/16/2020, and currently pending, the entire contents of each of which are specifically incorporated by reference.

Technical Field

The present invention relates to methods and compositions for expressing and purifying products, such as peptides and proteins, in microorganisms. In particular, the preproduct is expressed recombinantly, wherein the cytoplasm of the microorganism alters the expressed preproduct to produce the product in its final or useable form. Alterations include cytoplasmic redox state transitions and site-directed cleavage and/or ligation.

Description of the background

Coli is a widely used host to produce recombinant proteins for research and therapeutic purposes. The recombinant protein may be expressed in the cytoplasm or periplasm of E.coli. One limitation of cytoplasmic recombinant protein expression in E.coli is that, in order to initiate expression of the recombinant protein in E.coli, the coding sequence for the protein should start with the ATG codon, which is translated into formylmethionine, and then processed by formylmethionine deformylase to become the N-terminal methionine. Thus, for recombinant protein expression in E.coli, the ATG codon is added to the native or mature protein sequence. During intracellular expression of recombinant proteins, the N-terminal methionine is usually cleaved off by the endogenous E.coli Methionine Aminopeptidase (MAP). This process is not necessarily effective for recombinant proteins, even if the adjacent residues are best suited for cleavage, which may be due to overexpression of the recombinant protein and the presence of a limited amount of MAP. As a result, a large number of recombinant proteins may have methionine as the first amino acid. This is undesirable for most proteins because the N-terminal methionine is not part of the mature protein sequence. The presence of the N-terminal methionine may also cause structural changes in the protein that affect its function. Existing methods to ensure efficient cleavage of the N-terminal methionine include in vitro treatment with recombinant MAP. Another approach is to add the MAP coding sequence to an expression vector and thus co-express MAP with the recombinant protein. In the latter case, co-expression of MAP may reduce expression of the desired recombinant protein. Both methods are time consuming and expensive to implement. Therefore, it is desirable to have an e.coli expression strain in which MAP is highly expressed without significantly inhibiting recombinant protein expression. This will allow for the production of increased amounts of the target protein with its native sequence in the cytoplasm.

Recombinant proteins expressed in the cytoplasm of E.coli may form insoluble inclusion bodies. Proteins in inclusion bodies can refold in vitro to form soluble proteins. These proteins will contain an N-terminal methionine, which is undesirable.

Coli cytoplasm has a reducing environment and recombinant proteins containing disulfide bonds are generally insoluble when expressed intracellularly. In contrast, the periplasm of E.coli has an oxidizing environment. Thus, many recombinant proteins containing disulfide bonds are secreted into the periplasm to ensure proper folding and solubility. The signal peptide that directs the recombinant protein into the periplasm is cleaved off during the secretion process, resulting in the production of a protein with the native amino acid sequence. However, the translocation machinery that directs proteins to the periplasm is limited and thus the periplasmic expression levels of recombinant proteins are generally low. On the other hand, expression in the cytoplasm of E.coli may result in several grams of recombinant protein per liter of cell culture. Thus, it would be desirable to be able to express soluble, correctly folded disulfide-bonded proteins in the cytoplasm. Furthermore, it would be desirable if these proteins could be produced without the N-terminal methionine.

Commercially available strains of E.coli, such as Origami ^® (EMDMillipore)、Shuffle ^® (New England Bio) (with gor-/trx-mutation) can produce soluble intracellular proteins containing disulfide bonds, but these cell strains are defective and cannot grow to high densities, limiting yield. Thus, although these strains are suitable for the production of research materials, their low growth levels make them difficult to use commercially. Thus, there is a need for strains expressing high levels of correctly folded intracellular disulfide-bonded proteins without an N-terminal methionine.

Summary of The Invention

The present invention overcomes the problems and disadvantages associated with current strategies and designs, and provides novel compositions and methods for producing recombinant peptides and proteins.

One embodiment of the present invention relates to methods of producing recombinant peptides and proteins in bacteria, comprising: expressing the protein in a bacterium comprising an expression vector encoding a protein sequence comprising a promoter, and further comprising a gene under the control of the promoter, which is integrated into the genome of the host, wherein the polypeptide expressed by the gene facilitates the expression, folding or solubilization of the recombinant protein in the cytoplasm, and isolating the protein.

Another embodiment of the invention relates to methods of producing recombinant peptides and proteins in bacteria comprising: expressing the protein in a bacterium containing an expression vector encoding a protein sequence including a promoter, and which further expresses a peptidase gene integrated into the genome of the host cell, wherein peptidase expression is under the control of the promoter such that the peptidase acts on the expressed protein and removes the formylmethionine group from the N-terminal portion of the protein; and isolating the protein. The peptidase which removes the N-terminal methionine may be referred to as Methionine Aminopeptidase (MAP). Preferably, the integrated gene contains a ribosome binding site, an initiation codon and an expression enhancer and/or repression region. Preferably, the recombinant cell has reduced activity of only one disulfide reductase or reduced activity of only two disulfide reductases. Preferably, the recombinant cell is an escherichia coli cell or a derivative or strain of escherichia coli, and preferably the recombinant protein expressed comprises tetanus toxin, tetanus toxin heavy chain protein, diphtheria toxoid, tetanus toxoid, pseudomonas exoprotein a, pseudomonas aeruginosa toxoid, bordetella pertussis toxoid, clostridium perfringens toxoid, escherichia coli thermolabile toxin B subunit, neisseria meningitidis outer membrane complex, haemophilus influenzae protein D, flagellin Fli C, cytokines, single chain antibodies, camelid antibodies (camelids), nanobodies and fragments, derivatives and modifications thereof. Also preferably, the recombinant protein may be preprotein relaxin, insulin and a member of the insulin-like family. Preferably, the integrated gene and/or expression vector contains an inducible promoter for the peptidase. Expression includes inducing the inducible promoter with a first inducing agent and contains an expression vector encoding a recombinant peptide or protein that can be induced with a second inducing agent. Preferably, the first and second inducers are the same, although they may be different. Preferably, the first integrated gene or expression vector contains an inducible second promoter and expressing the peptidase includes inducing the inducible second promoter with the first inducing agent. Preferably, the separation comprises chromatography, wherein chromatography comprises a sulfate resin, a gel resin, an active sulfated resin, a phosphate resin, a heparin resin or a heparin-like resin. Preferably, the isolated expressed protein is conjugated to polyethylene glycol and/or a polyethylene glycol derivative or to a polymer such as, for example, a polysaccharide, a peptide, an antibody or a portion of an antibody, a lipid, a fatty acid, a small molecule, a hapten or a combination thereof.

Another embodiment of the invention relates to a method of producing a soluble or insoluble peptide comprising: expressing a peptide having a formylmethionine group at the N-terminus of the peptide from a recombinant cell containing an expression vector encoding the peptide, and expressing a peptidase acting on the expressed peptide and removing the formylmethionine group from the N-terminus of the peptide from the integrated gene of the recombinant cell; and isolating the peptide.

Another embodiment of the present invention relates to a method of producing a peptide comprising: expressing a peptide having a formylmethionine group at the N-terminus of the peptide from a recombinant cell containing an expression vector encoding the peptide, wherein the recombinant cell has reduced activity of one or more disulfide reductase enzymes, and the expression vector comprises a promoter functionally linked to a coding region of the peptide, wherein the reduced activity of the one or more disulfide reductase enzymes results in a transition from a redox state in the cytoplasm to a higher oxidation state as compared to a recombinant cell in which the activity of the one or more disulfide reductase enzymes is not reduced, and expressing a peptidase from an integrating gene in the recombinant cell which acts on the expressed peptide and removes the formylmethionine group from the N-terminus of the peptide; and isolating the peptide. Preferably, the expression vector contains a ribosome binding site, an initiation codon and an expression enhancer/repressor region. Preferably, the recombinant cell has reduced activity of only one disulfide reductase or only two disulfide reductases. Preferably, the one or more disulfide reductase enzymes comprise one or more of an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase or a glutathione reductase. Preferably, the recombinant cell is an escherichia coli cell or a derivative or strain of escherichia coli, and the peptide or protein includes tetanus toxin, tetanus toxin heavy chain protein, diphtheria toxoid, tetanus toxoid, pseudomonas exoprotein a, pseudomonas aeruginosa toxoid, bordetella pertussis toxoid, clostridium perfringens toxoid, botulinum toxin, escherichia coli thermolabile toxin B subunit, neisseria meningitidis outer membrane complex, haemophilus influenzae protein D, flagellin Fli C, cytokines, single chain antibodies, camelid antibodies, nanobodies, and fragments, derivatives, and modifications thereof. Preferably, the promoter is an inducible promoter and expression comprises inducing the inducible promoter with an inducing agent.

Preferably, the separation comprises chromatography, wherein chromatography comprises a sulphate resin, a gel resin, an active sulphated resin, a phosphate resin, a heparin resin or a heparin-like resin. Preferably, the isolated peptide is conjugated to polyethylene glycol (PEG) and/or a derivative of PEG or to a polymer such as, for example, a polysaccharide, a peptide, an antibody or a portion of an antibody, a lipid, a fatty acid, a small molecule, a hapten or a combination thereof.

Another embodiment of the invention relates to an E.coli cell line containing a gor mutation. Preferably, the cell line comprises cells obtained or derived from ATCC deposit No. PTA-126975.

Another embodiment of the present invention relates to a method of producing a protein comprising: the proprotein is expressed in recombinant cells containing the recombinantly engineered protease gene. Preferably, the protease gene and/or the preprotein gene contain a promoter and/or a translation inducing sequence. The promoters may be the same or different, and the translation inducing sequences (if present) may be the same or different for the genes. Preferably, after expression of the pre-protein, expression of the protease gene is induced such that the pre-protein is cleaved to form a protein; and harvesting the protein. Preferably, the preprotein is selected from the group consisting of proinsulin, proinsulin-like protein, prorelaxin, pro-opiomelanocortin, zymogen, prohormone, proangiotensinogen (proangiotensinogen), protrypsinogen (protrypsinogen), prochymotrypsinogen (prochloryprogen), proproteinogen, prothrombin of the coagulation system, prothrombin, plasminogen, proproteinogen of the complement system, procaspases (procaspases), propacifastin, elastase, lipogen, carboxypeptidase, proteins containing cleavable leaders, cleavable tag sequences, proteins containing cleavable additional N-or C-terminal amino acids (e.g., met). Preferably, the protease gene is integrated into the genome of the recombinant cell. Also preferably, a methionine aminopeptidase gene is integrated into the genome of the recombinant cell, wherein expression of the methionine aminopeptidase gene removes the N-terminal methionine from the pre-protein or the protein. The expression of the methionine aminopeptidase gene is preferably under the control of an inducer sequence, and the inducer sequence of the methionine aminopeptidase and the translation inducing sequence of the preprotein may be the same or different. Also preferably, the recombinant cell, which may be an e.coli having a gor mutation, has reduced activity of one or more disulfide bond reductase enzymes.

Another embodiment of the invention relates to a recombinant cell line containing a methionine aminopeptidase gene and a protease gene, both of which are integrated. Preferably, further, the cell has a decreased activity of one or more disulfide reductase enzymes, which may be attributable to gor mutation.

Additional embodiments and advantages of the invention are set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.

Description of the invention

Proteins in E.coli are usually expressed with a methionine at the N-terminus, since the corresponding ATG codon is required for translation initiation. Thus, proteins expressed in cells contain an N-terminal methionine that is not part of the native amino acid sequence (unless the native sequence begins with methionine). Removal of the N-terminal methionine by MAP may be important for the function and stability of the protein. Endogenous Methionine Aminopeptidase (MAP) can cleave the N-terminal methionine of newly synthesized proteins, usually up to 60-70%. However, in highly expressed recombinant proteins, the level of activity of endogenous MAP may not be sufficient to remove the desired amount of the N-terminal methionine. Removal of the N-terminal methionine is an important issue in the production of intracellular recombinant proteins in E.coli. Therefore, escherichia coli strains which can efficiently cleave the undesired N-terminal Met are highly desired.

Solubility and proper folding of recombinant proteins expressed in cells are issues of concern in E.coli expression systems. The E.coli cytoplasm has a reducing environment which is unfavorable for disulfide bond formation. As a result, recombinant proteins containing disulfide bonds are generally insoluble when expressed in cells. Purification of these insoluble proteins can be difficult, expensive, and time consuming. High cell densities are preferred for the production of recombinant proteins, particularly for commercial use.

It has surprisingly been found that microorganisms genetically engineered to express a large number of appropriately configured recombinant proteins, which also effectively remove the N-terminal formylmethionine, have been developed. These microorganisms were genetically engineered to express soluble recombinant proteins containing disulfide bonds in the cytoplasm and to remove the N-terminal methionine. These microorganisms produce large quantities of correctly folded intracellular recombinant proteins containing disulfide bonds and no methionine at the N-terminus of the protein.

Preferably, the microorganism contains one or more Methionine Aminopeptidase (MAP) genes incorporated into the bacterial genome. Peptidases that remove the N-terminal methionine include, but are not limited to, E.coli MAP, yeast MAP, and human MAP, and mutants thereof, all of which are available. The coding sequence for MAP is inserted into the bacterial genome under the control of an inducible promoter, preferably in a manner that prevents genome disruption. Having an inducible promoter allows for initiation of expression of additional MAP at a selected time, preferably only when more MAP is required to effectively remove formylmethionine from the overexpressed recombinant protein. The promoter of the MAP gene may be the same as or different from the promoter used for the recombinant protein. In one example, the tac promoter is used as a lactose/IPTG inducible promoter for both the MAP gene and the recombinant protein, and thus can induce expression of MAP and the recombinant protein simultaneously. Different combinations of inducible promoters for expression of MAP and for expression of recombinant proteins can be used to regulate the timing of the respective expression.

Incorporation of additional MAP into the genome is particularly desirable because the resulting stable bacterial expression strains can be used for intracellular production of recombinant proteins in which the unwanted f-Met is cleaved in vivo. Previously, the unwanted N-terminal methionine has been removed by post-translational in vitro digestion with purified MAP or by co-expression of MAP in the same vector as the recombinant protein or using additional vectors. The process is lengthy and complex. These microorganisms greatly simplify the protein expression and purification process by using cell lines that can cleave f-met in vivo as desired.

MAP cleaves the N-terminal methionine with special requirements for adjacent amino acids. Intracellular proteins devoid of an N-terminal methionine may be more efficiently produced using at least one additional MAP. If more than one MAP is inserted, they may be the same or different MAP genes. The transcription of these MAP genes may be under the same or different inducible promoters. These promoters may be the same as or different from the promoter used to express the recombinant protein. Combinations of inducible promoters can be used to control the timing and amount of production of recombinant proteins without the N-terminal methionine expressed in the cell.

Coli strains capable of expressing soluble, correctly folded disulfide-containing intracellular recombinant proteins have been described (see U.S. Pat. nos. 10,597,664 and 10,093,704). An example of such a strain is E.coli (BL 21 Gor-). BL21 Gor-has been used to express soluble, correctly folded disulfide-containing intracellular recombinant proteins at high levels, including the vaccine carrier protein CRM ₁₉₇ A genetically detoxified diphtheria toxin. Approximately 60% CRM expressed in these cells ₁₉₇ Contains an N-terminal methionine. Insertion of an additional MAP gene under the control of a promoter into this strain allows the production of soluble, correctly folded intracellular recombinant proteins containing disulfide bonds and no N-terminal methionine. An example of such a strain is the E.coli (BL 21 Gor/Met) strain. The strain can produce intracellular soluble proteins with disulfide bonds and no N-terminal methionine in amounts of grams per liter of cell culture. CRM expressed in BL21 Gor/Met cells ₁₉₇ Containing very low levels of the N-terminal methionine. In addition, the induced methionine aminopeptideIncorporation of the enzyme gene into the E.coli genome did not significantly affect CRM ₁₉₇ The level of expression.

The MAP gene is inserted into the genome by homologous recombination, although there are several other options that facilitate insertion. The method for generating Gor/Met cell lines is an example of gene insertion. For Gor/Met cells, the MAP gene was inserted into the Gor locus in BL21 Gor-cells using the red recombinase system. The MAP gene was excised from the BL21 genome using PCR and placed under the control of the Tac promoter and downstream of the Chloramphenicol Acetyltransferase (CAT) gene flanked by two short Flippase Recognition Target (FRT) sequences. Together, the MAP and CAT genes form a transgene cassette. Fifty bases of each of the sequences flanking the upstream and downstream of the original Gor locus were added by PCR to the transfer cassette upstream and downstream, respectively. The final PCR product was used for transformation into BL21 Gor-cell line that had been transformed with Red recombinase. Expression of red recombinase in the cell promotes homologous recombination of sequences flanking the Gor locus with the transfer cassette. Bacterial colonies demonstrating resistance to chloramphenicol were successfully inserted into the transfer cassette. The confirmed bacteria were then transformed with a flippase gene whose expression recognized the FRT sequences flanking the CAT gene and the gene was spliced out to leave the inserted MAP. Thus, gor/Met cell lines have a MAP gene located at the Gor locus without interfering with other parts of the genome.

For production of large amounts of protein from E.coli host cells, such as CRM ₁₉₇ The f-Met present at the N-terminus of the protein is enzymatically removed in the cytoplasm. The production is usually quantified in mg/L of bacterial cell culture. The protein yield can reach 25 mg/L or more, 50mg/L or more, 100 mg/L or more, 200 mg/L or more, 300 mg/L or more, 400 mg/L or more, 500 mg/L or more, 600 mg/L or more, 700 mg/L or more, 800 mg/L or more, 900 mg/L or more, 1,000 mg/L or more, 1,500 mg/L or more, or 2,000 mg/L or more. As desired, the expressed protein includes full-length and/or truncated proteins, as well as modified amino acid sequences of the protein. Modifications include one or more of conservative amino acid deletions, substitutions and/or additions. Conservative modifications are modifications that maintain the functional activity and/or immunogenicity of the molecule, althoughThe activity and/or immunogenicity may be increased or decreased. Examples of conservative modifications include, but are not limited to, amino acid modifications (e.g., single, double, and other short amino acid additions, deletions, and/or substitutions), modifications outside the active or functional sequence, residues that can be used in conjugation to form a vaccine, modifications due to serological variations, modifications that increase immunogenicity or increase conjugation efficiency, modifications that do not substantially alter binding to heparin, modifications that maintain proper folding or three-dimensional structure, and/or modifications that do not significantly alter the immunogenicity of the protein or protein portion that provides protective immunity.

The recombinant cells used are preferably escherichia coli bacteria, and preferably escherichia coli genetically engineered to convert the cytoplasmic redox state to a higher oxidation state, such as, for example, by mutation of one or more disulfide reductase genes (such as, for example, oxidoreductase, dihydrofolate reductase, thioredoxin reductase, glutamate cysteine lyase, disulfide reductase, protein reductase and/or glutathione reductase). Preferably, one or more disulfide reductase genes are mutated and rendered non-functional or functionally limited such that the redox state of the cytoplasm of the cell is converted to a higher oxidation state compared to the wild type without impairing viability. Oxidized protein folding involves disulfide bond formation and isomerization and includes CRM ₁₉₇ Play a key role in the stability and solubility of many proteins. Disulfide bond formation and cleavage is typically catalyzed by thiol-disulfide redox enzymes. These enzymes are characterized by one or more Trx folds, which consist of four-stranded β -sheets surrounded by three α -helices, with a CXXC redox active site motif. The assembly of various Trx modules has been used to construct different thiol oxidoreductase enzymes found in prokaryotes and eukaryotes. In the bacterial periplasm, the protein is maintained in the appropriate oxidation state by the combined action of the DsbB-DsbA and DsbD-DsbC/DsbE/DsbG pairs. Protein expression systems are well known in the art and are commercially available. Also preferred are E.coli expression strains that constitutively express chromosomal copies of disulfide isomerase DsbC. DsbC facilitates the correction of the wrongly oxidized protein to its correct form.Cytoplasmic DsbC is also a chaperone that can assist in protein folding without the need for disulfide bonds.

The recombinant bacteria contain an expressible protein sequence in which the f-Met present at the N-terminus of the newly expressed protein is enzymatically removed. Preferred host cells include, but are not limited to, cells genetically engineered to convert the cytoplasmic redox state to a higher oxidation state, which contain and express an inducible MAP gene. Preferred cells are prokaryotes such as E.coli expression systems, bacillus subtilis expression, and other bacterial cell expression systems. Preferably, the cell contains a protein expression system for expressing the exogenous or non-native sequence. Also preferably, the sequence to be expressed consists of an expression vector containing one or more of: inducible promoters (e.g., preferably induced with a particular medium), an initiation codon (e.g., ATG), a ribosome binding site, and/or a modified sequence between the ribosome binding site and the ATG initiation codon or between the initiation codon and the sequence to be expressed. Preferred modified or spacer sequences include, for example, a number of nucleotides greater or less than 9 (e.g., 7-12 nucleotides), and preferably not 9 nucleotides.

It has also been surprisingly found that recombinant cells can be developed which contain additional proteases which efficiently cleave one or more different preproteins and/or proproteins from an inactive to an active configuration. These proteins are often referred to as zymogens (zymogens) that require post-translational modifications (e.g., proenzymes). When an active protein is detrimental but requires expression, cells often use protein precursors. By integrating these proteins into recombinant cells, expression can be achieved safely and economically and in large quantities. The protease gene expressing the protease performing the specific cleavage from inactive to active may be integrated into the cell genome or transformed with a vector containing the protease gene of interest, all as described herein. The introduced protease gene may be placed under the control of a promoter together with the recombinant gene to be expressed and collected and the methionine-cleaving protease gene, or the protease gene may have a different promoter. Similarly, the gene may be inducible alone or substantially simultaneously with the recombinant gene to be expressed and collected and the methionine cleaving protease gene. Preferably, when expression of the recombinant protein is sufficiently complete, the second protease will be activated and process the proprotein into an active state. Preproteins that would be effective in saving time and cost include, but are not limited to, preproinsulin to insulin, preproinsulin-like protein to insulin-like protein, preprocesulin to relaxin, proopiomelanocortin to opiomelanocortin, proenzyme to enzyme, and prohormone to hormone, as well as removal of signal peptides, leader sequences, tags, and the like from proteins. Generally, the introduced protease is specific for the protein to be cleaved. Additional proteins that may be efficiently produced herein include, but are not limited to, angiotensinogen, trypsinogen, chymotrypsinogen, pepsinogen, proteins of the coagulation system (e.g., prothrombin, plasminogen), proteins of the complement system, procaspase, pacifastin, elastase, lipogen, carboxypeptidase. In addition, certain genes may be modified to include a portion (e.g., a leader or tag or internal sequence) that allows the protein to be expressed in an inactive form that is converted to an active form only upon cleavage by a protease whose gene is also introduced into the cell and subsequently activated.

For example, a gene of interest is inserted into a genome with a different promoter. Inducing cells to express genes with disulfide bonds, and methionine peptidase to trim methionine. Once an appropriate amount of the pruned protein is produced in the cytoplasm, the second promoter is induced, which processes the protein into its final or active form. Expression of active proteins during growth, such as trypsin, phagocytose large quantities of the desired protein in the cytoplasm and interfere with expression of the recombinant protein. This approach would avoid the need for in vitro processing of the expressed proprotein.

Another embodiment of the present invention relates to a recombinant protein expressed in E.coli or another host cell in which f-met present at the N-terminus of the recombinant protein is enzymatically removed using an expression vector having an inducible promoter and/or a modified sequence between a ribosome binding site and the ATG initiation codon. PreferablyThe expression vector comprises a lactose/IPTG inducible promoter, preferablytacA promoter, and a sequence between the ribosome binding site and the ATG initiation codon.

Another embodiment of the invention includes expression constructs of nucleotide or amino acid sequences with or without regulatory regions. The regulatory region regulates protein expression by adding one or more sequences that facilitate nucleic acid recognition to increase expression (e.g., initiation codon, enzyme binding site, translation or transcription factor binding site) or inhibit expression (e.g., operon). Preferably, the regulatory element of the present invention comprises a ribosome binding site and an initiation codon upstream of the coding sequence of the recombinant protein and a coding sequence which is different from the coding sequence of the recombinant protein.

Another embodiment of the invention relates to proteins and peptides and their parts and domains that can be made according to the methods disclosed herein. Proteins and peptides include, but are not limited to, those that can be expressed cytoplasmic without a leader or tag sequence and at commercially significant levels, e.g., according to the methods disclosed and described herein. Preferably, these proteins and peptides show correct folding after expression in the recombinant cells of the invention. The recombinant cells of the invention preferably exhibit reduced activity of one or more disulfide reductase enzymes, preferably less than five disulfide reductase enzymes, preferably less than four disulfide reductase enzymes, and preferably less than three disulfide reductase enzymes. Preferably, expression of proteins and peptides is increased in the recombinant cells of the invention, but may not be reduced or not significantly reduced as compared to expression in recombinant cells in which the activity of one or more disulfide reductase enzymes is not reduced. Proteins and peptides that can be expressed in the methods disclosed herein include, but are not limited to, for example, tetanus toxin heavy chain protein, diphtheria toxoid, CRM, tetanus toxoid, pseudomonas exoprotein a, pseudomonas aeruginosa toxoid, bordetella pertussis toxoid, clostridium perfringens toxoid, escherichia coli heat labile toxin B subunit, neisseria meningitidis outer membrane complex, haemophilus influenzae protein D, flagellin Fli C, horseshoe crab hemocyanin, and fragments, derivatives, and modifications thereof.

Another embodiment of the invention relates to portions and domains of proteins that are expressed, genetically fused or fused to another molecule by chemical modification or conjugation (e.g., carbodiimide, 1-cyanodimethylaminopyridinium tetrafluoroborate (CDAP)). Preferred other molecules are molecules such as, but not limited to, other proteins, peptides, lipids, fatty acids, carbohydrates and/or polysaccharides, including molecules that prolong half-life (e.g., PEG, antibody fragments, such as Fc fragments), stimulate and/or increase immunogenicity or reduce or eliminate immunogenicity.

Many proteins contain an N-terminal serine or threonine, or it is possible that a gene is expressed with an N-terminal serine or threonine. The N-terminal serine or threonine can be selectively activated, making it available for conjugation. The presence of the N-terminal methionine blocks the ability of these amino acids to be selectively activated. The method described in this patent allows cleavage of the N-terminal methionine, allowing the production of a protein with the desired N-terminal amino acid. Typical conjugation partner molecules include, but are not limited to, polymers such as, for example, bacterial polysaccharides, polysaccharides derived from yeast, parasites, and/or other microorganisms, polyethylene glycol (PEG) and PEG derivatives and modifications, dextran and derivatives, dextran-modified fragments and derivatives. An example of a conjugated compound is PEGASYS (polyethylene glycol interferon alpha-2 a). Other polymers, such as dextran, also increase the half-life of the protein and reduce the immunogenicity of the conjugate partner. The polymers may be randomly linked or targeted by site-specific conjugation, such as, for example, by modification of N-terminal serine and/or threonine. In addition, selective oxidative chemical groups can be used for modification of site-specific conjugation.

Another embodiment of the present invention relates to a method of producing a peptide comprising a domain, fragment and/or portion, comprising: expressing the peptide from a recombinant cell containing an expression vector encoding the peptide, wherein the recombinant cell has reduced activity of one or more disulfide bond reductase enzymes, and the expression vector contains a promoter functionally linked to a coding region for the peptide, wherein the one or more disulfide bond reductase enzymes comprises one or more of an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase, or a glutathione reductase; and isolating the expressed peptide, wherein the expressed peptide is soluble, and wherein the protein or peptide is expressed at the N-terminus with f-met, which is removed by a peptidase also expressed in the recombinant cell. Preferably, the expression vector contains a ribosome binding site, an initiation codon and optionally an expression enhancer/repressor region. Preferably, the recombinant cell has reduced activity of only one disulfide reductase, only two disulfide reductases, or two or more disulfide reductases. Preferably, the reduced disulfide reductase activity results in a transition from the cytoplasmic redox state to a higher oxidative state as compared to a recombinant cell in which the activity of the one or more disulfide reductase enzymes is not reduced. Preferably, the recombinant cell is an E.coli cell or a derivative or strain of E.coli. Preferably, the expressed soluble peptide comprises a naturally folded protein or a domain of a protein. The promoter may be a constitutive or inducible promoter, whereby expression comprises induction of the inducible promoter with an inducing agent. Preferred inducers include, for example, lactose (PLac), isopropyl beta-D-1-thiogalactoside (IPTG), substrates and derivatives of the substrates. In a preferred embodiment, the genome of the recombinant cell contains an additional gene, preferably containing a coding region for a peptidase, which preferably acts on and selectively cleaves peptides or proteins expressed from the expression vector. Preferably, the recombinant protein expression vector contains an inducible promoter that is the same as or different from the MAP gene that has been inserted into the genome. The additional genes and the genes in the expression vector may be induced together with the same inducing agent, or with different inducing agents, optionally at different times, depending on the promoter. Preferably, the peptidase acts on and cleaves peptides co-expressed with the peptidase. Preferably, the expressed peptide is conjugated to a polymer such as, for example, dextran, a bacterial capsular polysaccharide, polyethylene glycol (PEG), or a fragment, derivative or modification thereof. Preferably, the expressed peptide is coupled to a polymer comprising, for example, a polysaccharide, a peptide, an antibody or a portion of an antibody, a lipid, a fatty acid, or a combination thereof.

Another embodiment of the invention includes conjugates of proteins expressed and cleaved according to the disclosure herein, including fragments, domains, and portions thereof as disclosed and described herein.

Another embodiment of the present invention includes fusion molecules, including fragments, domains and portions thereof, of the proteins as disclosed and described herein.

Another embodiment of the present invention includes a protein vaccine as disclosed and described herein, including fragments, domains and portions thereof.

The following examples illustrate embodiments of the present invention but should not be construed as limiting the scope of the invention.

Example 1. The MAP gene was inserted into various loci of the bacterial genome.

Efficient removal of the N-terminal methionine from the overexpressed intracellular recombinant protein was achieved by inserting a MAP gene with a promoter, preferably an inducible promoter, into the E.coli genome. In this way a permanent cell line was generated which expresses the MAP gene under the control of the inducer. The recombinant gene is cloned into the cell, also under the control of an inducer. Therefore, the MAP gene can be expressed at a desired time to efficiently cleave the N-terminal methionine from the expressed recombinant protein. Many MAP enzymes other than E.coli MAP are known or have been designed. MAPs from other species may have different selectivities for adjacent amino acids. Some MAP has been genetically altered to place less stringent requirements on non-bulky amino acids adjacent to the N-terminal methionine. Inserting one or more of these MAP's with inducible promoters into the E.coli genome will expand the range of N-terminal sequences that can be efficiently processed.

Insertion of recombinant genes into the genome may disrupt the structure of the E.coli genome and may impair cell growth. One safe method of inserting the MAP gene into the genome of E.coli is to use a live strain in which the gene has been deleted and replace the deleted gene with the MAP gene. In this way, by replacing one gene with another, the probability of disrupting the genome can be reduced. Two strains of E.coli are widely used for the manipulation of genes and the expression of recombinant proteins: k12 strain and B strain. Coli strains, including K12 and B strains, can be used as host cells for insertion of the MAP gene.

Insertions at erroneous sites can be lethal to the bacterium, such as indels in the open reading frame of the essential gene or at sites that disrupt the control elements. Three illustrative protocols for inserting a MAP gene with an inducible promoter and terminator are:

(1) The MAP gene is inserted upstream or downstream of the already defined gene. For example, a recombinant MAP gene may be inserted downstream of an endogenous MAP gene. The E.coli MAP gene has been studied and the gene structure has been defined. Downstream insertion of recombinant genes does not interfere with the expression of other genes.

(2) The MAP gene was inserted into the site of the gene that had been previously deleted. The generation of Gor/Met cell lines is an example of the insertion of MAP genes into deleted gene sites. Gor-cells were generated by deleting the Gor gene in BL21 cells. Gor/Met cell lines were generated by inserting the MAP gene into the Gor locus of BL21 Gor-cells.

(3) Insertion/replacement of non-essential genes. As an example of the third method, a MAP gene was inserted to replace T7 RNA polymerase in BL21 (DE 3) cells. BL21 (DE 3) encodes a very active T7 RNA polymerase in the DE3 fragment, which can transcribe recombinant genes under the control of the T7 promoter. The T7 polymerase gene may be replaced by the MAP gene if the recombinant gene is transcribed by an intrinsic RNA polymerase under the control of the T5 promoter.

The above insertion site selection requires site-directed mutagenesis techniques. Many methods are known for manipulating bacterial genomes, and two examples are disclosed herein. The most commonly used method for insertion/deletion of a gene is Red recombination. This technique has been widely used for mutations in bacterial as well as eukaryotic genomes, and PCR was begun to be used to introduce short DNA sequences complementary upstream and downstream of the selected insertion site flanking the gene of interest. The PCR product is then electroporated into E.coli which has expressed the red recombinase in a previously transformed temperature-sensitive vector. The red recombinase facilitates homologous recombination of the inserted gene at the selected site. Red recombinase can be removed by culturing the bacteria at 42 ℃ because the Red recombinase gene is on a temperature sensitive plasmid.

To enhance the selection of positive clones, a marker gene may be introduced and later removed after confirmation of positive clones. In one approach, a flippase recognition signal is introduced to flank a marker gene, such as an antibiotic gene, which is cloned downstream of the inserted gene. The PCR product used for gene insertion will then include the marker gene. After a recombination event has occurred, the marker gene can be used for positive clone selection. Once positive clones were confirmed, flippase expression was introduced into the bacteria to remove the marker gene between the two flippase recognition sites. This insertion is marked with a scar (scar) that contains a flippase recognition sequence at the insertion site. This method was used to produce Gor/Met escherichia coli strains by inserting Met gene into the deleted Gor gene, and is described in example 2 below.

When combined with Red recombining, CRISPR technology can be used in e. In CRISPR-assisted red recombianing, two plasmids and one oligonucleotide are utilized. One plasmid encodes a constitutively expressed Red recombinase and a Cas9 protease. Another encodes CRISPR guide RNA in which the insertion site is cloned. Both plasmids have two compatible origins of replication. The oligonucleotide is made to contain the gene of interest flanked by insertion site sequences. Coli cells were transformed with these three elements, and the surviving colonies should be colonies into which the gene had been inserted: the Cas9 protease will bind to the CRISPR guide RNA to scan the insertion site. Once the insertion site is located, cas9 will cleave its double stranded DNA and allow red recombinase access for homologous recombination with the target protein between the cleavage site and the oligonucleotide. Those colonies with the original sequence will be identified and eliminated. Only colonies with the gene of interest will survive. CRISPR assisted Red recombienting has a 65% success rate, while the other 35% comes from Cas9 failing to localize the insertion site. Therefore, the screening of positive clones is fast and simple.

Example 2 construction of E.coli cell lines with Gene replacement for cytoplasmic expression of recombinant proteins without the N-terminal methionine

The construction of E.coli cell strain BL21 Gor-is described in U.S. Pat. Nos. 10,093,704 and 10,597,664. The described strain lacks Gor gene and has oxidized cytoplasm, so that protein is expressed cytoplasm, has correctly folded disulfide bond, and has high level. For these strains, an additional E.coli MAP gene was inserted into the Gor locus, and the new strain was named Gor/Met E.coli. The addition of the Tac promoter (with the Lac operon) upstream of the MAP gene allows the expression of the MAP gene to be regulated by the timing of the addition of IPTG. This is done as follows: additional E.coli MAP genes were inserted into the genome by homologous recombination. In the case of Gor/Met cells, the red recombinase system is used to facilitate the insertion of the MAP gene at the Gor locus in BL21 Gor-cells. The MAP gene was PCR amplified from the BL21 Gor-genome and placed under the control of the Tac promoter. This gene was then cloned downstream of the Chloramphenicol Acetyltransferase (CAT) gene flanked by two short Flippase Recognition Target (FRT) sequences. Together, the MAP and CAT genes form a transgene cassette. PCR was used to introduce fifty bases each of the sequences flanking the upstream and downstream of the original Gor locus into the transfer cassette upstream and downstream, respectively. The final PCR product was used for transformation into BL21 Gor-cells previously transformed with Red recombinase. Expression of Red recombinase in the cell accelerates homologous recombination of sequences flanking the Gor locus with the transfer cassette sequences. Bacterial colonies that demonstrated resistance to chloramphenicol had a transfer cassette insert. The confirmed bacteria were then transformed with a flippase gene whose expression recognized the FRT sequences flanking the CAT gene and the gene was spliced out so that the MAP was still inserted. Thus, gor/Met cell lines insert the MAP gene at the Gor locus without interfering with other parts of the genome. The cell line is a deposited strain and deposited at the American type culture Collection at 2021, 2 months and 9 days as accession number PTA-126975. Successful insertion of the MAP gene was confirmed by sequencing using primers designed to flank the Gor locus.

To demonstrate the function of this strain, several genes were expressed in BL21 Gor/Met cells and demonstrated to efficiently cleave the N-terminal methionine to produce the native protein.

Example 3 CRM ₁₉₇ Expression in Gor/Met E.coli

CRM ₁₉₇ Is an enzymatically inactive and non-toxic form of diphtheria toxin which contains a single amino acid substitution G52E. CRM like DT ₁₉₇ Has two disulfide bonds. One disulfide bond links Cys186 to Cys201, linking fragment a to fragment B. The second disulfide bond links Cys461 to Cys471 in fragment B. CRM ₁₉₇ Are commonly used as carrier proteins for carbohydrate-, peptide-and hapten-protein conjugates. As carrier protein, CRM ₁₉₇ There are many advantages over diphtheria toxoid and other toxoided (toxoided) proteins.

Despite CRM ₁₉₇ Has been produced in the original host corynebacterium (a slow growing bacterium with doubling times of hours rather than minutes), but with low yields, generally<50mg/L. Corynebacterium strains have been engineered to produce higher levels of CRM ₁₉₇ (see, for example, U.S. Pat. No. 5,614,382). CRM ₁₉₇ Also expressed at high levels in Pseudomonas fluorescens strains. However, CRM was produced in a strain that was safe for BL1 and inexpensive to culture and propagate ₁₉₇ It would be advantageous. Soluble, correctly folded intracellular CRM ₁₉₇ At BL21Gor-Expression in strains has been successful with one liter of fermentor cell culture>2g CRM ₁₉₇ . However, it was found that most of the CRM produced ₁₉₇ Having an N-terminal f-methionine. CRM with tac promoter ₁₉₇ The genes were cloned into Gor/Met escherichia coli strains (for Gor-strains, see, e.g., U.S. Pat. nos. 10,093,704 and 10,597,664). Thus, MAP genes and recombinant CRM ₁₉₇ Both genes are under the control of the same tac promoter, and are capable of simultaneous expression following IPTG induction.

Comparison of CRM in BL21 Gor-and BL21 Gor/Met E.coli ₁₉₇ Expression of (2). The yields of the two strains were found to be similar-2 g/L, indicating that co-expression of the MAP gene did not significantly affect CRM ₁₉₇ Expression of (2). CRM purified from BL21 Gor-and BL21 Gor/Met strains by MALDI-TOF mass spectrometry ₁₉₇ And is combined withThe results are summarized in table 1. Batch No. NO21p114 was expressed in Gor-strain and batch NO21p221 was expressed in Gor/Met strain. CRM expressed in BL21Gor- ₁₉₇ CRM expressed in Gor/Met E.coli and containing N-terminal Met ₁₉₇ Do not, indicating that the method described in the present invention is successful.

Example 4 expression of the cytokine IL10 from Epstein-Barr Virus in the Gor/Met strain.

The IL10 gene from Epstein-Barr virus was cloned and expressed as a soluble intracellular protein in Gor/Met E.coli. A metal affinity tag was included at the C-terminus to facilitate purification. IL10 purified by IMAC and ion exchange chromatography was subjected to mass spectrometry to determine the sequence of the N-terminal peptide. After enzymatic digestion with trypsin, the sample was analyzed by LC-MS/MS, which found that the protein had no N-terminal methionine.

The procedure was carried out using the following protocol: the samples were trypsinized and analyzed by LC-MS/MS on LTQ Orbitrap Velos (ThermoFisher Scientific, bremen, germany) interconnected with Proxeon 1200 nanolcs (Proxeon Biosystems). Chromatography was performed on a 75 μm i.d. Self-Pack PicoFrit fused silica capillary column 15 cm length (New Objective, woburn, MA). The stationary phase was a reversed phase C18 Jupiter column (5 μm, 300A) (Phenomenex, torrance, CA). The mass resolution was set to 30 000 in MS mode for maternal mass determination and to 7500 in MS/MS mode for acquisition of fragmentation spectra. The MS/MS spectra obtained during the LC-MS/MS run were submitted to a Mascot search against the expected protein sequence. Selecting a urethanyl group (C) asFixed decorationAnd oxidation (M) is selected asVariable decoration. The aim was to obtain the peptide corresponding to the first trypsin cleavage from the digestion solution. In the submitted sequence, the peptide will include an arginine at position 13. If methionine is present at the N-terminus, the result will be the amino acid sequences 1 to 13 (MTDQCDNFPQMLR; SEQ ID NO: 1), whereas if methionine is presentThe acid will be absent and the result will be the amino acid sequences 2 to 13 (TDQCDNFPQMLR; SEQ ID NO: 2).

Underlined and bolded amino acids in table 2 identify sequences identified by Mascot from MS/MS sequencing data. This indicates that the finding of an ion confirms the 2-13 sequence (no methionine at the N-terminus), and that no ion is found for the 1-13 sequence (methionine at the N-terminus).

The 2-13 sequence (no methionine at the N-terminus) is traced by the MS with the following ions present: 762.8m/z (+2)、508.9 m/z (+3)、770.8 m/z (+ 2) and its corresponding fragmentation pattern from the MS/MS trace were confirmed. All three ions identified contained alkylated cysteines, but 770.8m/zThe ion also contains oxidized methionine (position 11 of the submitted sequence). In conclusion, IL10 expressed in Gor/Met is produced in the absence of the N-terminal methionine.

Example 4 expression of the Gene detoxified tetanus toxin (8 MTT) in Gor/Met E.coli

Tetanus toxin is known as one of the most potent toxins in humans and is known as a spasmolytic toxin or TeNT. The LD50 of the toxin was measured to be approximately 2.5-3 ng/kg. Tetanus toxin is produced by clostridium tetani, an anaerobic bacterium commonly found in soil, as a single polypeptide chain that is cleaved into two chains upon translation by trypsin-like proteases to form the active protein. Light Chain (LC), 50kDa domain, containing N-terminal endopeptidase, heavy Chain (HC) containing the 50kDa receptor-binding domain (HCC) at the C-terminus, and 50kDa LC translocation domain located at the N-terminus (HCN). The two chains are linked by a single disulfide bond. Tetanus toxin enters peripheral motor neurons by binding gangliosides and synaptoproteins on its surface via the C-terminal domain of the heavy chain (HCC). The toxin is transported to synapses in interneurons in the soma and central nervous system and is endocytosed into inhibitory neurons in synaptic vesicles. In inhibitory neurons, the translocation domain of the Heavy Chain (HCN) undergoes pH-mediated conformational changes and transports the LC through the membrane of synaptic vesicles into the cytoplasm, where it is released into the cell cytosol and cleaves the vesicle-associated membrane protein 2 (VAMP 2), a vesicle soluble NSF attachment protein receptor (SNARE). VAMP2 cleavage in inhibitory neurons blocks neurotransmitter exocytosis, preventing the release of inhibitors of neuromuscular synaptic function, leading to continuous neuromuscular activation and spastic paralysis.

Chemical inactivation of tetanus toxoid (TTxd), formed by treatment of the toxin with formaldehyde, was used as an effective vaccine against tetanus. TTxd is also used as a conjugate vaccine carrier for polysaccharide antigens. Conjugate vaccines that use TTxd as a carrier protein include vaccines against haemophilus influenzae type b and neisseria meningitidis. However, many amines of TTxd are used for conjugation, blocked by the toxoidization process. Furthermore, TTxd is a heterogeneous product and contains aggregates along with clostridia and media contaminants. TTxd vaccines require further purification for use in conjugate vaccines. More importantly, the production and purification of TT from clostridium is time consuming and expensive. A genetically inactivated homogeneous recombinant tetanus toxin produced in a low cost host such as E.coli would be desirable.

Different strategies for producing inactivated recombinant TT proteins as vaccines or carrier proteins have been explored. One is the use of the heavy chain fragment (TTHC). Another is the use of genetically inactivated tetanus toxin (U.S. patent application publication No. 2020/03841201). TTHC is part of TT that does not carry a catalytic domain. Neutralizing antibodies against TTHC subunit vaccines are said to be superior to whole toxoid vaccine antibodies. TTHC is expressed at high levels (> 400 mg/L) in the BL21 Gor-system (see, e.g., U.S. patent nos. 10,597,664 and 10,093,704).

8MTT is a gene detoxified Tetanus Toxin (TT) with 8 amino acid mutations. Like tetanus toxin, 8MTT has 5 disulfide bonds. LD50 is more than 5000 ten thousand times less toxic than native TT. The 8MTT vaccination elicits a strong immune response IgG antibody response in mice, is a major candidate for a novel tetanus vaccine, and has great potential as a carrier protein for conjugate vaccines, similar to the broad spectrumCRM for use ₁₉₇ .8MTT was originally cloned into pET28 expression vector and expressed in BL21 (DE 3) cells with an attached His tag to facilitate purification. The amount expressed in the shake flask was about 10 mg/L. To produce a protein without tag and without N-terminal methionine, the 8MTT gene was subcloned into an expression vector with tac promoter (with lac operator) and T7 terminator and then expressed in BL21 Gor/Met cells in fed-batch fermenters. The expressed M8TT protein was found to be soluble and could be purified at over 500 mg/L. M8TT was purified to over 99% purity using a combination of anion exchange column, HIC column and TFF diafiltration/concentration. Purified M8TT was analyzed by MALDI-ISD (matrix assisted laser desorption/ionization-attenuation in source) to obtain terminal fragmentation. ISD allowed identification of the ladder band of the N-terminal fragment and confirmed that the sequence is free of the N-terminal methionine and that the first residue of the sequence is the expected proline. Therefore, gor/Met E.coli strains efficiently express large amounts of soluble M8TT without the N-terminal methionine.

Example 5 CRM with N-terminal serine ₁₉₇ Mutants

CRM containing an N-terminal serine ₁₉₇ The gene (CRM-Ser) was cloned into Gor/Met E.coli strain and grown and expressed in a bioreactor. In the absence of any optimized fermentation conditions, express>1g/L of soluble CRM-Ser, showing that the expression of the protein is excellent. Cells were harvested and CRM-Ser was purified. CRM-Ser was analyzed by MALDI-ISD as described in example 4. ISD allowed identification of the ladder of the N-terminal fragment and confirmed that the sequence does not have an N-terminal methionine and that the first residue of the sequence is the expected serine. Therefore, gor/Met E.coli strains efficiently expressed large amounts of soluble CRM-Ser without the N-terminal methionine. Serine can be selectively oxidized and used for conjugation.

Example 6: possible insertion of the MAP gene into the bacterial genome.

Removal of the N-terminal methionine by MAP may be important for the normal function and stability of the protein. Most recombinant proteins expressed in E.coli still have a methionine start codon at the N-terminus, even though intrinsic MAP is active. When an overexpressed recombinant protein is produced, insufficient MAP or its cofactor may be present. To ensure processing of the N-terminal methionine in the recombinant protein, an additional MAP gene was inserted under a strong inducible promoter as necessary to facilitate the methionine cleavage process.

1. MAP Gene of Escherichia coli

Gor/Met cells are an example of the insertion of additional e.coli MAP genes into the bacterial genome and under the same inducible promoter as that of the recombinant gene on the expression vector. When not induced, the MAP gene remains silent and the bacteria reproduce without the burden of additional gene expression. Since only recombinant proteins are induced to express (additional MAP proteins are also induced), MAP proteins can be designed to turn on as needed. A potential disadvantage of this system is that not all proteins with an N-terminal methionine can be efficiently cleaved by E.coli MAP. Coli MAP functions when a small amino acid (G, A, S, C, P, T, V) (P1' position) is adjacent to the N-terminal methionine. Preferably, the P2' position of the amino acid (the C-terminal amino acid of P1) is not proline. To treat those proteins with bulky amino acids next to the N-terminal methionine, it may instead be possible to insert other MAP genes with different P1 and P2 amino acid requirements.

2. Two different MAP genes are connected in series in different inducible promoters

The yeast gene is processed by two MAP genes. Yeast MAP1 and MAP2 show different cleavage efficiencies in vivo for the same substrate. When the second residue is V, both MAPs are less efficient, whereas when the second residue is G, C or T, MAP2 is less efficient than MAP1. Humans also have two types of MAPs: MAP1 and MAP2. They can all process proteins containing A, C, G, P or S at the P1' position. When the P1' residue is T or V, the removal of the N-terminal Met is catalyzed primarily by MAP2, and the extent of cleavage depends on the sequence at the P2' -P5' position. When the P2' residue is not A, G or P, N-terminal processing is expected to be complete. When A, G or P is a P2' residue, methionine removal is either incomplete or does not occur. Since different MAPs have different substrate specificities, two or more MAP genes from different or the same species may be able to cover the process of methionine removal from more recombinant proteins. The (e.g. inducible) promoters controlling gene transcription may be different, so that the two MAP genes can be turned on at different times, or one on/one off, as required.

3. The mutated MAT gene was inserted, which is capable of cleaving all N-terminal methionine without limitation of the subsequent amino acids.

Current MAP dislikes the N-terminal structure of some proteins and does not catalyze the removal of their N-terminal methionine. The general rule for predicting whether the initial methionine will be processed by MAP is based on the size of the amino acid at the P1' position. Typically, methionine is cleaved if the amino residue has a radius of gyration of 1.29 a or less. For human MAP, they have even more stringent requirements for the substrate, with acidic residues at the P2 'and P5' positions. To broaden the substrate specificity of existing MAP, the E.coli MAP gene was mutated so that its product could cleave 85-90% of the N-terminal methionine on proteins listed in the protein database. This MAP has three mutations in its substrate binding pocket, thus allowing the removal of the N-terminal methionine from proteins that have not only small amino acids, but also bulky amino acids or acidic amino acids (e.g., at the P2' position, M, H, D, N, E, Q, L, I, Y and W). These enzymes can also cleave the amino acid at the P1 'position if the amino acid residue at the P2' position is small. Insertion of this MAP gene processed proteins with a broader N-terminal structure. Also, the addition of an inducible promoter would be beneficial for controlling the activity of this powerful mutant gene expression.

Example 7: protein capable of being produced in Gor/Met cell line without disulfide bond

Although Gor/Met cell lines were developed for expressing disulfide-linked proteins without an N-terminal methionine, proteins without disulfide linkage can be expressed in Gor/Met cells without an N-terminal methionine. These proteins can also be expressed in E.coli with Met but not necessarily gor-. An example of such a protein is Staphylococcal Protein A (SPA) which is widely used in antibody purification. SPA is a 42kDa protein that is originally present in the cell wall of the bacterium Staphylococcus aureus. This protein consists of five homologous Ig-binding domains that can bind proteins from many mammalian species, particularly IgG, and bind heavy chains in the Fc region of most immunoglobulins and in the Fab region of the human VH3 family. SPA does not contain any disulfide bonds. Commercially, SPA comes from two sources: mutant staphylococcus aureus (Sigma) containing lesions in the cell wall secreting SPA; escherichia coli (Sigma-Aldrich, sinoBiological, thermoFisher, deNovo Biopharma, etc.) expressing SPA as a recombinant protein inside or outside the cell. SPA production in Pichia pastoris is likely to be at a high level. Most SPAs are expressed intracellularly in E.coli. Since the mature SPA sequence starts with alanine instead of methionine, for SPA to be produced in cells, either a methionine needs to be added to the gene at the N-terminus, or it needs to be expressed at the C-terminus of the fusion protein, which can be cleaved in vitro to release the mature protein. The former is not the true form of the mature protein, the latter is not cost and time efficient.

Expression of SPA is effective in the bacterial strains disclosed herein with inducible MAP gene insertion. SPA is expressed in large amounts in E.coli strains and functions similarly to Gor/Met cell lines and can also be expressed in MAP cell lines. The advantage of using MAP cell lines over other cell lines is that the f-methionine can be cleaved off at will. By design, the inducible promoter of the MAP gene can be expressed at any time simultaneously with or after expression of the SPA. The N-terminal methionine is removed without in vitro manipulation.

Other embodiments and uses of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. All references cited herein, including all publications, U.S. and foreign patents and patent applications, are specifically and entirely incorporated by reference. Wherever used, the term comprising is intended to include the term consisting of and consisting essentially of. Furthermore, the terms including, comprising, and containing are not intended to be limiting. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims.

Claims

1. A method of producing a protein containing one or more sulfur bonds, comprising:

expressing a protein from a recombinant cell comprising a genome and an expression vector encoding a protein sequence, wherein the recombinant cell has reduced activity of one or more disulfide reductase enzymes and the N-terminus of the protein comprises a methionine;

expressing a peptidase from the gene of the recombinant cell, wherein the peptidase removes methionine from the N-terminus of the expressed protein; and

isolating the protein.

2. The method of claim 1, wherein the expressed protein comprises tetanus toxin, tetanus toxin heavy chain protein, diphtheria toxoid, tetanus toxoid, pseudomonas exoprotein A, pseudomonas aeruginosa toxoid, bordetella pertussis toxoid, clostridium perfringens toxoid, E.coli (E.coli), (E.coli) A, B.pertussis toxoid, B.coli (E.coli) B.E. coli) Thermolabile toxin B subunit, neisseria meningitidis outer membrane complex, haemophilus influenzae protein D, flagellin Fli C, horseshoe crab hemocyanin, or fragments, derivatives or modifications thereof.

3. The method of claim 1, wherein the recombinant cell has reduced activity of only one disulfide reductase.

4. The method of claim 1, wherein the activity of more than one disulfide reductase is reduced.

5. The method of claim 1, wherein the recombinant cell is an E.coli cell or a derivative or strain of E.coli.

6. The method of claim 5, wherein the recombinant cells are obtained or derived from ATCC accession number PTA-126975.

7. The method of claim 1, wherein the peptidase comprises methionine aminopeptidase.

8. The method of claim 1, wherein the expression vector contains a ribosome binding site, an initiation codon and/or an expression enhancer region.

9. The method of claim 1, wherein the expression vector contains an inducible first promoter and expressing the protein comprises inducing the inducible first promoter with a first inducing agent.

10. The method of claim 1 wherein said gene contains an inducible second promoter and expressing said peptidase comprises inducing said inducible second promoter with a second inducing agent.

11. The method of claim 1 wherein the expression vector contains an inducible first promoter and expressing the protein comprises inducing the inducible first promoter with a first inducing agent, the gene contains an inducible second promoter and expressing the peptidase comprises inducing the inducible second promoter with a second inducing agent, and the first inducing agent and the second inducing agent are the same.

12. The method of claim 1 wherein said peptidase gene is integrated into the genome of said recombinant cell.

13. The method of claim 1, wherein the separating comprises chromatography.

14. The method of claim 12, wherein the chromatography comprises a sulfate resin, a gel resin, an activated sulfate resin, a phosphate resin, a heparin resin, or a heparin-like resin.

15. The method of claim 1, further comprising conjugating or coupling the isolated protein to a chemical compound.

16. The method of claim 14, wherein the chemical compound comprises a polysaccharide, a polymer, a polyethylene glycol derivative, a peptide, an antibody or a portion of an antibody, a lipid, a fatty acid, or a combination thereof.

17. A method of producing a peptide comprising:

expressing the peptide in a recombinant cell containing a gene encoding a peptidase,

wherein the gene encoding the peptidase is integrated into the genome of the recombinant cell,

wherein the recombinant cell has reduced activity of one or more disulfide reductase enzymes,

wherein a decrease in the activity of the one or more disulfide reductase enzymes results in a transition from the redox state of the cytoplasm to a higher oxidation state compared to a recombinant cell in which the activity of the one or more disulfide reductase enzymes is not decreased, and

wherein the peptide contains an N-terminal methionine;

expressing a peptidase that removes the N-terminal methionine from the peptide; and

isolating the peptide.

18. The method of claim 16, wherein the peptide comprises tetanus toxin, tetanus toxin heavy chain protein, diphtheria toxoid, tetanus toxoid, pseudomonas exoprotein a, pseudomonas aeruginosa toxoid, bordetella pertussis toxoid, clostridium perfringens toxoid, escherichia coli (e.coli: (a)E. coli) Thermolabile toxin B subunit, neisseria meningitidis outer membrane complex, haemophilus influenzae protein D, flagellin Fli C, horseshoe crab hemocyanin, or fragments, derivatives or modifications thereof.

19. The method of claim 16, wherein the recombinant cell has reduced activity of only one disulfide reductase.

20. The method of claim 16, wherein the recombinant cell has reduced activity of two or more disulfide reductase enzymes.

21. The method of claim 16, wherein the one or more disulfide reductase enzymes comprises one or more of an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase, or a glutathione reductase.

22. The method of claim 16, wherein the recombinant cell is an E.coli cell or a derivative or strain of E.coli.

23. The method of claim 16, wherein the gene encoding the peptide comprises a first inducible promoter and/or the gene encoding the peptidase comprises a second inducible promoter.

24. The method of claim 16 wherein the gene encoding said peptide comprises a first inducible promoter and the gene encoding said peptidase comprises a second inducible promoter, and said first and second inducible promoters are the same.

25. The method of claim 16, wherein the separating comprises chromatography.

26. The method of claim 24, wherein the chromatography comprises a sulfate resin, a gel resin, an activated sulfate resin, a phosphate resin, a heparin resin, or a heparin-like resin.

27. The method of claim 16, further comprising conjugating or coupling the isolated peptide to a chemical compound.

28. The method of claim 26, wherein the chemical compound comprises a polysaccharide, a polymer, a polyethylene glycol derivative, a peptide, an antibody or a portion of an antibody, a lipid, a fatty acid, or a combination thereof.

29. The method of claim 16, wherein said peptide is oxidized with an oxidizing agent.

30. The method of claim 28, wherein the oxidizing agent comprises a hydrazide, hydrazine, aminooxy, N-terminal 1-amino, 2-alcohol amino acid, or a combination thereof.

31. A method of producing a peptide comprising a disulfide bond, comprising:

wherein the peptide is encoded in an expression vector,

wherein the recombinant cell has reduced activity of one or more disulfide bond reductase enzymes,

wherein the recombinant cell is Escherichia coli, and

wherein the peptide contains an N-terminal methionine;

isolating the peptide from the cytoplasm of the recombinant cell, wherein the isolated peptide is soluble.

32. A recombinant cell obtained or derived from ATCC accession No. PTA-126975.

33. A method of producing a protein comprising:

expressing the proprotein in a recombinant cell containing a recombinantly engineered protease gene containing a translation inducing sequence;

inducing expression of the protease gene such that the pre-protein is cleaved to form a protein; and

and (5) harvesting the protein.

34. The method of claim 33, wherein said proprotein is selected from the group consisting of proinsulin, proinsulin-like protein, prorelaxin, proopiomelanocortin, zymogen, prohormone, proangiotensinogen, preprotapsin, procymotrypsin, proprotein of the coagulation system, prothrombin, plasminogen, proprotein of the complement system, caspase, propacin, elastase, lipogen, and carboxypeptidase.

35. The method of claim 33, wherein the protease gene is integrated into the genome of the recombinant cell.

36. The method of claim 33, wherein the methionine aminopeptidase gene is integrated into the genome of the recombinant cell.

37. The method of claim 36, wherein expression of the methionine aminopeptidase gene removes the N-terminal methionine from the preprotein or the protein.

38. The method of claim 37, wherein the expression of the methionine aminopeptidase gene is under the control of an inducer sequence.

39. The method of claim 38, wherein the inducer sequence of the methionine aminopeptidase and the translation inducing sequence of the preprotein are different.

40. The method of claim 38, wherein the inducer sequence of the methionine aminopeptidase and the translation inducing sequence of the preprotein are the same.

41. The method of claim 33, wherein the recombinant cell has reduced activity of one or more disulfide reductase enzymes.

42. The method of claim 41, wherein the recombinant cell is an E.coli comprising a gor mutation.

43. A recombinant cell line comprising a methionine aminopeptidase gene and a protease gene, both of which are integrated.

44. The recombinant cell of claim 43 having reduced activity of one or more disulfide reductase enzymes.

45. The recombinant cell of claim 44 which contains a gor mutation.

46. A method of producing a peptide comprising:

expressing the peptide in a recombinant cell, wherein the expressed peptide contains an N-terminal methionine and the recombinant cell contains a gene encoding a peptidase;

expressing the peptidase gene such that an N-terminal methionine is cleaved from the expressed peptide; and

isolating the peptide;

and (5) harvesting the protein.

47. The method of claim 46, wherein said peptide is expressed from another gene integrated into the genome of said recombinant cell.

48. The method of claim 46 wherein said peptidase gene is integrated into the genome of said recombinant cell.

49. The method of claim 46, wherein said peptidase is methionine aminopeptidase.

50. The method of claim 46, wherein the recombinant cell is an E.coli cell.