WO2025035093A1 - Cytoplasmic expression of soluble proteins - Google Patents

Abstract

The invention is directed to recombinant cell lines, methods and compositions for expressing and purifying products such as peptides and proteins in microorganisms. Products are expressed recombinantly, wherein the cytoplasm of the microorganism is altered to produce products in a final or usable form. Alterations include modifying the cell's chaperone composition or concentration to improve the solubility of heterologous proteins expressed in the cells.

Description

CYTOPLASMIC EXPRESSION OF SOLUBLE PROTEINS Reference to Related Applications This application claims priority to U.S. Provisional Application No. 63/531,838 filed August 10, 2023, the entirety of which is incorporated by reference. 1. Field of Invention The invention is directed to recombinant cell lines, methods and compositions for expressing and purifying products such as peptides and proteins in microorganisms. Products are expressed recombinantly, wherein the cytoplasm of the microorganism is altered to produce products in a final or usable form. Alterations include modifying the cell’s chaperone composition or concentration to improve the solubility of heterologous proteins expressed in the cells. 2. Background of the Invention Escherichia coli is an excellent microorganism to express heterologous (recombinant) proteins: around 30% of approved therapeutic proteins are currently being produced using these bacteria as the host. One major issue of the production of recombinant proteins using an E. coli host is the accumulation of heterologous proteins, mainly as insoluble aggregates in the form of inclusion bodies. Extraction of the recombinant protein from inclusion bodies is a tedious and cumbersome process that requires denaturation and several renaturing steps to obtain a soluble and properly folded protein. A need exists to produce native structures of recombinant proteins at desired expression levels both efficiently and inexpensively and with minimal accumulation of insoluble heterologous proteins. Summary of the Invention The present invention overcomes the problems and disadvantages of current strategies and designs and provides new compositions and methods for producing soluble recombinant peptides and proteins in prokaryotic cells. One embodiment of the invention is directed to recombinant bacterial cells whose genome contains one or more chaperone sequences integrated into a genome of the bacterial cell under the control of a promoter. Preferably the one or more chaperone sequences encode a cytosolic chaperone system. Such as, for example, a ribosome-associated trigger factor, a DnaK system, a DnaJ chaperone, a GrpE chaperones; a KJE chaperone, a GroEL chaperone system, a GroES chaperone, an ELS chaperone, a ClpB chaperone, an IbpAB chaperone, or a combination thereof. 1 | P a g e Preferably, the recombinant bacterial cell comprises a recombinant protein or polypeptide and, also preferably, the genetic sequence of the recombinant protein or polypeptide is integrated into the genome of the bacterial cell. Preferably, the one or more chaperon sequences encode one or more of the chaperones DnaK, DnaJ or GrpE, and the recombinant protein or polypeptide comprises an antibody or antibody fragment. Another preferred alternative comprises one or more chaperon sequences that encode a trigger factor and the recombinant protein or polypeptide comprises a lysozyme, a mouse endostatin, or an ORP150 protein. Preferably the one or more chaperone sequences and/or the sequence which encodes the recombinant protein are controlled by inducing agents, and also preferably, the controlling inducing agent is the same for both. Preferably, the recombinant cell comprises an oxidative cytoplasm that allows a recombinant protein to form disulfide bonds in the cytoplasm. With an oxidative cytoplasm, preferably the one or more chaperones comprise chaperones selected from the group consisting of DsbD, DsbE, DsbG, SurA, FkpA, Skp, and protein disulfide isomerase. Also preferably, the bacterial cell contains a recombinant peptidase sequence that expresses a peptidase and, optionally, that peptidase sequence may be integrated into the genome of the bacterial cell. Preferable, the recombinant bacterial cell is an E. coli expression strain containing a modified genome with a Gor gene mutation or deletion. Another embodiment of the invention is directed to a recombinant bacterial cell containing: one or more chaperone sequences integrated into a genome of the bacterial cell under the control of a first inducible promoter; a recombinant sequence that encodes a recombinant protein containing one or more disulfide bonds under the control of a second inducible promoter; and an oxidative cytoplasm that allows the recombinant protein to form the one or more disulfide bonds in the cytoplasm. Preferably the one or more chaperone sequences encode a cytosolic chaperone system selected from the group consisting of a ribosome-associated Trigger Factor, a DnaK system, a DnaJ chaperone, a GrpE chaperones; a KJE chaperone, a GroEL chaperone system, a GroES chaperone, an ELS chaperone, a ClpB chaperone, an IbpAB chaperone, or a combination thereof. Preferably, expression of the first and second inducible promoters are controlled by a same inducing agent. Also preferably, the bacterial cell comprises a recombinant peptidase sequence that expresses a peptidase and, optionally, that recombinant peptidase sequence may be integrated into the genome. Another embodiment of the invention is directed to methods of expressing a recombinant protein containing one or more disulfide bonds in the cytoplasm of a bacterial cell comprising: 2 | P a g e providing the bacterial cell containing one or more chaperone sequences that express chaperone proteins integrated into a genome of the bacterial cell and a recombinant protein sequence; providing an oxidative cytoplasm to the bacterial cell that allows the recombinant protein to form the one or more disulfide bonds in the cytoplasm; and expressing the recombinant protein in the bacterial cell. Preferably the expression of the one or more chaperone sequences and the recombinant protein are controlled by one or more inducing agents. Optionally, expression of the one or more chaperone sequences and the recombinant protein are controlled by the same inducing agent. Preferably, the recombinant protein is purified and/or isolated from heterologous and/or insoluble proteins of the bacterial cell. Also preferably, the chaperone proteins do not trigger undesired proteolytic activity of the bacterial cell as occurs in a bacterial cell in the absence of chaperones. Preferably, the recombinant protein expressed is in a a native formation with one or more disulfide bonds. Another embodiment of the invention is directed to recombinant bacterial cells whose genome contains: one or more chaperone sequences integrated into a genome of the bacterial cell under the control of a first inducible promoter; and a genomic sequence that encodes a recombinant protein or polypeptide under the control of a second inducible promoter; wherein the bacterial cell contains an oxidative cytoplasm that allows a recombinant protein to form disulfide bonds in the cytoplasm; wherein the one or more chaperon sequences encode one or more of the chaperones DnaK, DnaJ or GrpE, and wherein the recombinant protein or polypeptide comprises an antibody or antibody fragment. Preferably, expression of the first and second inducible promoters are controlled by a same inducing agent. Preferably, the bacterial cell contains a recombinant peptidase sequence that expresses a peptidase which may be integrated into the genome. Other embodiments and advantages of the invention are set forth in part in the description, which follows, and in part, may be obvious from this description, or may be learned from the practice of the invention. Description of the Figures Figure 1 depicts two SDS-PAGE of the total protein and the soluble fractions of Expression of the scFV and the chaperone operon induced with IPTG and arabinose. Figure 2 depicts cell cultures analysis by SDS-PAGE. Figure 3 depicts an SDS-PAGE analysis of clarified cell lysate/ Description of the Invention 3 | P a g e The present invention describes bacterial strains, methods, and recombinantly produced proteins whose expression is facilitated by chaperone assisted folding proteins. Microorganisms have been used and are currently in use to express recombinantly produced proteins for research and a host of therapeutic purposes. An important aspect in protein expression is the ability to properly express and isolate a desired protein product. Improperly folded proteins and proteins inextricably complexed with heterologous or undesired proteins are ever present problems. One strategy to improve protein solubility in E. coli is using molecular chaperones. Chaperones assist in folding newly synthesized proteins to the native state and provide a quality control system that refolds misfolded and aggregated proteins. The major cytosolic chaperone systems include the ribosome-associated Trigger Factor, the DnaK system (DnaK with its DnaJ and GrpE cochaperones; KJE), the GroEL system (GroEL with its GroES cochaperone; ELS), ClpB, and heat shock proteins such as IbpAB. Because recombinant proteins are often expressed at high levels, there may be an insufficient concentration of chaperones in the cell cytoplasm, resulting in misfolded and/or aggregated proteins. To address this limitation, E. coli chaperones have been co-expressed with the recombinant protein to assist with the correct folding. Currently, co-expression is achieved by a technique wherein additional chaperones are either expressed from the same vector as the recombinant heterologous protein or from a separate vector. From the industry perspective, these approaches have undesirable effects on the cost of production caused by increased expression vector instability due to the plasmid size or exhausting cellular resources due to maintaining two plasmids with different antibiotics. Furthermore, since there are multiple copies of the expression vector, plasmid-based expression may result in unbalanced amounts of chaperones that might trigger undesired proteolytic activities, which is detrimental to the stability, quality, and yield of the recombinant protein. Chaperones derived from E. coli or other bacteria can be used alone or in combination, as an operon under the control of a single promoter or a single gene with its own promoter. For example, the E. coli chaperones GroEL/GroES and Trigger Factor (TF) are expressed in E. coli cells by integrating extra copies of their genes into the bacterial genome. A preferred promoter is the arabinose promoter which can be utilized as an inducible promoter for integrated chaperons. Other inducible or constitutive promoters can be used. To integrate extra copies of GroEL/GroES and TF, a synthetic operon containing all three genes under the control of an arabinose promoter 4 | P a g e was synthesized and cloned into the E. coli genome. Optionally, the araC arabinose transcription factor is part of the operon. Many methods are known for inserting chaperone genes into the bacterial genome. Red Recombinase is one such technique that can be used to integrate the chaperone operon into the genome. Chaperons that help to form disulfide bonds include, but are not limited to: DsbD, DsbE, DsbG, other E. coli chaperons: SurA, FkpA, Skp, human Protein Disulfide Isomerase (PID). The genome of existing or new expression strains of E. coli or other prokaryotic systems, e.g., Bacillus subtilis, can be modified with extra copies of chaperones alone or in combination with other chaperones to improve the correct folding and solubility of a broader range of recombinant proteins. Some proteins may require a particular chaperone or a particular set of chaperones for correct folding and solubility. For example, co-expression with one or more of chaperons DnaK, DnaJ, and/or GrpE improves solubility of antibodies and antibody fragments (e.g., scFV). Co-expression with trigger factor, for example, improves solubility of lysozyme such as human lysozyme, endostatin such as murine endostatin, and ORP150 protein, such as human ORP150 protein. The additional chaperones integrated into the genome of a prokaryotic expression host with an unmodified genome (e.g., BL21). They can also integrate into the genome of a prokaryotic expression host with a modified genome, for example, the Gor∆ E. coli strain (U.S. Patent Nos. 10,093,704 and 10,597,664, which are incorporated by reference), a host mutated to have an oxidative cytoplasm. The suitability of chaperone combination can be target-protein specific. Fine tuning of the expression of both the target protein (e.g., expression rate, temperature) and chaperones (e.g., type of chaperone(s), levels, timing of expression, etc.) can often improve the amount of soluble protein. Using different promoters on the plasmid containing the target recombinant protein and on the chaperone gene which is inserted into the bacterial genome allows for independent control of expression of the chaperones and target protein. An embodiment of the invention disclosed herein is directed to bacterial cell lines containing integrated chaperone sequences. Preferably the bacterial cell line is E. coli, but other bacterial and non-bacterial cell lines can also be utilized, especially those which are known to be useful as recombinant microorganisms. Examples of cell lines containing integrated chaperone sequences are: (i) the E. coli cell line referred to as FinaGor A herein in which recombinant 5 | P a g e sequences of chaperone GroEL/GroES have been integrated; (ii) the E. coli cell line referred to as FinaGor B herein in which recombinant sequences of the chaperone trigger factor have been integrated; (iii) the E. coli cell line referred to as FinaGor A or B herein in which recombinant sequences of the chaperone DnaK or DnaJ have been integrated; (iv) the E. coli cell line referred to as FinaGor A or B herein in which recombinant sequences of chaperone GrpE have been integrated; (v) the E. coli cell line referred to as FinaGor A or B herein in which recombinant sequences of chaperone ClpB or IbpAB have been integrated; (vi) the E. coli cell line referred to as FinaGor A or B herein in which recombinant sequences of chaperone DspD, DspE, or DspG have been integrated; and (vii) the E. coli cell line referred to as FinaGor A or B herein in which recombinant sequences of chaperone SurA, FkpA, Skp, or PID have been integrated. Without wishing to be limited, it is believed that the ratio of recombinant protein to chaperone protein in a cell line functions well at a ratio of about 1 to about 1. Preferably the ratio would be about or less than 10 to 1 (protein to chaperone), also preferably about or less than 5 to 1 or about or less than 2 to 1. In a similar fashion, preferably the ratio would be about or less than 10 to 1 (chaperone to protein), also preferably about or less than 5 to 1 or about or less than 2 to 1. Another embodiment of the invention disclosed herein is directed to methods of producing recombinant peptides and proteins in bacteria comprising: expressing the protein from a recombinant cell containing an expression vector that encodes the protein sequence, wherein the cell cytoplasm has an increased concentration of chaperones in which the increased concentration of chaperones is achieved by integrating one or more copies of chaperone genes under the control of a promoter into the bacterial genome. Chaperones are preferably from E. coli, but chaperones from other bacteria can be used alone or in combination, as an operon under the control of a single promoter or a single gene with its own promoter. There may be more than one chaperone gene, each with the same promoter or with different promoters. Preferably the promoter is inducible. An inducible promoter allows one to increase chaperones concentration on an as-needed basis. The promoter driving integrated chaperone/chaperones expression can be the same or different from a promoter for recombinant protein expression. Another embodiment of the invention is directed to methods of producing soluble recombinant peptides and proteins in bacteria comprising: expressing the protein from a prokaryotic cell containing an expression vector that encodes the protein sequence, wherein the prokaryotic cell genome has one or more extra copies of chaperone genes and cells are capable 6 | P a g e of disulfide bonds formation in the cytoplasm.. Such cytoplasm can be oxidative or contain a recombinant sulfhydryl oxidase and a recombinant disulfide bond isomerase. The coexpression of a recombinant protein with sulfhydryl oxidase and a disulfide bond isomerase allows disulfide bond formation in reduced cytoplasm. The oxidative cytoplasm has reduced activity of one or more disulfide reductase enzymes, wherein the one or more disulfide reductase enzymes comprise one or more of an oxidoreductase, a dihydrofolate reductase, a thioredoxin reductase, or a glutathione reductase, wherein the reduced activity of one or more disulfide reductase enzymes results in a shift the redox status of the cytoplasm to a more oxidative state. Another embodiment of the invention is directed to methods of producing soluble recombinant peptides and proteins in bacteria comprising: expressing the protein from a prokaryotic cell containing an expression vector that encodes the protein sequence, wherein the prokaryotic cell has one or more extra copies of chaperone genes and elevated levels of N terminal methionine peptidase. The cell may also have an oxidative cytoplasm in addition to elevated levels of N terminal methionine peptidase. Another embodiment of the invention is directed to soluble recombinant peptides and proteins produced in bacteria or other cell strains with minimal accumulation of insoluble heterologous proteins. The following examples illustrate embodiments of the invention but should not be viewed as limiting the scope of the invention. Examples Example 1. Construction of an E. coli strain with enhanced chaperone production. Human interferon gamma (hIFN-γ) is not expressed as a soluble protein in E. coli. A synthetic operon containing GroEL/GroES and Trigger Factor (TF) genes under the control of an arabinose promoter and araC arabinose transcription factor is inserted into the genome of E. coli BL21 using the Lambda Red Recombinase method (e.g., see Brian J. Caldwell and Charles E. Bell, Prog. Biophys. Mol. Biol. Oct 147:33-46, 2019, which is specifically incorporated by reference). Although restriction enzyme cloning is the standard for recombinant engineering, sequence modifications can only be made at restriction enzyme sites, a significant limitation. Lambda Red Recombinase is not limited to restriction sites, but uses homologous recombination for cloning and, thus, permits direct modification of sequences within the microorganism of interest. Lambda Red Recombinase is derived from lambda red bacteriophage and its use generically referred to as 7 | P a g e homologous recombination. As such, a large variety of modifications are possible including but not limited to point mutations, base pair changes, and the addition of protein tags of chromosomes and plasmid sequences. The integration site utilized is fliT, one of the flagellar loci of E. coli, as deletion or mutation of flagellar genes do not affect E. coli vitality or growth rate. Other loci of nonessential genes can be used to insert copies of chaperone genes. The resulting strain, “BL21Plus”, along with parental strain BL21, are transformed with an expression vector containing the hIFN-γ gene. Each strain is evaluated for expression of soluble hIFN-γ. The cells are initially grown at 37°C. After induction, the cells are grown at 20°C for 6 hours. On harvest of the cell mass by centrifugation, the soluble fraction is obtained by breaking cells with glass beads and subsequently clarifying the lysate by centrifugation. SDS-PAGE analysis is used to confirm increased levels of soluble hIFN-γ expressed in BL21Plus compared to BL21 expression. This example provides a chaperone- assisted folding of a recombinant protein with the chaperone genes inserted into the bacterial genome under the control of a different chaperone than on the plasmid. Accordingly, ao-expressing chaperones encoded on a plasmid allows for the expression of soluble hIFN-γ in E. coli. Example 2. Construction of an E coli strain with an oxidative cytoplasm and enhanced chaperone production. Recombinant protein obtained by the insertion of additional chaperone genes under the control of a promoter into an E. coli expression strain with a modified genome have improved solubility. The BL21 Gor∆ strain (see U.S. Patent Nos. 10,093,704; 10,597,664; and 11,060,123; and U.S. Patent Application Publication No. 2023/0242961, each or which is specifically incorporated by reference) has the Gor gene deleted, resulting in oxidative cytoplasm that allows recombinant proteins to form disulfide bonds in the cytoplasm. A synthetic operon containing GroEL/GroES and Trigger Factor (TF) genes under the control of arabinose promoter and araC arabinose transcription factor was inserted into the Gor locus of the Gor∆ genome using the Red Recombinase method. The resulting strain, Gor∆Plus, along with the parental strain Gor∆, was transformed with an expression vector containing the gene for a single chain antibody fragment (scFv) under the control of an IPTG inducible promoter. The scFv construct was derived from the Cetuximab sequence. Expression of the scFV and the chaperone operon was induced with IPTG and arabinose. After 5-hour expression at 30C, the total protein and the soluble fractions were analyzed by SDS-PAGE (see Figure 1). 8 | P a g e A band corresponding to the scFv was detected only in the soluble fraction from Gor∆Plus cells but not in the soluble fraction from Gor∆ cells. The increased band intensity for Gor∆Plus indicates that there was increased expression level for scFv. This teaches that inducing expression of extra copies of GroEL/GroES and Trigger Factor resulted in both enhanced expression and solubility of scFv in Gor∆Plus. Example 3. Construction of an E. coli strain with an oxidative cytoplasm and enhanced chaperone production. Inducible overexpression of GroEL/GroES and Trigger Factor. The E. coli BL21 Gor∆ strain was genomically engineered to harbour one of two distinct overexpression cassettes within the gorA locus. Briefly, the cassettes were designed in silico and contain either GroEL/GroES gene or Trigger Factor (TF) gene under the control of tac-promoter. Then, each cassette was integrated into the genome of the parental strain using Lambda Red recombination and CRISPR selection. The correct clones were fully cured of genome engineering plasmids. Liquid and solid culture tests were performed to confirm complete plasmid loss. The resulting strains, FinaGorA (GroEL/GroES) and FinaGorB (TF), were subjected to genomic sequencing in order to confirm the proper integration. FinaGorA and FinaGorB were grown in antibiotic-free media, and expression of the chaperones was induced with 0.25mM IPTG. The Gor∆ strain was used as a negative control. After 4 hours of expression at 37°C, the cell cultures were analysed by SDS-PAGE. The bands corresponding to GroEL/GroEs and TF chaperones are indicated by the arrow (Figure 2). There was no band corresponding to either chaperone in the Gor∆ negative control. The density of the GroEL GroEs and TF bands indicate high expression levels of each protein. Thus, the integration of a chaperone or multiple chaperones into the genome under the control of a promoter resulted in high expression levels. Example 4. Construction of an E. coli strain with an oxidative cytoplasm and enhanced chaperone production. Soluble expression of recombinant proteins can depend on the presence or absence of a particular chaperone(s). Soluble expression of Target Protein 1 in FinaGorA and FinaGorB was evaluated. The expression vector containing the coding sequence of the recombinant protein (Target Protein 1), under the control of an IPTG promoter, was transformed into FinaGorA and FinaGorB overexpression bacterial chaperones GroEL/GroES and TF, respectively. Target Protein 1 is not soluble when expressed in either BL21 or Gor∆ E. coli strains. 9 | P a g e Selected colonies were grown in an expression media at 37°C. The temperature was reduced to 22°C and protein expression was induced by 0.25mM IPTG. After 16 hours of expression, the cells were harvested by centrifugation, and the lysis was performed using glass beads. 10µl of cell culture and 10µl of clarified lysate were analyzed by SDS-PAGE for total and soluble proteins, respectively. The FinaGorA and FinaGorB strains expressed similar total amounts of Target 1 protein but expression in FinaGorB resulted in significantly higher levels of soluble Target 1 protein. This demonstrates that integration of a chaperone into the genome, under the control of a promoter, can enhance the solubility of recombinant protein and that enhancement depended on the particular chaperone. 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. The term comprising, where ever used, is intended to include the terms consisting and consisting essentially of. Furthermore, the terms comprising, including, and containing are not intended to be limiting. It is intended that the specification and examples be considered exemplary only with the true scope and spirit of the invention indicated by the following claims. 10 | P a g e

Claims

Claims 1. A recombinant bacterial cell whose genome contains one or more chaperone sequences integrated into a genome of the bacterial cell under the control of a promoter.

2. The recombinant bacterial cell of claim 1, wherein the one or more chaperone sequences encode a cytosolic chaperone system.

3. The recombinant bacterial cell of claim 2, wherein the cytosolic chaperone system comprises a ribosome-associated trigger factor, a DnaK system, a DnaJ chaperone, a GrpE chaperones; a KJE chaperone, a GroEL chaperone system, a GroES chaperone, an ELS chaperone, a ClpB chaperone, an IbpAB chaperone, or a combination thereof.

4. The recombinant bacterial cell of claim 1, further comprising a recombinant protein or polypeptide.

5. The recombinant bacterial cell of claim 4, wherein a genetic sequence of the recombinant protein or polypeptide is integrated into the genome of the bacterial cell.

6. The recombinant bacterial cell of claim 4, wherein the one or more chaperon sequences encode one or more of the chaperones DnaK, DnaJ or GrpE, and the recombinant protein or polypeptide comprises an antibody or antibody fragment.

7. The recombinant bacterial cell of claim 4, wherein the one or more chaperon sequences encode a trigger factor and the recombinant protein or polypeptide comprises a lysozyme, a mouse endostatin, or an ORP150 protein.

8. The recombinant bacterial cell of claim 1, wherein expression of the one or more chaperone sequences is controlled by an inducing agent.

9. The recombinant cell of claim 4, wherein expression of the recombinant protein is controlled by an inducing agent.

10. The recombinant cell of claim 4, wherein expression of the one or more chaperone sequences and expression of the recombinant protein or polypeptide are controlled by a same inducing agent.

11. The recombinant cell of claim 1, which further comprises an oxidative cytoplasm that allows a recombinant protein to form disulfide bonds in the cytoplasm.

12. The recombinant bacterial cell of claim 11, wherein the one or more chaperones comprise chaperones selected from the group consisting of DsbD, DsbE, DsbG, SurA, FkpA, Skp, and protein disulfide isomerase. 11 | P a g e

13. The recombinant cell of claim 1, which further comprises a recombinant peptidase sequence that expresses a peptidase.

14. The recombinant cell of claim 13, wherein the recombinant peptidase sequence is integrated into the genome.

15. The recombinant bacterial cell of claim 1, which is an E. coli expression strain containing a modified genome with a Gor gene mutation or deletion.

16. A recombinant bacterial cell containing: one or more chaperone sequences integrated into a genome of the bacterial cell under the control of a first inducible promoter; a recombinant sequence that encodes a recombinant protein containing one or more disulfide bonds under the control of a second inducible promoter; and an oxidative cytoplasm that allows the recombinant protein to form the one or more disulfide bonds in the cytoplasm.

17. The recombinant bacterial cell of claim 16, wherein the one or more chaperone sequences encode a cytosolic chaperone system.

18. A recombinant bacterial cell of claim 17, wherein the cytosolic chaperone systems comprises a ribosome-associated Trigger Factor, a DnaK system, a DnaJ chaperone, a GrpE chaperones; a KJE chaperone, a GroEL chaperone system, a GroES chaperone, an ELS chaperone, a ClpB chaperone, an IbpAB chaperone, or a combination thereof.

19. The recombinant bacterial cell of claim 16, wherein expression of the first and second inducible promoters are controlled by a same inducing agent. 19. The recombinant cell of claim 16, which further comprises a recombinant peptidase sequence that expresses a peptidase.

20. The recombinant cell of claim 19, wherein the recombinant peptidase sequence is integrated into the genome.

21. A method of expressing a recombinant protein containing one or more disulfide bonds in the cytoplasm of a bacterial cell comprising: providing the bacterial cell containing one or more chaperone sequences that express chaperone proteins integrated into a genome of the bacterial cell and a recombinant protein sequence; 12 | P a g e providing an oxidative cytoplasm to the bacterial cell that allows the recombinant protein to form the one or more disulfide bonds in the cytoplasm; and expressing the recombinant protein in the bacterial cell.

22. The method of claim 21, wherein expression of the one or more chaperone sequences and the recombinant protein are controlled by one or more inducing agents.

23. The method of claim 22, wherein expression of the one or more chaperone sequences and the recombinant protein are controlled by the same inducing agent.

24. The method of claim 21, further comprising isolating the recombinant protein from heterologous proteins of the bacterial cell.

25. The method of claim 21, wherein the chaperone proteins do not trigger undesired proteolytic activity of the bacterial cell.

26. A recombinant protein expressed in the bacterial cell of claim 21, which contains a native formation of the one or more disulfide bonds.

27. A recombinant bacterial cell whose genome contains: one or more chaperone sequences integrated into a genome of the bacterial cell under the control of a first inducible promoter; and a genomic sequence that encodes a recombinant protein or polypeptide under the control of a second inducible promoter; wherein the bacterial cell contains an oxidative cytoplasm that allows a recombinant protein to form disulfide bonds in the cytoplasm; wherein the one or more chaperon sequences encode one or more of the chaperones DnaK, DnaJ or GrpE, and wherein the recombinant protein or polypeptide comprises an antibody or antibody fragment.

28. The recombinant bacterial cell of claim 16, wherein expression of the first and second inducible promoters are controlled by a same inducing agent.

29. The recombinant cell of claim 16, which further comprises a recombinant peptidase sequence that expresses a peptidase.

30. The recombinant cell of claim 19, wherein the recombinant peptidase sequence is integrated into the genome. 13 | P a g e