JP7449000B2 - Production of soluble recombinant proteins - Google Patents

Description

ATCC PTA-126975

Rights to the Invention This invention was made with support from the United States Government under Grant No. 1R43AI148018-01A1 FAIN: R43AI148018 awarded by the National Institutes of Health, and the United States Government has certain rights in this invention.

REFERENCES TO RELATED APPLICATIONS This application is filed in U.S. Provisional Application No. 63/152,954, filed February 24, 2021; U.S. Provisional Application No. 62/990,083, filed March 16, 2020; No. 16/819,775, filed and currently pending, US Application No. 16/819,775, each of which is specifically incorporated by reference in its entirety.

FIELD OF THE INVENTION The present invention relates to methods and compositions for expressing and purifying products such as peptides and proteins in microorganisms. In particular, the semi-product is expressed recombinantly and the cytoplasm of the microorganism modifies the expressed semi-product to produce the final or usable form of the product. Modifications include shifts in cytoplasmic redox states and site-specific cleavage and/or ligation.

Background Information Escherichia coli (E. coli) is a widely used host for producing recombinant proteins for research and therapeutic purposes. Recombinant proteins can be expressed in the E. coli cytoplasm or periplasm. One limitation of cytoplasmic recombinant protein expression in E. coli is that to initiate expression of a recombinant protein in E. coli, the coding sequence of the protein should begin with an ATG codon, which is a formylmethionine is translated into N-terminal methionine and then processed by formylmethionine deformylase to form N-terminal methionine. Thus, for recombinant protein expression in E. coli, an ATG codon is added to the native or mature protein sequence. During intracellular expression of recombinant proteins, the N-terminal methionine is usually excised by endogenous E. coli methionine aminopeptidase (MAP). This process is not always effective for recombinant proteins even when adjacent residues are optimal for cleavage, probably due to the overexpression of recombinant proteins and the limited amount of MAP present. As a result, a significant amount of recombinant proteins are likely to have methionine as the first amino acid. This is undesirable for the majority of proteins since the N-terminal methionine is not part of the mature protein sequence. The presence of the N-terminal methionine can also cause structural changes in the protein that affect its function. Existing methods to ensure effective 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 the MAP with the recombinant protein. In the latter case, co-expression of MAP may reduce expression of the desired recombinant protein. Both approaches are time-consuming and costly to implement. Therefore, it would be desirable to have an E. coli expression strain in which MAP is highly expressed without significantly inhibiting recombinant protein expression. This would allow production of increased amounts of the target protein with its native sequence in the cytoplasm.

Recombinant proteins expressed in the cytoplasm of E. coli can form insoluble inclusion bodies. Proteins within inclusion bodies can be refolded in vitro to form soluble proteins. These proteins will contain an undesirable N-terminal methionine.

The E. coli cytoplasm has a reducing environment, and recombinant proteins containing disulfide bonds are usually insoluble when expressed intracellularly. In contrast, the periplasm of E. coli has an oxidative 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 to the periplasm is excised during the secretion process, resulting in the production of a protein with the native amino acid sequence. However, the translocation mechanisms that direct proteins to the periplasm have limited capacity, so periplasmic expression levels of recombinant proteins are usually low. On the other hand, expression in the E. coli cytoplasm can yield several grams of recombinant protein per liter of cell culture. It would therefore be desirable to be able to express soluble, properly folded disulfide bond proteins in the cytoplasm. Additionally, it would be desirable if these proteins could be produced without the N-terminal methionine.

Commercially available E. coli strains such as Origami® (EMDMillipore) and Shuffle® (New England Bio) with gor-/trx- mutations can produce soluble intracellular proteins containing disulfide bonds; These cell lines are clipped and do not grow to high densities, which limits production yields. Therefore, although these strains are suitable for producing experimental materials, their low growth levels make them difficult to use commercially. Therefore, there is a need for strains that express high levels of properly folded, disulfide-bonded intracellular proteins that do not contain an N-terminal methionine.

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

One embodiment of the invention relates to a method of producing recombinant peptides and proteins in bacteria, comprising the steps of: expressing a protein in a bacterium containing an expression vector encoding a protein sequence that includes a promoter. The bacterium also contains a gene under the control of a promoter, which is integrated into the genome of the host, and the polypeptide expressed by the gene is responsible for the expression, folding, or solubility of the recombinant protein in the cytoplasm. and isolating the protein.

Another aspect of the invention relates to a method of producing recombinant peptides and proteins in bacteria, comprising the steps of: expressing the protein in a bacterium containing an expression vector encoding a protein sequence that includes a promoter. The bacterium also expresses a peptidase gene, which is integrated into the genome of the host cell, in which the peptidase acts on the expressed protein and extracts formylmethionine from the N-terminal part of the protein. peptidase expression is under the control of a promoter so as to remove the group; and isolating the protein. Peptidases that remove N-terminal methionine may be referred to as methionine aminopeptidases (MAPs). Preferably, the integrated gene contains a ribosome binding site, an initiation codon, and an expression enhancer and/or repressor region. Preferably, the recombinant cell has reduced activity of only one disulfide reductase enzyme or reduced activity of only two disulfide reductase enzymes. Preferably, the recombinant cell is an E. coli cell or a derivative or strain of E. coli, and preferably the recombinant protein expressed is tetanus toxin, tetanus toxoid heavy chain protein, diphtheria toxoid, tetanus toxoid, Pseudomonas exoprotein A, Pseudomonas aeruginosa toxoid, Bordetella pertusis toxoid, Clostridium perfringens toxoid, Escherichia coli heat-labile toxin B subunit, Neisseria meningitidis outer membrane complex, Includes Hemophilus influenzae protein D, flagellin Fli C, cytokines, single chain antibodies, camelids, nanobodies, and fragments, derivatives, and modifications thereof. Also preferably, the recombinant protein may be a member of the preprotein prorelaxin, insulin, and insulin-like families. Preferably, the integrated gene and/or expression vector contains an inducible promoter for the peptidase. The expressing step includes inducing an inducible promoter with a first inducing agent, containing an expression vector encoding a recombinant peptide or protein that may be inducible with a second inducing agent. Preferably, the first and second inducing agents are the same, but may be different. Preferably, the first integrated gene or expression vector contains a second inducible promoter and the step of expressing the peptidase comprises inducing the second inducible promoter with the first inducing agent. Preferably, the step of isolating comprises chromatography, the chromatography comprising a sulfate resin, a gel resin, an activated sulfate resin, a phosphate resin, a heparin resin, or a heparin-like resin. Preferably, the expressed and isolated protein is combined with polyethylene glycol and/or derivatives of polyethylene glycol, or with polymers such as polysaccharides, peptides, antibodies or parts of antibodies, lipids, fatty acids, small molecules, haptens, or conjugated with a combination of

Another aspect of the invention relates to a method of producing a soluble or insoluble peptide, comprising the steps of: producing a peptide having a formylmethionine group at the N-terminus of the peptide into a vector containing an expression vector encoding the peptide. expressing a peptidase that acts on the expressed peptide and removes the formylmethionine group from the N-terminus of the peptide from an integrated gene of the recombinant cell; The step of isolating.

Another aspect of the invention relates to a method of producing a peptide, comprising the steps of: 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. a step in which the recombinant cell has reduced activity of one or more disulfide reductase enzymes, the expression vector contains a promoter operably linked to the coding region of the peptide, and the expression vector contains a promoter operably linked to the coding region of the peptide; The reduced activity of the one or more disulfide reductase enzymes causes a shift in the cytoplasmic redox state to a more oxidative state compared to recombinant cells that do not have the reduced activity of the one or more disulfide reductase enzymes. and expressing a peptidase from the integrated gene of the recombinant cell that acts on the expressed peptide and removes the formylmethionine group from the N-terminus of the peptide; and isolating the peptide. The process of doing. 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 enzyme or only two disulfide reductase enzymes. Preferably, the one or more disulfide reductase enzymes include one or more of oxidoreductase, dihydrofolate reductase, thioredoxin reductase, or glutathione reductase. Preferably, the recombinant cell is an E. coli cell or a derivative or strain of E. coli, and the peptide or protein is tetanus toxin, tetanus toxoid heavy chain protein, diphtheria toxoid, tetanus toxoid, Pseudomonas exoprotein A, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoids, Clostridium perfringens toxoids, botulism toxins, Escherichia coli heat-labile toxin B subunit, meningococcal outer membrane complex, Haemophilus influenzae protein D, flagellin Fli C, cytokines, single chain antibodies, camelids, nanobodies, and these including fragments, derivatives, and modifications of. Preferably, the promoter is an inducible promoter, and the step of expressing includes inducing the inducible promoter with an inducing agent.

Preferably, the step of isolating comprises chromatography, the chromatography comprising a sulfate resin, a gel resin, an activated sulfate resin, a phosphate resin, a heparin resin, or a heparin-like resin. Preferably, the isolated peptide is conjugated with polyethylene glycol (PEG) and/or derivatives of PEG, or with polymers such as polysaccharides, peptides, antibodies or parts of antibodies, lipids, fatty acids, small molecules. , a hapten, or a combination thereof.

Another aspect of the invention relates to E. coli cell lines containing gor mutations. Preferably, the cell line comprises cells obtained from or derived from ATCC Deposit No. PTA-126975.

Another aspect of the invention relates to a method of producing a protein comprising: expressing a pre-protein in a recombinant cell containing a recombinantly engineered protease gene. Preferably, the protease gene and/or preprotein gene contains a promoter and/or translation-directing sequence. Promoters may be the same or different, and translation-inducing sequences, if present, may be the same or different between genes. Preferably after expression of the pre-protein, expression of a protease gene is induced such that the pre-protein is cleaved to form a protein; the protein is recovered. Preferably, the preprotein is proinsulin, proinsulin-like protein, prorelaxin, proopiomelanocortin, proenzyme, prohormone, proangiotensinogen, protrypsinogen, prochymotrypsinogen, propepsinogen, proprotein of the coagulation system, prothrombin, proplasminogen, proprotein of the complement system, procaspase, propacifastin, proelastase, prolipase, procarboxypolypeptidase, protein containing a cleavable leader sequence, cleavable tag sequence, cleavable addition proteins containing an N- or C-terminal amino acid (eg, 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, where expression of the methionine aminopeptidase gene removes the N-terminal methionine from the pre-protein or protein. Expression of the methionine aminopeptidase gene is preferably under the control of an inducer sequence, and the methionine aminopeptidase inducer sequence and the translation inducer sequence of the preprotein may be the same or different. Also preferably, the recombinant cell has reduced activity of one or more disulfide reductase enzymes, which may be E. coli with a gor mutation.

Another aspect of the invention relates to a recombinant cell line containing a methionine aminopeptidase gene and a protease gene, both of which have been integrated. Preferably, the cell further contains reduced activity of one or more disulfide reductase enzymes, which may contribute to the gor mutation.

[Invention 1001]
A method of producing a protein containing one or more sulfide linkages, comprising the steps of:
expressing the protein from a recombinant cell containing an expression vector and a genome encoding the protein sequence, the recombinant cell having reduced activity of one or more disulfide reductase enzymes; the N-terminus of containing methionine;
expressing a peptidase from the gene of the recombinant cell, the peptidase removing methionine from the N-terminus of the expressed protein; and
A step of isolating the protein.
[Present invention 1002]
The expressed proteins are tetanus toxoid, 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) heat-labile toxin B subunit, Neisseria meningitidis outer membrane complex, Hemophilus influenzae protein D, flagellin Fli C, horseshoe crab hemocyanin, or these A method of the invention 1001, including a fragment, derivative, or modification.
[Present invention 1003]
The method of the invention 1001, wherein the recombinant cell has reduced activity of only one disulfide reductase enzyme.
[Present invention 1004]
Decreased activity of multiple disulfide reductase enzymes, the method of the invention 1001.
[Present invention 1005]
The method of the invention 1001, wherein the recombinant cell is an E. coli cell or a derivative or strain of E. coli.
[Present invention 1006]
The method of the invention 1005, wherein the recombinant cells are obtained or derived from ATCC Deposit No. PTA-126975.
[Present invention 1007]
1001, wherein the peptidase comprises methionine aminopeptidase.
[Present invention 1008]
The method of the invention 1001, wherein the expression vector contains a ribosome binding site, an initiation codon, and/or an expression enhancer region.
[Present invention 1009]
1001. The method of the invention 1001, wherein the expression vector contains a first inducible promoter and the step of expressing the protein comprises inducing the first inducible promoter with a first inducing agent.
[Present invention 1010]
1001. The method of the invention 1001, wherein the gene contains a second inducible promoter and the step of expressing the peptidase comprises inducing the second inducible promoter with a second inducing agent.
[Present invention 1011]
the expression vector contains a first inducible promoter, the step of expressing the protein comprises inducing the first inducible promoter with a first inducing agent, and the gene contains a second inducible promoter; , wherein the step of expressing the peptidase comprises inducing a second inducible promoter with a second inducing agent, the first inducing agent and the second inducing agent being the same.
[Invention 1012]
1001. The method of the invention 1001, wherein the peptidase gene is integrated into the genome of the recombinant cell.
[Present invention 1013]
1001. The method of the invention 1001, wherein the step of isolating comprises chromatography.
[Present invention 1014]
The method of the invention 1012, 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.
[Present invention 1015]
1001. The method of the invention 1001 further comprising conjugating or coupling the isolated protein with a chemical.
[Invention 1016]
1014. The method of the invention 1014, wherein the chemical entity comprises a polysaccharide, a polymer, polyethylene glycol, a derivative of polyethylene glycol, a peptide, an antibody or a portion of an antibody, a lipid, a fatty acid, or a combination thereof.
[Invention 1017]
A method of producing peptides, including the following steps:
Expressing a peptide in a recombinant cell containing a gene encoding a peptidase enzyme, the step comprising:
The gene encoding the peptidase enzyme is integrated into the genome of the recombinant cell,
the recombinant cell has reduced activity of one or more disulfide reductase enzymes;
The reduced activity of the one or more disulfide reductase enzymes results in oxidation of the cytoplasm to a more oxidative state compared to recombinant cells that do not have the reduced activity of the one or more disulfide reductase enzymes. bring about a shift in the reducing state, and
the peptide contains an N-terminal methionine;
expressing a peptidase enzyme that removes the N-terminal methionine from the peptide; and
A step of isolating the peptide.
[Invention 1018]
Peptides include tetanus toxoid, tetanus toxoid heavy chain protein, diphtheria toxoid, tetanus toxoid, pseudomonas exoprotein A, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, Clostridium perfringens toxoid, Escherichia coli heat-labile toxin B subunit, Neisseria meningitidis 1016. The method of the invention 1016, comprising a complex, Haemophilus influenzae protein D, flagellin Fli C, horseshoe crab hemocyanin, or a fragment, derivative, or modification thereof.
[Invention 1019]
1016. The method of the invention 1016, wherein the recombinant cell has reduced activity of only one disulfide reductase enzyme.
[Invention 1020]
1016. The method of the invention 1016, wherein the recombinant cell has reduced activity of two or more disulfide reductase enzymes.
[Invention 1021]
1016. The method of the invention 1016, wherein the one or more disulfide reductase enzymes include one or more of oxidoreductase, dihydrofolate reductase, thioredoxin reductase, or glutathione reductase.
[Invention 1022]
1016. The method of the invention, wherein the recombinant cell is an E. coli cell or a derivative or strain of E. coli.
[Invention 1023]
1016. The method of the invention 1016, wherein the gene encoding the peptide contains a first inducible promoter and/or the gene encoding the peptidase enzyme contains a second inducible promoter.
[Invention 1024]
The present invention, wherein the gene encoding the peptide contains a first inducible promoter, the gene encoding the peptidase enzyme contains a second inducible promoter, and the first and second inducible promoters are the same. 1016 ways.
[Invention 1025]
1016. The method of the invention 1016, wherein the step of isolating comprises chromatography.
[Invention 1026]
1024. The method of the invention 1024, 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.
[Invention 1027]
1016. The method of the invention further comprising conjugating or coupling the isolated peptide with a chemical entity.
[Invention 1028]
1026. The method of the invention 1026, wherein the chemical entity comprises a polysaccharide, a polymer, polyethylene glycol, a derivative of polyethylene glycol, a peptide, an antibody or a portion of an antibody, a lipid, a fatty acid, or a combination thereof.
[Invention 1029]
1016. The method of the invention 1016, wherein the peptide is oxidized by an oxidizing agent.
[Invention 1030]
1028. The method of the invention 1028, wherein the oxidizing agent comprises a hydrazide, hydrazine, an aminooxy group, an N-terminal 1-amino 2-alcohol amino acid, or a combination thereof.
[Present invention 1031]
A method of producing a peptide containing a disulfide bond, comprising the steps of:
Expressing a peptide in a recombinant cell containing a gene encoding a peptidase enzyme, the step comprising:
the peptide is encoded within the expression vector;
The gene encoding the peptidase enzyme is integrated into the genome of the recombinant cell,
the recombinant cell has reduced activity of one or more disulfide reductase enzymes;
the recombinant cell is E. coli, and
the peptide contains an N-terminal methionine;
expressing a peptidase enzyme that removes the N-terminal methionine from the peptide; and
A step of isolating the peptide from within the cytoplasm of a recombinant cell, the peptide being isolated being soluble.
[Invention 1032]
Recombinant cells obtained from or derived from ATCC Deposit No. PTA-126975.
[Present invention 1033]
A method of producing proteins, including the following steps:
expressing a pre-protein in a recombinant cell containing a recombinantly engineered protease gene containing a translation-directing sequence;
inducing expression of a protease gene such that the preprotein is cleaved to form a protein; and
A step of recovering the protein.
[Invention 1034]
Pre-proteins include proinsulin, proinsulin-like protein, prorelaxin, proopiomelanocortin, proenzyme, prohormone, proangiotensinogen, protrypsinogen, prochymotrypsinogen, propepsinogen, proprotein of the coagulation system, prothrombin, and proplasminol. 1033. The method of the invention 1033, wherein the method is selected from the group consisting of pro-proteins of the complement system, pro-caspases, pro-pacifastins, pro-elastases, pro-lipases, and pro-carboxy polypeptidases.
[Present invention 1035]
1033. The method of the invention 1033, wherein the protease gene is integrated into the genome of the recombinant cell.
[Invention 1036]
1033. The method of the invention 1033, wherein the methionine aminopeptidase gene is integrated into the genome of the recombinant cell.
[Present invention 1037]
1036. The method of the invention 1036, wherein expression of a methionine aminopeptidase gene removes the N-terminal methionine from said preprotein or said protein.
[Invention 1038]
1037. The method of the invention 1037, wherein the expression of the methionine aminopeptidase gene is under the control of an inducer sequence.
[Invention 1039]
1038. The method of the present invention, wherein the methionine aminopeptidase inducer sequence and the preprotein translation inducer sequence are different.
[Invention 1040]
1038. The method of the invention 1038, wherein the methionine aminopeptidase inducer sequence and the preprotein translation inducer sequence are the same.
[Present invention 1041]
1033. The method of the invention 1033, wherein the recombinant cell has reduced activity of one or more disulfide reductase enzymes.
[Present invention 1042]
1041. The method of the invention 1041, wherein the recombinant cell is E. coli containing a gor mutation.
[Invention 1043]
A recombinant cell line containing a methionine aminopeptidase gene and a protease gene that have both been integrated.
[Present invention 1044]
A recombinant cell of the invention 1043 having reduced activity of one or more disulfide reductase enzymes.
[Invention 1045]
A recombinant cell of the present invention 1044 containing a gor mutation.
[Invention 1046]
A method of producing peptides, including the following steps:
expressing a peptide in a recombinant cell, the expressed peptide containing an N-terminal methionine, and the recombinant cell containing a gene encoding a peptidase;
expressing a peptidase gene such that the N-terminal methionine is cleaved from the expressed peptide; and
isolating the peptide;
Process of recovering proteins.
[Invention 1047]
1046. The method of the invention 1046, wherein the peptide is expressed from another gene that is integrated into the genome of the recombinant cell.
[Invention 1048]
1046. The method of the invention 1046, wherein the peptidase gene is integrated into the genome of the recombinant cell.
[Invention 1049]
1046. The method of the invention 1046, wherein the peptidase is methionine aminopeptidase.
[Invention 1050]
1046. The method of the invention, wherein the recombinant cell is an E. coli cell.
Other aspects and advantages of the invention will, in part, be set forth in the description that follows, and in part may be obvious from the description, or may be learned from practice of the invention.

Description of the Invention In E. coli, proteins are typically expressed with a methionine at the N-terminus since the corresponding ATG codon is required for initiation of translation. Thus, proteins expressed within cells contain an N-terminal methionine that is not part of the natural amino acid sequence (unless the natural sequence begins with a methionine). Removal of the N-terminal methionine by MAP may be important for protein function and stability. Endogenous methionine aminopeptidase (MAP) can cleave the N-terminal methionine of newly synthesized proteins, typically up to 60-70%. However, with highly expressed recombinant proteins, the level of endogenous MAP activity may not be sufficient to remove the desired amount of N-terminal methionine. Removal of the N-terminal methionine would be of significant interest in producing intracellular recombinant proteins in E. coli. Therefore, an E. coli strain that can efficiently cleave undesired N-terminal Met would be highly desirable.

Solubility and proper folding of recombinant proteins expressed within cells are concerns with the E. coli expression system. The E. coli cytoplasm has a reducing environment that is not favorable for disulfide bond formation. As a result, recombinant proteins containing disulfide bonds are usually insoluble when expressed within cells. Purification of these insoluble proteins can be difficult, expensive, and time consuming. High cell densities are preferred, especially for the production of recombinant proteins for commercial use.

Genetically engineered microorganisms have surprisingly been developed to express large amounts of appropriately constructed recombinant proteins that also effectively remove N-terminal formylmethionine. These microorganisms are genetically engineered to express in the cytoplasm soluble recombinant proteins containing disulfide bonds and to remove the N-terminal methionine. These microorganisms produce large amounts of properly folded intracellular recombinant proteins containing disulfide bonds without methionine at the N-terminus of the protein.

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

Incorporation of additional MAPs into the genome is particularly desirable. This is because the stable bacterial expression strains created can be used for the intracellular production of recombinant proteins in which the unwanted f-Met is cleaved in vivo. Previously, removal of the undesired N-terminal methionine has been accomplished by post-translational in vitro digestion using purified MAP or by co-expression of the MAP in the same vector as the recombinant protein or using an additional vector. Ta. The process is long and complex. By using cell lines that can cleave f-Met in vivo on demand, these microorganisms greatly simplify the protein expression and purification process.

MAP cleaves the N-terminal methionine with specific requirements for adjacent amino acids. The use of at least one additional MAP may allow more efficient production of intracellular proteins without N-terminal methionine. If multiple MAPs are inserted, they can be the same or different MAP genes. Transcription of these MAP genes may be under the same or different inducible promoters. These promoters may be the same or different from the promoters used to express the recombinant protein. Combinations of inducible promoters can be used to control the timing and amount of production of intracellularly expressed recombinant proteins that do not have an N-terminal methionine.

E. coli strains capable of expressing soluble, properly folded intracellular recombinant proteins containing disulfide bonds have been described (see US Pat. Nos. 10,597,664 and 10,093,704). An example of such a strain is E. coli (BL21 Gor-). BL21Gor- to express soluble, properly folded intracellular recombinant proteins containing high levels of disulfide bonds, such as the genetically detoxified diphtheria toxin vaccine carrier protein CRM ₁₉₇ . It is used. Approximately 60% of CRM ₁₉₇ expressed in these cells contained an N-terminal methionine. Insertion of additional MAP genes under promoter control into such strains allows the production of soluble, properly folded intracellular recombinant proteins that contain disulfide bonds and lack an N-terminal methionine. Make it. An example of such a strain is the E. coli (BL21 Gor/Met) strain. This strain can produce soluble proteins with disulfide bonds and no N-terminal methionine intracellularly in amounts of several grams per liter of cell culture. CRM ₁₉₇ expressed in BL21 Gor/Met cells contained very low levels of N-terminal methionine. Furthermore, integration of the inducible methionine aminopeptidase gene into the E. coli genome did not significantly affect CRM ₁₉₇ expression levels.

Although the MAP gene was inserted into the genome by homologous recombination, several other options are available to facilitate insertion. The approach used to create Gor/Met cell lines is an example of gene insertion. In Gor/Met cells, the red recombinase system was used to insert the MAP gene into the Gor locus in BL21 Gor- cells. The MAP gene was excised from the BL21 genome using PCR and placed downstream of the chloramphenicol acetyltransferase (CAT) gene under the control of the Tac promoter and flanked by two short flippase recognition target (FRT) sequences. . The MAP and CAT genes together formed a transferred gene cassette. Fifty bases of flanking sequence upstream and downstream of the original Gor locus were added by PCR upstream and downstream of the import cassette, respectively. The final PCR product was used to transform the BL21 Gor- cell line, which had already been transformed with Red recombinase. Expression of red recombinase in cells facilitated homologous recombination of the import cassette with sequences flanking the Gor locus. Bacterial colonies that were resistant to chloramphenicol were confirmed to have successfully inserted the transfer cassette. The identified bacteria were subsequently transformed with the flippase gene, whose expression recognized the FRT sequence flanking the CAT gene and excised the gene, leaving the inserted MAP intact. Therefore, the Gor/Met cell line has the MAP gene located at the Gor locus without disturbing other parts of the genome.

To produce large amounts of proteins such as CRM ₁₉₇ from E. coli host cells, f-Met present at the N-terminus of the protein is enzymatically removed in the cytoplasm. Yield is typically quantified as mg per liter of bacterial cell culture. Protein production is 25 mg/L or more, 50 mg/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. 600mg/L or more, 700mg/L or more, 800mg/L or more, 900mg/L or more, 1,000mg/L or more, 1,500mg/L or more, or 2,000mg Achieved /L or above. The expressed protein includes both full-length and/or truncated proteins, as well as modified amino acid sequences of the protein, as desired. Modifications include one or more of conservative amino acid deletions, substitutions, and/or additions. Conservative modifications are those that maintain the functional activity and/or immunogenicity of the molecule, although the activity and/or immunogenicity may be increased or decreased. Examples of conservative modifications include amino acid modifications (e.g., single, double, and otherwise short amino acid additions, deletions, and/or substitutions), modifications outside of the active or functional sequence. , residues that are accessible for conjugation in forming the vaccine, modifications due to serotype variations, modifications that increase immunogenicity or increase conjugation efficiency, that substantially inhibit binding to heparin. modifications that do not alter the protein, maintain proper folding or three-dimensional structure, and/or do not significantly alter the immunogenicity of the protein or portion of the protein that provides protective immunity; Not limited to these.

The recombinant cells used are preferably Escherichia coli bacteria and preferably contain enzymes such as oxidoreductase, dihydrofolate reductase, thioredoxin reductase, glutamate cysteine lyase, disulfide reductase, protein reductase and/or glutathione reductase, such as e.g. E. coli that has been genetically engineered to shift the cytoplasmic redox state to a more oxidative state, such as by mutation of one or more disulfide reductase genes. Preferably, one or more disulfide reductase genes are mutated and non-functional so as to shift the cytoplasmic redox state of the cell to a more oxidative state compared to the wild type without reducing viability. to be functionally or slightly functional. Oxidative protein folding involves the formation and isomerization of disulfide bridges and plays a key role in the stability and solubility of many proteins, including CRM ₁₉₇ . Formation and cleavage of disulfide bridges is typically catalyzed by thiol-disulfide oxidoreductase. These enzymes are characterized by one or more Trx folds consisting of a four-stranded β-sheet surrounded by three α-helices with a CXXC redox active site motif. Assemblies of various Trx modules have been used to assemble various thiol oxidoreductases found in prokaryotes and eukaryotes. In the bacterial periplasm, proteins are kept 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 a chromosomal copy of the disulfide bond isomerase DsbC. DsbC facilitates the correction of incorrectly oxidized proteins to their correct form. Cytoplasmic DsbC is also a chaperone that can assist in protein folding without the need for disulfide bonds.

A recombinant bacterium containing 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 shift the cytoplasmic redox state to a more oxidative state, which contain and express an inducible MAP gene. Preferred cells are prokaryotes, such as E. coli expression systems, Bacillus subtillis expression systems and other bacterial cell expression systems. Preferably, the cells contain a protein expression system for expressing foreign or non-natural sequences. Preferably, the sequences to be expressed also include an inducible promoter (e.g. preferably inducible by a particular medium), an initiation codon (e.g. ATG), a ribosome binding site, and/or a ribosome binding site and ATG. It is constructed of an expression vector containing one or more of the modified sequences between the initiation codon or between the initiation codon and the sequence to be expressed. Preferred modified or spacer sequences include a number of nucleotides, for example more than or less than 9 (eg 7-12 nucleotides), preferably not 9 nucleotides.

Surprisingly, it is possible to develop recombinant cells containing additional proteases that effectively cleave one or more different preproteins and/or preproproteins from an inactive to an active conformation. has also been discovered. These proteins are commonly referred to as zymogens (eg, proenzymes) and require post-translational modification. Protein precursors are often used by cells when active proteins are harmful but need to be expressed. By incorporating these proteins into recombinant cells, expression can be achieved safely, at low cost, and in large quantities. A protease gene expressing a protease that performs a specific cleavage from inactive to active may be integrated into the genome of the cell or contain the protease gene of interest, all as described herein. It may also be transformed by a vector. The introduced protease gene can be placed under the control of a common promoter with the recombinant gene to be expressed and recovered and the methionine-cleaving protease gene, or the protease gene can have a different promoter. Similarly, the gene can be induced separately or essentially simultaneously with the recombinant gene and the methionine-cleaving protease gene to be expressed and recovered. Preferably, when expression of the recombinant protein is sufficient, a second protease is activated and processes the proprotein into an active state. This will save both time and cost Pre-proteins such as proinsulin to insulin, proinsulin-like protein to insulin-like protein, prorelaxin to relaxin, proopiomelanocortin to opiomelanocortin, proenzyme to enzyme, and hormones from prohormones, and signal peptides, leader sequences, tags, etc. from proteins. Generally, the protease introduced will be specific to the protein to be cleaved. Additional proteins that can be efficiently produced in this are angiotensinogen, trypsinogen, chymotrypsinogen, pepsinogen, proteins of the coagulation system (e.g. prothrombin, plasminogen), proteins of the complement system, procaspases, pacifastin, proelastase. , prolipase, and procarboxy polypeptidase. In addition, certain genes can be modified to include portions (e.g., leaders or tags or internal sequences) that allow the protein to be expressed in an inactive form, which means that the gene also It is also converted to its active form only when introduced into cells and subsequently cleaved by activated proteases.

As an example, a gene of interest is inserted into the genome with another promoter. Cells are induced to express the gene with disulfide bonds and also methionine peptidase, which trims methionine. Once a suitable amount of the protein to be trimmed is produced in the cytoplasm, a second promoter is induced, which processes the protein into its final or active form. Expression of active proteins such as trypsin during growth destroys many needed proteins in the cytoplasm and prevents expression of recombinant proteins. This approach would avoid the need for in vitro processing of the expressed proprotein.

Another embodiment of the present invention provides the use of expression vectors having an inducible promoter and/or a modified sequence between the ribosome binding site and the ATG start codon to express the N-terminus of a recombinant protein in E. coli or another host cell. relates to a recombinant protein expressed in cells in which f-met present in the cell is enzymatically removed. Preferably, the expression vector includes a lactose/IPTG inducible promoter, preferably the tac promoter, and a sequence between the ribosome binding site and the ATG initiation codon.

Another aspect of the invention involves expression constructs of nucleotide or amino acid sequences, with or without control regions. Adding one or more sequences that facilitate nucleic acid recognition for increased expression (e.g., initiation codon, enzyme binding site, translation or transcription factor binding site) or for inhibited expression (e.g., operator) The control region thereby controls protein expression. Preferably, the control element of the invention contains a ribosome binding site with an initiation codon upstream of and a different coding sequence from the coding sequence of the recombinant protein.

Another aspect of the invention relates to proteins and peptides and portions and domains thereof that can be produced according to the methods disclosed herein. Proteins and peptides include, for example, but are not limited to, proteins and peptides that can be expressed cytoplasmically without leader or tag sequences and at commercially significant levels according to the methods described and disclosed herein. Preferably, these proteins and peptides exhibit proper folding upon expression in the recombinant cells of the invention. Recombinant cells of the invention preferably have reduced activity of one or more disulfide reductase enzymes, preferably less than 5 disulfide reductase enzymes, preferably less than 4 disulfide reductase enzymes. activity, and preferably less than three disulfide reductase enzymes. Preferably, expression of proteins and peptides is increased, but not decreased or significantly, in recombinant cells of the invention compared to expression in recombinant cells that do not have reduced activity of one or more disulfide reductase enzymes. may not decrease. Proteins and peptides that can be expressed in the methods disclosed herein include, for example, tetanus toxoid, tetanus toxin heavy chain protein, diphtheria toxoid, CRM, tetanus toxoid, Pseudomonas exoprotein A, Pseudomonas aeruginosa toxoid, Bordetella pertussis toxoid, Bordetella pertussis toxoid, Including, but not limited to, bacterial toxoids, E. coli heat-labile toxin B subunit, meningococcal outer membrane complex, Haemophilus influenzae protein D, flagellin Fli C, horseshoe crab hemocyanin, and fragments, derivatives, and modifications thereof. Not done.

Another aspect of the invention is that the expressed protein is fused genetically or by chemical modification or conjugation with another molecule (e.g., carbodiimide, 1-cyanodimethylaminopyridinium tetrafluoroborate (CDAP)). Concerning parts and domains of proteins. Preferred other molecules are molecules such as, but not limited to, other proteins, peptides, lipids, fatty acids, sugars, and/or polysaccharides. For example, molecules that extend half-life (eg, PEG, antibody fragments, such as Fc fragments), stimulate and/or increase immunogenicity, or reduce or eliminate immunogenicity are included.

Many proteins contain or can be genetically expressed with an N-terminal serine or threonine. The N-terminal serine or threonine can be selectively activated, thereby making it useful for conjugates. The presence of the N-terminal methionine will prevent the ability of these amino acids to be selectively activated. The method described in this patent allows the N-terminal methionine to be cleaved, which allows proteins to be produced with the desired N-terminal amino acid. Typical conjugate partner molecules include polymers such as bacterial polysaccharides, polysaccharides derived from yeast, parasites, and/or other microorganisms, polyethylene glycol (PEG), and PEG derivatives and modifications, dextran, and dextran derivatives. Including, but not limited to, derivatives, modified fragments, and derivatives. One example of a conjugated compound is PEGASYS® (pegylated interferon alpha-2a). 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 linked randomly or may be guided by site-specific conjugation, such as by modification of the N-terminal serine and/or threonine. Modifications that selectively oxidize chemical groups can also be used for site-specific conjugation.

Another aspect of the invention relates to a method of producing a peptide containing a domain, fragment, and/or portion, comprising the steps of: expressing the peptide from a recombinant cell containing an expression vector encoding the peptide. a step in which the recombinant cell has reduced activity of one or more disulfide reductase enzymes, the expression vector contains a promoter operably linked to the coding region of the peptide, and the expression vector contains a promoter operably linked to the coding region of the peptide; the reductase enzyme comprises one or more of oxidoreductase, dihydrofolate reductase, thioredoxin reductase, or glutathione reductase; and isolating the expressed peptide, the expressed peptide being soluble. and the protein or peptide is expressed with f-Met at the N-terminus, which is removed by a peptidase also expressed in recombinant cells. 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 enzyme, only two disulfide reductase enzymes, or two or more disulfide reductase enzymes. Preferably, the reduced activity of the disulfide reductase enzyme results in a shift in the cytoplasmic redox state to a more oxidative state compared to recombinant cells that do not have the reduced activity of the one or more disulfide reductase enzymes. bring about. 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 the protein. The promoter may be a constitutive or inducible promoter, where expression involves inducing the inducible promoter with an inducing agent. Preferred inducing agents include, for example, lactose (PLac), isopropyl β-D-1-thiogalactopyranoside (IPTG), substrates, and derivatives of substrates. In one 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 the expressed peptide or protein from the expression vector. do. Preferably, the recombinant protein expression vector contains the same or a different inducible promoter as the MAP gene inserted into the genome. The additional genes and genes within the expression vector may be induced together by the same inducing agent or by different inducing agents, optionally at different times depending on the promoter. Preferably, the peptidase acts on and cleaves the peptide that is coexpressed with the peptidase. Preferably, the expressed peptide is conjugated to a polymer such as, for example, dextran, bacterial capsular polysaccharide, polyethylene glycol (PEG), or a fragment, derivative, or modification thereof. Preferably, the expressed peptides are coupled to polymers including, for example, polysaccharides, peptides, antibodies or portions of antibodies, lipids, fatty acids, or combinations thereof.

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

Another aspect of the invention includes protein vaccines, including fragments, domains, and portions thereof as disclosed and described herein.

The following examples illustrate embodiments of the invention but are not to be considered as limiting the scope of the invention.

Example 1. Insertion of MAP genes into various loci of the bacterial genome.
Efficient removal of the N-terminal methionine of overexpressed intracellular recombinant proteins was achieved by inserting the MAP gene into the E. coli genome, preferably with an inducible promoter. In this manner, permanent cell lines were created that expressed the MAP gene under the control of inducers. The recombinant gene, also under the control of the inducer, is also cloned into the cell. Therefore, the MAP gene can be expressed at the 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 devised. MAPs from other species may have different preferences for adjacent amino acids. Some MAPs have been genetically modified to be less stringent in their requirement for a non-bulky amino acid adjacent to the N-terminal methionine. Inserting one or more of these MAPs with an inducible promoter into the E. coli genome will expand the range of N-terminal sequences that can be efficiently processed.

Inserting recombinant genes into the genome can disrupt the E. coli genome structure and impair cell growth. A safe way to insert the MAP gene into the E. coli genome is to use a viable strain in which the gene has been deleted and to replace the deleted gene with the MAP gene. In this way, by replacing one gene with another, the probability of genome disruption can be reduced. Two strains of E. coli, strain K12 and strain B, are widely used to manipulate genes and express recombinant proteins. E. coli strains, including K12 and B strains, can be used as host cells for insertion of the MAP gene.

Insertions/deletions at the wrong site, such as within the open reading frame of an essential gene or at a site that disrupts regulatory elements, can be lethal to bacteria. Three illustrative protocols for inserting MAP genes with inducible promoters and terminators are:
(1) Insertion of a MAP gene upstream or downstream of a previously determined gene. For example, a recombinant MAP gene can be inserted downstream of an endogenous MAP gene. The E. coli MAP gene has been studied and the genetic structure has been determined. Insertion of downstream recombinant genes will not disrupt the expression of other genes.
(2) Insertion of the MAP gene into the previously deleted gene locus. The creation of Gor/Met cell lines is an example of insertion of a MAP gene at the site of a deleted gene. Gor- cells were generated by deleting the Gor gene in BL21 cells. The Gor/Met cell line was generated by insertion of the MAP gene into the Gor locus of BL21 Gor- cells.
(3) Insertion/replacement of non-essential genes. As an example of a third method, the MAP gene is inserted to replace T7 RNA polymerase in BL21(DE3) cells. BL21(DE3) encodes a highly active T7 RNA polymerase within the DE3 fragment that can transcribe recombinant genes under the control of the T7 promoter. If the recombinant gene is transcribed by the native RNA polymerase under the control of the T5 promoter, the T7 polymerase gene can be replaced by the MAP gene.

Site-directed mutagenesis techniques are required for the above-mentioned insertion site selection. Many methods are known for manipulating bacterial genomes, and the present invention discloses two examples. The most common method for gene insertion/deletion is Red recombineering. This technique, widely used for mutations in bacterial and eukaryotic genomes, introduces short sequences of DNA that are complementary upstream and downstream of a selected insertion site adjacent to the gene of interest. Begin by using PCR for The PCR product is then electroporated into E. coli already expressing the red recombinase in the previously transformed temperature sensitive vector. red recombinase assists in homologous recombination to insert genes at selected sites. Since the Red recombinase gene is on a temperature sensitive plasmid, Red recombinase can be removed by growing the bacteria at 42°C.

To improve selection of positive clones, marker genes can be introduced and then removed after positive clones are confirmed. In one method, a flippase recognition signal is introduced adjacent to a marker gene, such as an antibiotic gene, that is cloned downstream of the inserted gene. The PCR product used for gene insertion will then include the marker gene. After the recombination event has occurred, the marker gene can be used to select positive clones. Once a positive clone is identified, flippase expression is introduced into the bacteria to remove the marker gene between the two flippase recognition sites. This type of insertion is marked with a trace containing a flippase recognition sequence at the insertion site. This method was used to create Gor/Met E. coli strains by inserting the Met gene over the deleted Gor gene, and is described in Example 2 below.

CRISPR technology can be used in E. coli when combined with Red recombineering. CRISPR-assisted red recombineering utilizes two plasmids and one oligo. One plasmid encodes constitutively expressed Red recombinase and Cas9 protease. The other encodes a CRISPR guide RNA whose insertion site has been cloned. These two plasmids have two compatible origins of replication. The oligo is made to contain the gene of interest adjacent to the insertion site sequence. E. coli cells will be transformed with these three elements, and surviving colonies should be those with the inserted gene: Cas9 protease will bind to the CRISPR guide RNA and scan the insertion site. Once the insertion site is located, Cas9 cuts the double-stranded DNA, allowing red recombinase to function to perform homologous recombination with the protein of interest between the cut site and the oligo. Will. Those colonies with the original sequence will be recognized and eliminated. Only those with the gene of interest will survive. CRISPR-assisted Red recombineering has a 65% success rate, with the remaining 35% coming from Cas9 failure to locate the insertion site. Therefore, screening for positive clones is quick and simple.

Example 2. Construction of an E. coli cell line with gene replacement for cytoplasmic expression of recombinant proteins without N-terminal methionine Construction of the E. coli cell line BL21 Gor- is described in US Pat. Nos. 10,093,704 and 10,597,664. The described strain has a deleted Gor gene and an oxidative cytoplasm such that the protein is expressed with properly folded disulfide bonds and at high levels in the cytoplasm. For these strains, an additional E. coli MAP gene was inserted into the Gor locus and the new strains were designated Gor/Met E. coli. A Tac promoter (with a Lac operator) was added upstream of the MAP gene so that the expression of the MAP gene could be controlled by the timing of IPTG addition. This was accomplished as follows: Additional E. coli MAP genes were inserted into the genome by homologous recombination. In the Gor/Met cell example, the red recombinase system was used to help insert the MAP gene into 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. The gene was then cloned downstream of the chloramphenicol acetyltransferase (CAT) gene flanked by two short flippase recognition target (FRT) sequences. The MAP and CAT genes together formed a transferred gene cassette. Using PCR, 50 bases each of the upstream and downstream flanking sequences of the original Gor locus were introduced upstream and downstream of the import cassette, respectively. The final PCR product was used to transform BL21 Gor- cells previously transformed with Red recombinase. Expression of Red recombinase in cells accelerated homologous recombination of sequences flanking the Gor locus with sequences of the import cassette. Bacterial colonies that are resistant to chloramphenicol were confirmed to have an import cassette insertion. The identified bacteria were then transformed with a flippase gene whose expression recognizes the FRT sequence flanking the CAT gene and excises the gene, leaving the MAP inserted. Therefore, the Gor/Met cell line has the MAP gene inserted into the Gor locus without disturbing the rest of the genome. This cell line is a deposited strain and was deposited with the American Type Culture Collection on February 9, 2021 under deposit number PTA-126975. Successful insertion of the MAP gene was confirmed by sequencing results using primers designed to flank the Gor locus.

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

Example 3. Expression of CRM ₁₉₇ in Gor/Met E. coli
CRM ₁₉₇ is an enzymatically inactive and non-toxic form of diphtheria toxin that contains a single amino acid substitution, G52E. Like DT, CRM ₁₉₇ has two disulfide bonds. One disulfide connects Cys186 to Cys201 and connects fragment A to fragment B. A second disulfide bridge connects Cys461 in fragment B to Cys471. CRM ₁₉₇ is commonly used as a carrier protein for carbohydrate-, peptide-, and hapten-protein conjugates. As a carrier protein, CRM ₁₉₇ has several advantages compared to diphtheria toxoid and other toxoidated proteins.

CRM ₁₉₇ is produced in its original host Corynebacterium, a slow-growing bacterium with doubling times of hours rather than minutes, but yields are low, typically <50 mg /L. Corynebacterium strains have been engineered to produce higher levels of CRM ₁₉₇ (see, eg, US Pat. No. 5,614,382). CRM ₁₉₇ is also expressed at high levels in strains of Pseudomonas fluorescens. However, production of CRM ₁₉₇ in a strain that is at the BL1 safety level and cheap to culture and propagate would be advantageous. Expression of soluble, properly folded intracellular CRM ₁₉₇ in the BL21 Gor- strain was successful, with >2 g CRM ₁₉₇ per liter of fermenter cell culture. However, the majority of CRM ₁₉₇ produced was found to have an N-terminal f-methionine. The CRM ₁₉₇ gene with the tac promoter was cloned into the Gor/Met E. coli strain (see, eg, US Pat. Nos. 10,093,704 and 10,597,664 for gor- strains). Therefore, both the MAP gene and the recombinant CRM ₁₉₇ gene were under the control of the same tac promoter and could be expressed simultaneously upon IPTG induction.

The expression of CRM ₁₉₇ in BL21 Gor- and BL21 Gor/Met E. coli was compared. Similar yields of approximately 2 g/L were found for both strains, indicating that co-expression of the MAP gene did not significantly affect the expression of CRM ₁₉₇ . CRM ₁₉₇ purified from BL21 Gor- and BL21 Gor/Met strains was analyzed by MALDI-TOF mass spectrometry, and the results are summarized in Table 1. Lot NO21p114 was expressed in the Gor- strain and lot NO21p221 was expressed in the Gor/Met strain. CRM ₁₉₇ expressed in BL21 Gor- contained an N-terminal Met, whereas CRM ₁₉₇ expressed in Gor/Met E. coli did not, making the method described in this invention a success. It was proven that there is.

(Table 1) CRM197 that did not decrease (n=3, 1SD)

Example 4. Expression of the cytokine IL10 from Epstein-Barr virus in the Gor/Met strain.
The IL10 gene derived from Epstein-Barr virus was cloned and expressed as a soluble intracellular protein in Gor/Met E. coli. A metal affinity tag was included on 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 and it was found that the protein does not have an N-terminal methionine.

The method was performed using the following protocol: samples were digested with trypsin and analyzed by LC-MS/MS on an LTQ Orbitrap Velos (ThermoFisher Scientific, Bremen, Germany) connected to a Proxeon 1200 nanoLC (Proxeon Biosystems). . Chromatography was performed on a 75 μm i.d. Self-Pack PicoFrit fused silica capillary column (New Objective, Woburn, MA) with a length of 15 cm. The stationary phase was a reverse phase C18 Jupiter column (5 μm, 300 Å) (Phenomenex, Trans, CA). Mass resolution was set to 30 000 for parent mass determination in MS mode and 7 500 for acquisition of fragmentation spectra in MS/MS mode. MS/MS spectra obtained during the LC-MS/MS run were subjected to a Mascot search against predicted protein sequences. Carbamidomethyl (C) was selected as the Fixed modification and oxidation (M) was selected as the Variable modification. The aim was to retrieve the peptide corresponding to the first tryptic cleavage from the digestion solution. This peptide on the submitted sequence will include an arginine at position 13. If methionine was present at the N-terminus, the result would be amino acid sequence 1-13 (MTDQCDNFPQMLR; SEQ ID NO: 1); if methionine was absent, the result would be amino acid sequence 2-13 ( TDQCDNFPQMLR; SEQ ID NO: 2).

(Table 2)

Underlined and bolded amino acids in Table 2 identify sequences identified from MS/MS sequencing data by Mascot. This means that the ions were found to have a 2-13 sequence (no methionine at the N-terminus), and that no ions were found for the 1-13 sequence (methionine at the N-terminus). means.

2-13 sequence (without N-terminal methionine) is associated with the presence of the following ions in the MS trace: 762.8 m/z (+2), 508.9 m/z (+3), 770.8 m/z (+2). , confirmed by their corresponding fragmentation patterns from MS/MS traces. All three identified ions contain alkylated cysteine, but the ion at 770.8 m/z also contains oxidized methionine (position 11 on the submitted sequence). In conclusion, IL10 expressed in Gor/Met was produced without N-terminal methionine.

Example 4. Expression of genetically neutralized tetanus toxin (8MTT) in Gor/Met E. coli Tetanus toxin is known as one of the most potent toxins in humans and is referred to as spastic toxin or TeNT. The LD50 of this toxin has been determined to be approximately 2.5-3 ng/kg. Tetanus toxin is produced as a single polypeptide chain by Clostridium tetani, an anaerobic bacterium normally found in soil, which is posttranslationally cleaved into two chains by a trypsin-like protease to form the active protein. form. The light chain (LC) of the 50kDa domain contains an N-terminal endopeptidase, the heavy chain (HC) contains a 50kDa receptor-binding domain at the C-terminus (HCC), and a 50kDa LC translocation domain located at the N-terminus (HCN). It will be done. The two chains are connected by a single disulfide bond. Tetanus toxin enters peripheral motor neurons by binding to gangliosides and synaptic proteins on their surface through the C-terminal domain of the heavy chain (HCC). The toxin passes through the cell bodies and synapses of interneurons in the central nervous system and is transcytosed into inhibitory neurons in synaptic vesicles. In inhibitory neurons, the heavy chain translocation domain (HCN) undergoes a pH-mediated conformational change and transports the LC through the membrane of synaptic vesicles into the cytoplasm, where it is transported within the cytosol of the cell. is released to cleave the vesicle-associated membrane protein 2 (VAMP2), a soluble NSF-binding protein receptor (SNARE) on vesicles. VAMP2 cleavage in inhibitory neurons prevents neurotransmitter exocytosis, which prevents the release of inhibitors of neuromuscular synaptic function, leading to continued neuromuscular activation and spastic paralysis.

Chemically inactivated tetanus toxoid (TTxd), formed by treating the toxin with formaldehyde, is used as an effective vaccine against tetanus. TTxd is also used as a conjugate vaccine carrier for polysaccharide antigens. Conjugate vaccines using TTxd as a carrier protein include vaccines against Haemophilus influenzae type b and Neisseria meningitidis. However, TTxd has many amines used in the conjugate that are blocked by the toxoidation process. Furthermore, TTxd is a heterogeneous product, containing aggregates along with Clostridium and media contaminants. TTxd vaccine needs to be further purified for use in conjugate vaccines. More importantly, the production and purification of TT from Clostridium is time-consuming and costly. A genetically inactivated homogeneous recombinant tetanus toxin produced in a low cost host such as E. coli would be desirable.

Various strategies to generate inactivated recombinant TT proteins as vaccines or carrier proteins have been considered. One is the use of heavy chain fragments (TTHC). Another is the use of genetically inactivated tetanus toxoid (US Patent Application Publication No. 2020/03841201). TTHC is part of TT and does not have a catalytic domain. It was claimed that neutralizing antibodies against TTHC subunit vaccines were more efficient than full-length toxoid vaccine antibodies. TTHC was expressed at high levels (>400 mg/L) in the BL21 Gor-line (see, eg, US Pat. Nos. 10,597,664 and 10,093,704).

8MTT is a genetically detoxified tetanus toxin (TT) with eight amino acid mutations. Like tetanus toxin, 8MTT has five disulfide bonds. LD50 has a toxicity that is 50 million times less than natural TT. 8MTT vaccination induces a strong immune response IgG antibody response in mice and is a lead candidate for a new tetanus vaccine and is used as a conjugate vaccine carrier protein, similar to the widely used CRM ₁₉₇ . He has great abilities. 8MTT was originally cloned into the pET28 expression vector and expressed in BL21(DE3) cells with a His tag attached to facilitate purification. Expression was approximately 10 mg/liter in shake flasks. To produce a protein without tags and without N-terminal methionine, the 8MTT gene was subcloned into an expression vector with a tac promoter (with a lac operator) and a T7 terminator, and then BL21 Gor/Met in a fed-batch fermenter. Expressed intracellularly. The expressed M8TT protein was found to be soluble and could be purified in excess of 500 mg per liter. M8TT was purified to >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-in-source decomposition) to obtain terminal fragmentation. ISD confirmed that the sequence does not have an N-terminal methionine and the first residue of the sequence is the predicted proline, allowing the identification of a ladder of N-terminal fragments. Therefore, the Gor/Met E. coli strain efficiently expressed large amounts of soluble M8TT without the N-terminal methionine.

Example 5. CRM ₁₉₇ variant with N-terminal serine
The CRM ₁₉₇ gene containing an N-terminal serine (CRM-Ser) was cloned into the Gor/Met E. coli strain and grown and expressed in a bioreactor. >1 g/L of soluble CRM-Ser was expressed without any optimization of fermentation conditions, indicating excellent expression of the protein. Cells were collected and CRM-Ser was purified. CRM-Ser was analyzed by MALDI-ISD as described in Example 4. ISD enabled the identification of a ladder of N-terminal fragments 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, the Gor/Met E. coli strain efficiently expressed large amounts of soluble CRM-Ser without the N-terminal methionine. Serine can be selectively oxidized and used in conjugates.

Example 6: Possible MAP genes for insertion into bacterial genomes.
Removal of the N-terminal methionine by MAP may be important for proper function and stability of the protein. Despite the activity of the native MAP, the majority of recombinant proteins expressed in E. coli retain an N-terminal methionine initiation codon. When overexpressed recombinant proteins are produced, insufficient MAP or its cofactors may be present. To ensure processing of the N-terminal methionine in the recombinant protein, an additional MAP gene is inserted under a strong inducible promoter to facilitate the methionine cleavage process when necessary.

1. E. coli MAP gene
Gor/Met cells are an example for inserting an additional E. coli MAP gene into the bacterial genome under the same inducible promoter as that of the recombinant gene on the expression vector. When not induced, this MAP gene remains silent and the bacterium grows without the burden of additional gene expression. MAP proteins can be designed to be turned on as needed, since only recombinant proteins are induced to be expressed, and additional MAP proteins are also induced. A potential drawback of this system is that not all proteins with an N-terminal methionine can be efficiently cleaved by E. coli MAP. E. coli MAP works when small amino acids (G, A, S, C, P, T, V) (P1' position) are adjacent to the N-terminal methionine. Preferably, the P2' position of the amino acid (the C-terminal amino acid to P1) is not proline. Other MAP genes with different P1 and P2 amino acid requirements may be inserted instead to process proteins with bulky amino acids next to the N-terminal methionine.

2. Two different MAP genes in tandem in different inducible promoters Yeast genes are processed by two MAP genes. Yeast MAP1 and MAP2 exhibit different cleavage efficiencies for the same substrate in vivo. Both MAPs were less efficient when the second residue was V, and MAP2 was less efficient than MAP1 when the second residue was G, C, or T. Humans also have two MAPs, MAP1 and MAP2. They can both process proteins containing A, C, G, P, or S at the P1' position. When the P1' residue is T or V, N-terminal Met removal is primarily catalyzed by MAP2, and the extent of cleavage depends on the sequence at the P2' to P5' positions. When the P2' residue is not A, G, and P, N-terminal processing is expected to be complete. When A, G, or P is a P2' residue, methionine removal is incomplete or not occurring. Since different MAPs have different substrate specificities, two or more MAP genes from different or the same species can cover methionine removal processing from more recombinant proteins. The (eg, inducible) promoters that control gene transcription may be different so that the two MAP genes can be turned on or one turned on/one turned off at different times as desired.

3. Insert a mutant MAT gene that can cleave all N-terminal methionines without constraints on the following amino acids.
Current MAPs are inconvenient for the N-terminal structure of some proteins and will not catalyze the removal of their N-terminal methionine. A universal rule for predicting whether an initiating methionine will be processed by MAP is based on the size of the amino acid at the P1' position. Generally, methionine is cleaved if the amino residue has a radius of gyration of 1.29 Å or less. Human MAP has even more stringent requirements for substrates with acidic residues at the P2' and P5' positions. To expand the substrate specificity of existing MAPs, the E. coli MAP gene was mutated so that its product could cleave 85-90% of the N-terminal methionines on proteins listed in protein databases. This MAP has three mutations in its substrate binding pocket, thus allowing not only small amino acids but also bulky or acidic amino acids (e.g. M, H, D, N, E, Q, L, I at the P2' position , Y, and W) allowing the removal of the N-terminal methionine from proteins. These enzymes can cleave the amino acid at the P1' position even when the amino acid residue at the P2' position is small. This MAP gene insertion processes proteins with a broader N-terminal structure. Again, it would be beneficial to add an inducible promoter to control the activity of this strong mutant gene expression.

Example 7: Proteins that can be produced without disulfide bonds in Gor/Met cell lines
Although Gor/Met cell lines have been developed to express disulfide bond proteins without N-terminal methionine, proteins without disulfide bonds can be expressed in Gor/Met cells without N-terminal methionine. These proteins can be expressed in E. coli with Met, but not necessarily with gor-. An example of such a protein is Staphylococcal protein A (SPA), which is widely used for antibody purification. SPA is a 42kDa protein originally found in the cell wall of the Staphylococcus aureus bacterium. This protein consists of five homologous Ig-binding domains that can bind to proteins from many mammalian species, especially IgG, and is found to overlap within the Fc region of most immunoglobulins and within the Fab region of the human VH3 family. Bind to the chain. SPA does not contain any disulfide bonds. Commercially, SPA is derived from two sources: a Staphylococcus aureus mutant strain (Sigma) that contains lesions in the cell wall from which SPA is secreted, and SPA as a recombinant protein intracellularly or intracellularly. expressed in E. coli (Sigma-Aldrich, SinoBiological, ThermoFisher, and DeNovo Biopharma, among others). SPA production can be at high levels in Pichia pastoris. The majority of SPAs are expressed within E. coli cells. The mature SPA sequence begins with alanine rather than methionine, so for SPA to be produced intracellularly, a methionine must be added to the gene at the N-terminus or to release the mature protein in vitro. Therefore, it needs to be expressed at the C-terminus of the cleavable fusion protein. The former is not the true form of the mature protein, and the latter is not cost-effective and time-efficient.

Expression of SPA is efficient in the bacterial strains disclosed herein that have an inducible MAP gene insertion. SPA is abundantly expressed in E. coli strains, functions similarly in 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 f-methionine can be cleaved at will. The inducible promoter of the MAP gene can be expressed in a controlled manner at the same time that SPA is expressed, or at any time thereafter. No in vitro manipulation is required to remove the N-terminal methionine.

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, United States and foreign patents and patent applications, are specifically and entirely incorporated by reference. The term comprising wherever used is intended to encompass the terms consisting of and consisting essentially of. Furthermore, the terms including, including, 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 (10)

A method of producing a protein containing one or more sulfide linkages, comprising the steps of:
expressing the protein from a recombinant cell containing an expression vector and a genome encoding the protein sequence, the recombinant cell having reduced activity of one or more disulfide reductase enzymes; the N-terminus of the protein contains methionine, the expression vector contains a first inducible promoter, and the step of expressing the protein comprises inducing the first inducible promoter with a first inducing agent. , process;
a step of expressing a peptidase from a gene in the recombinant cell , the gene containing a second inducible promoter, and a step of expressing the peptidase, wherein the gene contains a second inducible promoter; inducing, the peptidase gene is integrated into the genome of the recombinant cell, the peptidase removes methionine from the N-terminus of the expressed protein , and the first inducing agent and the first the two inducing agents are the same ; and isolating the protein. The expressed proteins are tetanus toxoid, 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) heat-labile toxin B subunit, Neisseria meningitidis outer membrane complex, Hemophilus influenzae protein D, flagellin Fli C, horseshoe crab hemocyanin, or these 2. The method of claim 1, comprising a fragment, derivative, or modification. 2. The method of claim 1, wherein the recombinant cell is an E. coli cell or a derivative or strain of E. coli. 4. The method of claim 3, wherein the recombinant cell is E. coli containing the gor mutation. 4. The method of claim 3, wherein the recombinant cell is obtained from or derived from ATCC Deposit No. PTA-126975. 2. The method of claim 1, wherein the step of isolating comprises chromatography comprising a sulfate resin, a gel resin, an activated sulfate resin, a phosphate resin, a heparin resin, or a heparin-like resin. conjugating or coupling the isolated protein with a chemical entity including a polysaccharide, a polymer, polyethylene glycol, a derivative of polyethylene glycol, a peptide, an antibody or a portion of an antibody, a lipid, a fatty acid, or a combination thereof. 2. The method of claim 1, comprising: 2. The method of claim 1, wherein the peptide is oxidized with an oxidizing agent comprising hydrazide, hydrazine, an aminooxy group, an N-terminal 1-amino 2-alcohol amino acid, or a combination thereof. Proteins include proinsulin, proinsulin-like protein, prorelaxin, proopiomelanocortin, proenzyme, prohormone, proangiotensinogen, protrypsinogen, prochymotrypsinogen, propepsinogen, proprotein of the coagulation system, prothrombin, proplasminogen , proprotein of the complement system, procaspase, propacifastin, proelastase, prolipase, and procarboxy polypeptidase. Recombinant cells obtained from ATCC deposit number PTA-126975.