Protein Purification Methods

Abstract

The present invention provides methods for purifying proteins that are expressed in the form of solubilizable complexes that, upon solubilization, yield properly folded, active proteins. A further aspect of the invention relates to polypeptides that can be used in the expression and purification of proteins. [Selected Figure] Figure 1

Description

[Technical field]

[0001] REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 62 / 735,861, filed Sep. 25, 2018, the entire disclosure of which is incorporated herein by reference.

[0002] Reference to sequence listing This application includes a Sequence Listing that was submitted electronically in a file entitled "AbSci-005PCT_ST25.txt," created on September 20, 2019, and having a size of 63 kilobytes (KB), which is incorporated herein by reference.

[0003] FIELD OF THEINVENTION The present invention is in the integrated technical field of molecular biology and biotechnological manufacturing, more particularly in the technical field of recombinant protein production. [Background technology]

[0004] Efficient expression of recombinant proteins or other gene products requires the use of a system whose various aspects, i.e., expression construct(s), host cell line, growth conditions, and purification methods, all cooperate to produce the desired product in sufficient quantities while minimizing expenditure of materials and time.

[0005] Many expression systems currently used for industrial production of recombinant products rely on expensive mammalian cell culture or utilize secretion of proteins into the periplasm of bacterial cells, which has limited amounts of product per cell and is more time-consuming than expression of gene products in the bacterial cytoplasm. In many expression systems developed by others in which the bacterial cytoplasm is used as the preferred cellular compartment for recombinant expression, the desired protein is typically produced as insoluble inclusion bodies (see, e.g., Chung et al., "Recombinant production of biologically active giant grouper (Epinephelus lanceolatus) growth hormone from inclusion bodies of Escherichia coli by fed-batch culture", Protein Expr Purif 2015 Jun;110:79-88;doi:10.1016 / j.pep.2015.02.012;Epub 2015 Feb 19). To recover some soluble and correctly folded proteins from inclusion bodies, further refolding steps need to be performed (Yamaguchi and Miyazaki, "Refolding techniques for recovering biologically active recombinant proteins from inclusion bodies", Biomolecules 2014 Feb 20;4(1):235-251; doi:10.3390 / biom4010235; Review). For proteins that contain disulfide bonds, these refolding steps usually involve the use of reducing agents to convert any improperly formed disulfide bonds, especially intermolecular disulfides that may contribute to aggregation into insoluble inclusion bodies, into thiol groups. Summary of the Invention [Problem to be solved by the invention]

[0006] There is a clear need for improved expression systems and methods of using them to more efficiently produce gene products, such as recombinant proteins, in a properly folded, soluble form and in a manner that can be scaled up to commercial production levels. [Means for solving the problem]

[0007] The present invention provides a method for purifying proteins and other gene products expressed in the form of solubilizable complexes that, when solubilized, result in properly folded, active gene products without the need for the use of reducing agents. An advantage of the present invention is that the gene product solubilizable complexes can be collected in the form of a solubilizable pellet, allowing undesirable components of the host cell lysate to be discarded in the supernatant. If the gene product solubilizable complexes are present, for example, in a host cell lysate, the mixture can be considered a suspension in that the solubilizable complexes can be sedimented into a pellet by centrifugation or other means and separated from the predominantly liquid fraction of the host cell lysate. As used herein, the term "solution" encompasses mixtures that can exhibit the properties of a suspension. A further aspect of the present invention relates to polypeptide prosequences that can be used in the expression and purification of proteins.

[0008] An aspect of the invention is a method of producing one or more gene products, comprising the steps of providing a first solution comprising at least one gene product expressed in a host cell, wherein at least some of the at least one gene product in the first solution can be sedimented by centrifugation (at pH 7.4 and 4° C., salt conditions of 200 mM NaCl) at a force of 900×g, or between 900×g and 7,000×g, or at 7,000×g to form a solubilizable pellet, and placing at least some of the at least one gene product in a solubilization solution. The above methods of the invention may be utilized in any combination thereof according to any of the aspects of the methods of the invention as presented in the following paragraphs: The method of the present invention, wherein the at least one gene product is a polypeptide that forms at least one disulfide bond.

[0009] The method of the present invention, wherein said at least one gene product is a polypeptide lacking a single peptide. The method of the present invention, wherein the at least one gene product comprises a polypeptide selected from the group consisting of: (a) a polypeptide comprising the amino acid sequence of the mature chain of leptin, metreleptin, growth hormone, human growth hormone, or insulin, and (b) a fragment of any of the polypeptides of (a).

[0010] The method of the present invention, wherein the at least one gene product comprises a polypeptide comprising an amino acid sequence selected from the group consisting of the amino acid sequence of a mature insulin chain and amino acid sequences sharing at least 70% (or at least 80%, or at least 90%) amino acid sequence identity to at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%) of the length of any of the amino acid sequences in (a).

[0011] The method of the present invention, wherein the at least one gene product comprises a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) any of SEQ ID NOs: 27 to 36; and (b) amino acid sequences sharing at least 70% (or at least 80%, or at least 90%) amino acid sequence identity to at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%) of the length of any of the amino acid sequences in (a).

[0012] The method of the invention, wherein at least one gene product comprises a polypeptide comprising an Asp-Pro amino acid sequence, further comprising cleavage of said propeptide at the Asp-Pro amino acid sequence.

[0013] The method of the present invention, wherein the first solution is a lysate of the host cells, wherein the lysate of the host cells has been produced by contacting the host cells with lysozyme, or wherein the lysate of the host cells has been produced by mechanical lysis.

[0014] The method of the invention, wherein the host cell is a prokaryotic cell, wherein the host cell is an Escherichia coli cell. The method of the present invention, wherein the host cell has been modified to have a more oxidized cytoplasm, said modification of said host cell resulting in a loss of expression of at least one gene selected from the group consisting of trxB, gor, gshA, and gshB, and said host cell further comprises a mutation in the ahpC gene.

[0015] 1. The method of the invention, wherein a host cell comprises one or more expression constructs, wherein at least one of said expression constructs comprises at least one inducible promoter, wherein said at least one inducible promoter is selected from the group consisting of an arabinose-inducible promoter, a propionate-inducible promoter, a rhamnose-inducible promoter, a xylose-inducible promoter, a lactose-inducible promoter, and a promoter inducible by phosphate depletion, and / or wherein said host cell has a reduced level of gene function of at least one gene encoding a protein that metabolizes at least one inducer of said at least one inducible promoter, wherein the at least one gene is selected from the group consisting of araA, araB, araD, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB.

[0016] The method of the present invention further comprises the step of subjecting the first solution to centrifugation, the centrifugation being at a force of 900 x g, or 900 x g to 25,000 x g, or 900 x g to 7,000 x g, or 2,000 x g to 20,000 x g, or 3,300 x g, or 3,300 x g to 20,000 x g, or 7,000 x g, or 7,000 x g to 20,000 x g, and the first solution is separated into a soluble fraction and a pellet, the pellet comprising at least some of the at least one gene product, and the method of the present invention further comprises the step of recovering at least some of the at least one gene product from the pellet, wherein at least some of the at least one gene product present in the pellet is disposed in a solubilization solution.

[0017] The method of the present invention, wherein the solubilization solution contains at least one chaotropic agent, and the at least one chaotropic agent is selected from the group consisting of n-butanol, ethanol, guanidinium chloride, guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea, and the at least one chaotropic agent is selected from the group consisting of urea at a concentration of 2M to 10M and guanidine hydrochloride at a concentration of 2M to 8M, or is urea at a concentration of 7M to 8M.

[0018] The method of the present invention further comprises a step of reducing the concentration of the at least one chaotropic agent in the solubilization solution, wherein the concentration of the at least one chaotropic agent in the solubilization solution is reduced to 50% or less of its initial concentration in the solubilization solution, and / or the initial concentration of the at least one chaotropic agent in the solubilization solution is urea at a concentration of 7M to 8M and the concentration of the at least one chaotropic agent is reduced to urea at a concentration of 3M to 4M and the at least one chaotropic agent in the solubilization solution is reduced to 50% or less of its initial concentration in the solubilization solution. The method of the present invention, wherein the reduction of the concentration of the at least one chaotropic agent is achieved by a method selected from the group consisting of dialysis, dilution, and diafiltration, and further comprises the step of incubating the solubilization solution containing the reduced concentration of said at least one chaotropic agent for a period of time selected from the group consisting of at least 1 hour, 2 hours, 5 hours, 10 hours, 12 hours, 15 hours, 12 hours to 24 hours, 24 hours, 24 hours to 72 hours, 36 hours, 48 ​​hours, 72 hours, 72 hours to 120 hours, and 120 hours.

[0019] The method of the invention further comprising the step of recovering at least some of the at least one gene product from the solubilization solution, wherein the amount of the at least one gene product recovered from the solubilization solution is at least 50%, or at least 60%, or at least 70%, or at least 80% of the total amount of the at least one gene product present in the first solution, and / or at least some of the at least one gene product recovered from the solubilization solution has a characteristic selected from the group consisting of properly formed disulfide bonds and gene product activity, and at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 85%, or at least 90% of the at least one gene product recovered from the solubilization solution has properly formed disulfide bonds.

[0020] The method of the present invention further comprising chromatographic purification of said at least one gene product, wherein the chromatographic purification is immobilized metal affinity chromatography (IMAC), and wherein the chromatographic purification utilizes a Ni-NTA column.

[0021] The method of the invention, wherein the at least one gene product is not contacted with a reducing agent. A further aspect of the present invention is a polypeptide comprising an amino acid sequence selected from the group consisting of: (a) any of SEQ ID NOs: 12 to 14 and 27 to 36, and (b) amino acid sequences sharing at least 70% (or at least 80%, or at least 90%) amino acid sequence identity over at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%) of the length of any of the amino acid sequences in (a). [Brief description of the drawings]

[0022] [Figure 1] FIG. 1 is a flow chart outlining a method for purifying the solubilizable gene product complex produced by the methods of the present invention. [Diagram 2] Figure 2 shows that CPBpro_lispro proinsulin expressed and solubilized according to the method of the invention contains disulfide bonds. CPBpro_lispro proinsulin solubilized with 8 M urea was analyzed by polyacrylamide gel electrophoresis on a 12% Bis-Tris gel. Host cells were lysed at a concentration 5-fold ("5X") or 10-fold ("10X") higher than the concentration of the host cell culture medium.

[0023] [Table 1]

[0024] Treatment of solubilized CPBpro_lisproinsulin with the reducing agent DTT caused the solubilized CPBpro_lisproinsulin to migrate at a slightly slower rate on the gel, indicating that DTT treatment reduced the disulfide bonds present in the non-reduced solubilized CPBpro_lisproinsulin. Figure 2 also shows that pelleting the solubilizable CPBpro_lisproinsulin complex removes most of the potentially contaminating proteins present in the host cell lysate (lanes 1 and 2) from the solubilizable pellet, resulting in a highly purified preparation of solubilized CPBpro_lisproinsulin (lanes 3 to 6). [Diagram 3] Figure 3 is a schematic diagram of the CPBpro_glargine proinsulin polypeptide. The amino acids of the A and B chains are shown as light and dark grey circles, respectively. The N-terminal CPBpro propeptide is shown as a dashed line, and the C-peptide (or "connecting peptide") that connects the A and B chains is shown as a grey arch. The dark grey solid lines between the cysteine ​​residues in the A and B chains, and the dark grey solid line connecting the two cysteines in the A chain, represent the disulfide bonds present in correctly folded insulin glargine. [Figure 4] 4 is a schematic diagram depicting digestion of purified CPBpro_glargine proinsulin with trypsin and glutamyl endopepsidase ("Glu-C") to generate cross-linked peptide fragments for mass spectrometry characterization. Disulfide bonds are represented by solid dark grey lines connecting cysteine ​​residues. [Diagram 5]Figure 5 is a set of three mass spectrometry chromatograms showing that 93% of CPBpro_glargine proinsulin purified only by solubilization from pelleted solubilizable complex has the correct formation of disulfide bonds and is therefore properly folded. Panel A: base peak chromatogram (non-reduced); Panel B: extracted ion chromatogram (non-reduced, + / - 5 ppm) showing peaks corresponding to peptide fragments with two disulfide bonds; Panel C: extracted ion chromatogram (non-reduced, + / - 5 ppm) showing peaks corresponding to peptide fragments with one disulfide bond. Arrows indicate peaks corresponding to the indicated peptide fragments as determined by comparison to chromatograms generated by insulin glargine standards. The arrow labeled "swapped form" indicates a minor peak corresponding to a conformation in which the cysteines at positions A6 and A7 of mature insulin (see Figure 4) have "swapped" disulfide bond partners. *: Asterisks indicate peaks that cannot be attributed to CPBpro_glargine proinsulin in the correct charge state. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] The problem of producing gene products such as recombinant proteins on a commercial scale and in an active form is addressed by providing the protein expression and purification methods described herein. The inventors have found that when gene products such as polypeptides are expressed in a host cell to a sufficient gene product density and in a manner that allows the gene product to be properly folded when expressed and any disulfide bonds to be properly formed, the gene products such as polypeptides form solubilizable complexes that are easily purified from other cellular components and subsequently solubilized to produce a properly folded, putatively active gene product. These methods of the invention for producing gene products in the form of such solubilizable complexes and subsequent purification of the properly folded gene product have the advantage that they do not require a procedure that involves contacting the gene product with a reducing agent.

[0026] As another aspect of the present invention, a method is provided which relates to the direct solubilization of polypeptides produced by host cells in the form of solubilizable complexes, without an initial centrifugation step to separate insoluble and soluble fractions after cell lysis, and allows the purification of correctly folded and / or active polypeptides which form disulfide bonds, without the need to contact such polypeptides with a reducing agent.

[0027] Proper folding of a gene product, such as a gene product comprising one or more polypeptides, is consistent with any disulfide bonds in the gene product being formed at the correct positions within the gene product. Thus, determining whether a gene product is properly folded can involve characterizing any disulfide bonds present in the gene product to assess whether they are properly formed, as further described in Example 2C and Example 8. A properly formed disulfide bond, when assayed, is one that is a covalent bond linking two sulfur atoms and, if present within a polypeptide or between two polypeptides, is one that is a covalent bond linking (or joining) the sulfur atoms of two sulfur-containing amino acid residues (cysteine ​​or Cys residues) that are linked by a disulfide bond in the desired form of the gene product comprising the polypeptide(s). For example, for glargine proinsulin as shown in Figure 3, the three properly formed disulfide bonds are those linking the Cys residues at positions 6 and 11 of SEQ ID NO:6, the Cys residues at positions 7 of SEQ ID NO:6 and 7 of SEQ ID NO:7, and the Cys residues at positions 20 of SEQ ID NO:6 and 19 of SEQ ID NO:7.

[0028] An active gene product includes any gene product that has a measurable form of a type that is associated with a desired form of the gene product. For example, an active insulin gene product can have measurable insulin receptor binding activity, or measurable anti-insulin antibody binding activity, or any other type of activity that is associated with a desired form of the insulin gene product.

[0029] Recovery of properly folded and / or active forms of proteins from inclusion bodies usually involves treatment with at least one reducing agent. The term "reducing agent" as used herein encompasses chemicals (non-protein) with a reduction potential more negative than -0.26 V at pH 7.0 and 25°C, such as DTE (dithioerythritol), DTT (dithiothreitol), and TCEP (tris(2-carboxyethyl)-phosphine), and thus the term "reducing agent" does not include L-cysteine ​​("L-cys") or glutathione. Unlike recovery of gene products from inclusion bodies, the solubilization methods disclosed herein that do not involve the use of reducing agents result in substantially higher recovery of gene products, e.g., recovery of at least 50%, 60%, 70%, or 80% of the total gene product material present in the host cell lysate, as calculated by the method described in Example 7. Given the high yields of gene product achieved in cell lysates (5 g / L-20 g / L), the solubilization methods described herein (as seen in Examples 1-3) can result in gene product yields (4 g / L-16 g / L).

[0030] For the host cell to produce the gene product(s) in the form of a solubilizable complex, it is most suitable to utilize an appropriate combination of the following aspects I to IV of gene product expression, as detailed herein: I. The gene product(s) to be produced, including any transporters, cofactors, chaperones, and / or tags or propeptides to be used in the expression of the desired gene product(s).

[0031] II. The expression construct(s) to be used to express the gene product(s). III. The host cell to be used to express the expression construct(s) encoding the gene product(s).

[0032] IV. Conditions for host cell growth and induction of expression. Section V. describes solubilization and purification methods of the present invention. The following patent publications and patent application(s), all of which are expressly incorporated herein by reference, provide further examples of gene products, expression constructs, host cells, and growth and induction conditions that may be used to produce solubilizable complexes suitable for the purification methods of the present invention: US9617335B2, "Inducible Coexpression System," WO2016205570A1, "Vectors for Use in an Inducible Coexpression System," and International Application PCT / US2016 / 067064, "Cytoplasmic Expression System."

[0033] I. Products produced by the methods of the invention In many expression applications, and in product properties, great versatility is seen in utilizing the gene expression and gene product purification methods of the present invention.

[0034] Gene products produced by the methods of the invention may include any one or more of the following: 1-antitrypsin; 2C4; activin; addressin; alkaline phosphatase; anti-CD11a; anti-CD18; anti-CD20; anticoagulants such as protein C; anti-HER-2 antibodies; anti-IgE; anti-IgG; anti-VEGF; antibodies and antibody fragments; 2C4 (WO 01 / 00245, Hybridoma ATCC 611449) which binds to a region in the extracellular domain of ErbB2 (including any one or more residues in the region from about residue 22 to about residue 584 of ErbB2, including extremities). Antibodies against ErbB2 domain(s), such as HB-12697; Apo2 ligand (Apo2L); atrial natriuretic factor; BDNF; beta-lactamase; bombesin; bone morphogenetic protein (BMP); botulinum toxin; brain IGF-I; calcitonin; cardiotrophins (cardiac hypertrophy factors), such as cardiotrophin-1 (CT-1); CD proteins, such as CD-3, CD-4, CD-8, and CD-19; clotting factors, such as factor VIIIC, factor IX, tissue factor, and von Willebrand factor; colony-stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; cytokines; decay-accelerating factors; des(1-3)-IGF-I (brain IGF-I); DNase; enkephalinase; epidermal growth factor (EGF); erythropoietin; fibroblast growth factor (GF) such as aFGF and bFGF. Follicular growth factors; follicle-stimulating hormone; glucagon; gp120; ghrelin; growth hormones, including human growth hormone or bovine growth hormone; growth hormone releasing factor; hematopoietic growth factors; homing receptors; HSA; IGF-I; IGF-II; immunotoxins; inhibin; insulin chains (insulin A chain, insulin B chain) or proinsulin; insulin-like growth factor binding proteins; insulin-like growth factors I and II (IGF-I and IGF-II); integrins; interferons, such as interferon-alpha, interferon-beta, and interferon-gamma; interleukins (IL), e.g., IL-1 to IL-10; leptin; lipoproteins; pulmonary surfactant; progesterone, metreleptin; mouse gonadotropin-related peptide; Müllerian inhibitory substance; nerve growth factor (NGF);Neurotrophic factors such as brain-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5, or neurotrophin-6 (NT-3, NT-4, NT-5, or NT-6); bone morphogenetic factors; parathyroid hormone; plasminogen activators such as urokinase or human urinary or tissue-type plasminogen activator (t-PA); platelet-derived growth factor (PDGF); prorelaxin; protein A or D; receptors for hormones or growth factors; regulatory proteins; relaxin A chain; relaxin B chain; rennin; rheumatoid factors; serum albumins such as human serum albumin (HSA) or bovine serum albumin (BSA); superoxide dismutase; surface membrane proteins; T cell receptors; TGF-beta; thrombin; thrombopoietin; thyroid stimulating hormone; TGF-alpha and TGF-beta, including TGF-1, TGF-2, TGF-3, TGF-4, or TGF-5. transforming growth factors (TGFs) such as α, β, β-glucosamine ...

[0035] The gene product produced by the invention may comprise any or more than one of the insulin polypeptides described below. Insulin polypeptides produced by the methods of the invention, in some embodiments, comprise the amino acid sequence of the mature A chain or mature B chain of insulin, and in other embodiments, comprise both the mature A chain and the mature B chain. Proinsulin polypeptides comprise the mature A chain of insulin and the mature B chain of insulin. Insulin polypeptide chains, in certain embodiments, comprise one or more of any of the naturally occurring amino acid sequences of insulin, or fragments thereof, in other embodiments, comprise one or more insulin analog amino acid sequences, or fragments thereof, and in further embodiments, comprise a combination of naturally occurring insulin amino acid sequences and / or insulin analog amino acid sequences. Examples of naturally occurring insulin amino acid sequences and insulin analog amino acid sequences are shown in Table 1.

[0036] [Table 2]

[0037] A preproinsulin polypeptide may comprise the following components, preferably in the following N-terminal to C-terminal order: a prepeptide, which may be a signal peptide that is cleaved during protein expression by a host cell signal peptidase; a propeptide; a B-chain; a C-peptide (or "linking peptide"); and an A-chain. A preproinsulin polypeptide may also comprise an A-chain and a B-chain, in a different N-terminal to C-terminal order, for example: a prepeptide; a propeptide; an A-chain; a C-peptide; and a B-chain. For proinsulin polypeptides that are to be expressed in the cytoplasm of a host cell, the prepeptide, including the signal sequence, is not present. A diagram of a proinsulin glargine polypeptide is shown in FIG. 3. Examples of C-peptides include the C-peptide of human insulin (amino acids 55 to 89 of the NCBI reference sequence NP_001278826.1, SEQ ID NO:10) and the artificial C-peptide RRYPGDVKR (SEQ ID NO:11) (Chang et al., "Human insulin production from a novel mini-proinsulin which has high receptor-binding activity", Biochem J 1998 Feb 1;329(Pt 3):631-635). Further C-peptide amino acid sequences that may be used in the proinsulin polypeptide are artificial variants of the human C-peptide (SEQ ID NO:12 and SEQ ID NO:13) and the artificial C-peptide RRDDNLER (SEQ ID NO:14). C-peptide amino acid sequences are generally presented herein as including the terminal arginine and lysine acid groups that are normally cleaved when the proinsulin polypeptide is converted to mature insulin by the process of trypsin digestion. The exception is the C-peptide of proinsulin glargine as shown in FIG. 3: because the mature B-chain of insulin glargine has two arginine (R) residues, these arginine residues are represented in FIG. 3 as part of the mature B-chain of insulin glargine, not as part of the C-peptide.

[0038] Gene products produced by the methods of the invention may include leptin and / or metreleptin polypeptides. An example of a leptin polypeptide, also referred to as metreleptin, is set forth in SEQ ID NO: 15 and corresponds to mature human leptin having a methionine residue at its N-terminus. Other examples of leptin polypeptides include amino acid sequences that lack an N-terminal methionine residue, such as amino acids 2 through 147 of SEQ ID NO: 15. A common isoform of leptin has a methionine residue at position 74 of SEQ ID NO: 15, instead of a valine residue. A leptin polypeptide produced by the methods of the invention includes the amino acid sequence of a leptin polypeptide that, in some embodiments, has a methionine residue at its N-terminus (metreleptin), or in other embodiments, has a tag, linker, or other propeptide amino acid sequence (as described further below) added to the N-terminus of the leptin polypeptide, in some embodiments, including a methionine at the N-terminus of the metreleptin amino acid sequence, and in other embodiments, does not include a methionine at the N-terminus of the metreleptin amino acid sequence.

[0039] Signal peptides. Polypeptide gene products produced by the methods of the invention may have or lack a signal peptide. In certain embodiments of the invention, the polypeptide gene products lack a signal peptide, since such gene products are preferably retained in the oxidizing cytoplasm of the host cell. Signal peptides (also called signal sequences, leader sequences, or leader peptides) are structurally characterized by a stretch of hydrophobic amino acids, approximately 5 to 20 amino acids long, often approximately 10 to 15 amino acids long, that tend to form a single alpha-helix. This hydrophobic stretch is often immediately preceded by a shorter stretch rich in positively charged amino acids, particularly lysines. Signal peptides that are to be cleaved from the mature polypeptide usually end with a stretch of amino acids that is recognized and cleaved by a signal peptidase. The signal peptide, sometimes called a signal anchor sequence, that directs the insertion of the polypeptide gene product into the membrane may lack an amino acid sequence that is cleaved by a signal peptidase, and is then retained in the polypeptide gene product. A signal peptide may often be functionally characterized by its ability to direct the co-translational or post-translational transport of a polypeptide out of the cytoplasm and, for example, through the plasma membrane of prokaryotes (or the inner membrane of gram-negative bacteria such as E. coli), or directly into the endoplasmic reticulum of eukaryotic cells. The extent to which a signal peptide enables a polypeptide to be transported into the periplasmic space of a host cell, such as E. coli, can be determined by separating periplasmic proteins from proteins retained in the cytoplasm, using methods such as those provided in Example 9 below. Tags and other polypeptide sequences that can be used with gene products Tags. Gene products to be expressed by the methods of the invention can be designed to contain molecular moieties that aid in the purification and / or detection of the gene product. Many such moieties are known in the art, and as an example, a polypeptide gene product can be designed to contain a polyhistidine "tag" sequence at its N- or C-terminus of six or more consecutive histidines, preferably six to ten histidine residues, most preferably six histidines ("6xHis"). The presence of the polyhistidine sequence on the end of the polypeptide allows it to be bound and separated from other polypeptides by cobalt or nickel-based affinity media. The polyhistidine tag sequence can be removed by exopeptidases.

[0040] Additional tags expressed at the N-terminus of the amino acid sequence of the polypeptide gene product produced by the methods of the invention can, in certain embodiments, be (1) a self-cleaving N-terminal portion (N-terminal portion) of the polyprotein from a pestivirus, such as classical swine fever virus (Alfort strain) (SEQ ID NO: 16), also known as classical swine fever virus (CSFV), Border disease virus (BDV) and Bovine viral diarrhea virus (BVDV). pro ), and fragments thereof, and / or (2) small ubiquitin-like modifier (SUMO) (SEQ ID NO: 17, SwissProt P55853.1). Any N-terminal tag may itself be further tagged at its N-terminus with a polyhistidine tag, such as 6xHis, followed by initial purification of the tagged polypeptide on a nickel column, followed by purification of each of the N-terminal tags. proor enzymatic cleavage of the SUMO N-terminal tag by a SUMO protease and elution of the released polypeptide from the column. In one embodiment of this method, the SUMO protease polypeptide is also a fusion protein containing a 6xHis tag, allowing for a two-step purification where in a first step the expressed 6xHis-SUMO tagged polypeptide is purified by binding to a nickel column followed by elution from the column. In a second step, the SUMO tag on the purified polypeptide is cleaved by a 6xHis tagged SUMO protease and the SUMO protease-polypeptide reaction mixture is loaded onto a second nickel column, which retains the SUMO protease but allows for the loading of untagged polypeptides at this stage.

[0041] As another example, a fluorescent protein sequence can be expressed as part of a polypeptide gene product, with the amino acid sequence for the fluorescent protein being preferably added at the N- or C-terminus of the amino acid sequence of the polypeptide gene product. The resulting fusion protein will fluoresce when exposed to certain wavelengths of light, allowing the presence of the fusion protein to be visually detected. A well-known fluorescent protein is the green fluorescent protein of Aequorea victoria, and many other fluorescent proteins are commercially available along with the nucleotide sequences that encode them.

[0042] Linkers. A linker is a polypeptide used to connect two other polypeptides. Examples of linker polypeptides that form alpha-helices are provided as SEQ ID NO: 18 and SEQ ID NO: 19 (Amet et al., "Insertion of the designed helical linker led to increased expression of Tf-based fusion proteins", Pharm Res 2009 Mar;26(3):523-528;doi:10.1007 / s11095-008-9767-0;Epub 2008 Nov 11).

[0043] Cleavage sequences. A cleavage sequence is a distinct amino acid sequence that can be acted upon by a chemical reagent or enzyme to achieve cleavage of a polypeptide containing the cleavage sequence. One or more of these sequences can be introduced between a tag or propeptide sequence and the amino acid sequence of a polypeptide gene product to allow the tag or propeptide to be cleaved during the purification process of the gene product. Examples of cleavage sequences include the amino acid sequences DP and GGDPGGG (SEQ ID NO:20, which is the bond between D (Asp) and P (Pro) and can be cleaved by treatment with formic acid). Certain acid-cleavable sequences are present within certain propeptides described below (SEQ ID NO:33-SEQ ID NO:35). Further examples are amino acid sequences cleavable by proteases such as TEV (tobacco etch virus) protease (cleavage sequence ENLYFQGG (SEQ ID NO:21)), enterokinase (cleavage sequence DDDDKG (SEQ ID NO:22)), and thrombin (cleavage sequence LVPRGS (SEQ ID NO:23)).

[0044] Propeptides. The propeptides described herein are attached to the polypeptide gene product either N-terminally or C-terminally, or both, to the amino acid sequence of the polypeptide gene product, and may be attached directly to the amino acid sequence of the polypeptide gene product or using other polypeptide sequences, such as linkers or tags, disposed between the propeptide and the polypeptide gene product. Examples of polypeptides that can be used as propeptides include mammalian carboxypeptidase B precursor protein (described further below) and maltose binding protein or "MBP" with a signal sequence (UniProtKB / Swiss-Prot:P0AEX9.1, SEQ ID NO:24), in which amino acids 2 through 26 of SEQ ID NO:24 can be removed to generate a propeptide that remains localized in the cytoplasm. Another polypeptide that has been used as a propeptide is the family 9 carbohydrate-binding module or "CBM9" from Thermotoga maritima xylanase 10a (SEQ ID NO:25, amino acids 700 to 868 of UniProtKB / Swiss-Prot:Q60037; Notenboom et al., "Crystal structures of the family 9 carbohydrate-binding module from Thermotoga maritima xylanase 10A in native and ligand-bound forms", Biochemistry 2001 May 29;40(21):6248-6256).

[0045] Carboxypeptidase B Propeptide (CPBpro). A typical mammalian carboxypeptidase B precursor protein has a signal peptide at its N-terminus followed by a 95 amino acid propeptide with an arginine residue at its C-terminus; this propeptide, also called the carboxypeptidase B activation domain, is cleaved from the rest of the carboxypeptidase B enzyme (EC 3.4.17.2) by trypsin hydrolysis to activate the enzyme (Coll et al., "Three-dimensional structure of porcine procarboxypeptidase B: a structural basis of its inactivity", EMBO J 1991 Jan;10(1):1-9). The amino acid sequence of human carboxypeptidase B precursor protein or CPBpro is provided as SEQ ID NO:26.

[0046] The terms "CPBpro" and "CPBpro propeptide" are used herein to refer to carboxypeptidase B propeptides, including novel variants disclosed herein. CPBpro propeptides can be used to produce recombinant polypeptides, e.g., fused at the C-terminal arginine residue of the CPBpro propeptide to a desired N-terminal residue of a polypeptide of interest, and after expression of the CPBpro_polypeptide, the CPBpro propeptide can be cleaved from the polypeptide of interest by trypsin to generate the desired N-terminus. Examples of variant CPBpro propeptides include SEQ ID NO: 27 to SEQ ID NO: 36, and further propeptides having the amino acid sequence of SEQ ID NO: 37 are provided.

[0047] Formation of solubilizable gene products. In certain embodiments, expression of gene products using the expression methods described herein results in the formation of solubilizable gene product complexes without the need for the addition of tags or other polypeptides to the gene products. For example, in small volume experiments, co-expression of metreleptin (SEQ ID NO: 15) with Erv1p sulfhydryl oxidase (SEQ ID NO: 38, described below) according to the expression methods described herein results in the majority (about 70%) of expressed metreleptin forming solubilizable complexes, and similar co-expression of Erv1p with the gene product (SEQ ID NO: 39) formed by the addition of CPBpro variant propeptide (SEQ ID NO: 27) to metreleptin (SEQ ID NO: 15) results in a greater proportion (about 84%) of the gene product forming solubilizable complexes. With regard to optimizing expression of gene product(s) in solubilizable complexes, the gene product(s) can first be expressed unmodified and the amount of solubilizable complex produced can be determined. The effect of the addition of various polypeptide sequences (tags, propeptides, optionally in combination, and optionally in further combination with linkers and / or cleavage sequences) to the polypeptide gene product(s) can then be assessed, preferably in small volume expression experiments, to determine whether a greater proportion of the desired gene product(s) is subsequently expressed as a solubilizable complex.

[0048] Disulfide bonds. The gene products produced by the methods of the invention are, in some cases, polypeptides that form disulfide bonds. The number and location of disulfides formed by a polypeptide can be determined by methods such as those in Example 8 below. The number of disulfides for a gene product such as a polypeptide is the total number of intra- and intermolecular bonds formed by the gene product when it is present in a functional product. For example, the light chain of a human IgG antibody typically has three disulfide bonds (two intramolecular bonds and one intermolecular bond) and the heavy chain of a human IgG antibody has seven disulfide bonds (four intramolecular bonds and three intermolecular bonds). In certain embodiments of the invention, the gene product produced by the methods of the invention is a polypeptide that forms at least 1 and fewer than 12 disulfide bonds, or at least 2 and fewer than 17 disulfide bonds, or at least 17 and fewer than 50 disulfide bonds, or at least 3 and fewer than 10 disulfide bonds, or at least 3 and fewer than 8 disulfide bonds, or a polypeptide that forms disulfide bonds selected from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, and 9 disulfide bonds.

[0049] Glycosylation. The gene products produced by the present invention may be glycosylated or non-glycosylated. In one embodiment of the present invention, the gene product is a polypeptide. A glycosylated polypeptide is a polypeptide that contains covalently attached glycosyl groups, including all glycosyl groups normally attached to a particular residue of the polypeptide (fully glycosylated polypeptides), partially glycosylated polypeptides, polypeptides with glycosylation at one or more residues where glycosylation does not normally occur (altered glycosylation), and polypeptides glycosylated with at least one glycosyl group that differs in structure from the glycosyl group normally attached to one or more designated residues (modified glycosylation). An example of modified glycosylation is the production of a "defucosylated" or "fucose-deficient" polypeptide, which is a polypeptide lacking a fucosyl moiety at the glycosyl group attached to the fucosyl moiety, by expression of the polypeptide in a host cell lacking the ability to fucosylate the polypeptide. A non-glycosylated polypeptide is a polypeptide that does not contain a covalently bound glycosyl group. A non-glycosylated polypeptide can be the result of deglycosylation of a polypeptide or the production of an aglycosylated polypeptide. A deglycosylated polypeptide can be obtained by enzymatically deglycosylating a glycosylated polypeptide, whereas an aglycosylated polypeptide can be produced by expressing the polypeptide in a host cell that does not have the ability to glycosylate the polypeptide, such as a prokaryotic cell or a cell in which the function of at least one glycosylation enzyme has been eliminated or reduced. In a particular embodiment, the expressed polypeptide is aglycosylated, and in a more particular embodiment, the aglycosylated polypeptide is expressed in a prokaryotic cell, such as E. coli.

[0050] Other modifications of gene products. The gene products produced by the methods of the invention may be covalently linked to other types of molecules. Without limiting the scope of the invention, examples of molecules that may be covalently linked to gene products include polypeptides (e.g., receptors, ligands, cytokines, growth factors, polypeptide hormones, DNA binding domains, protein interaction domains such as PDZ domains, kinase domains, antibodies, and fragments of any such polypeptides); water-soluble polymers (e.g., polyethylene glycol (PEG), carboxymethylcellulose, dextran, polyvinyl alcohol, polyoxyethylated polyols (e.g., glycerol), polyethylene glycol propionaldehyde and similar compounds, derivatives, or mixtures thereof); and cytotoxic agents (e.g., chemotherapeutic agents, growth inhibitory agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), and radioisotopes).

[0051] Chaperones. In some embodiments, the desired gene product is co-expressed with other gene products, such as chaperones, that are beneficial to the production of the desired gene product. Chaperones are proteins that aid in the non-covalent folding or unfolding and / or assembly or disassembly of other gene products, but are not found in the resulting monomeric or multimeric gene product structures when the structures are performing their normal biological functions (intended folding and / or assembly processes). Chaperones can be expressed from inducible or constitutive promoters within the expression construct, or expressed from the host cell chromosome, and preferably, expression of the chaperone protein(s) in the host cell is at a high enough level to produce the co-expressed gene products that are properly folded and / or assembled into the desired product. Examples of chaperones present in E. coli host cells are the folding factors DnaK / DnaJ / GrpE, DsbC / DsbG, GroEL / GroES, IbpA / IbpB, Skp, Tig (inducer), and FkpA, which are used to prevent protein aggregation of cytoplasmic or periplasmic proteins. It has been shown that DnaK / DnaJ / GrpE, GroEL / GroES, and ClpB can function synergistically to facilitate protein folding, and thus expression of these chaperones, in combination, is beneficial for protein expression (Makino et al., "Strain engineering for improved expression of recombinant proteins in bacteria", Microb Cell Fact 2011 May 14;10:32). When expressing a eukaryotic protein in a prokaryotic host cell, a eukaryotic chaperone protein, such as protein disulfide isomerase (PDI) from the same or a related eukaryotic species, is in certain embodiments of the invention co-expressed or inducibly co-expressed with the desired gene product.

[0052] One chaperone that can be expressed in a host cell is the protein disulfide isomerase from the soil hyphomycete (soft rot fungus) Humicola insolens. The amino acid sequence of Humicola insolens PDI is shown as SEQ ID NO: 40, and it lacks the signal peptide of the native protein so that it remains in the host cytoplasm. The nucleotide sequence encoding PDI has been optimized for expression in E. coli, and the expression construct for PDI is shown as SEQ ID NO: 41. SEQ ID NO: 41 contains a GCTAGC NheI restriction site at its 5' end, an AGGAGG ribosomal binding site at nucleotides 7-12, the PDI coding sequence at nucleotides 21-1478, and a GTCGAC SalI restriction site at its 3' end. The nucleotide sequence of SEQ ID NO: 41 was designed to be inserted immediately downstream of a promoter, such as an inducible promoter. The NheI and SalI restriction sites in SEQ ID NO:41 can be used to insert it into a vector multiple cloning site, such as the vector multiple cloning site of the pSOL expression vector (SEQ ID NO:42), described in U.S. Patent Application No. US2015353940A1, which is incorporated by reference in its entirety. Other PDI polypeptides, including PDI polypeptides from various species (Saccharomyces cerevisiae (UniProtKB P17967), Homo sapiens (UniProtKB P07237), Mus musculus (UniProtKB P09103), Caenorhabditis elegans (UniProtKB Q17770 and Q17967), Arabidopsis thaliana (UniProtKB O48773, Q9XI01, Q9SRG3, Q9LJU2, Q9MAU6, Q94F09, and Q9T042), Aspergillus niger (UniProtKB Q12730)), as well as modified forms of such PDI polypeptides, can also be expressed in host cells.In certain embodiments of the invention, a PDI polypeptide expressed in a host cell of the invention shares at least 70%, or 80%, or 90%, or 95% amino acid sequence identity to at least 50% (or at least 60%, or at least 70%, or at least 80%, or at least 90%) of the length of SEQ ID NO:40, where the amino acid sequence identity is determined according to Example 11.

[0053] Cellular Transport of Cofactors. When using the expression systems of the present invention to produce gene products that require a cofactor for function, it is useful to use host cells that are capable of synthesizing the cofactor from available precursors or harvesting the cofactor from the environment. Common cofactors include ATP, coenzyme A, flavin adenine dinucleotide (FAD), NAD + Polynucleotides encoding cofactor transporting and / or cofactor synthesis polypeptides can be introduced into host cells, and such polypeptides can be constitutively expressed or inducibly co-expressed with the gene products to be produced by the methods of the invention.

[0054] II. Expression constructs. An expression construct is a polynucleotide designed for the expression of one or more gene products of interest. Certain gene products of interest are "heterologous" gene products that are derived from a species different from that of the host cell in which they are expressed, and / or are heterologous gene products that are not naturally expressed from the promoter(s) utilized in the gene construct, and / or are modified gene products that are designed to include differences from the naturally occurring form of such gene products. Expression constructs that contain polynucleotides encoding heterologous and / or modified gene products, or that contain combinations of polynucleotides derived from different species of organisms, or that contain polynucleotides that are modified differently from naturally occurring polynucleotides, are non-naturally occurring molecules. Expression constructs can be integrated into a host cell chromosome or can be maintained in the host as polynucleotide molecules that replicate independently of the host cell chromosome, such as plasmids or artificial chromosomes. An example of an expression construct is a polynucleotide resulting from the insertion of one or more polynucleotide sequences into a host cell chromosome, where the inserted polynucleotide sequence alters the expression of a chromosomal coding sequence. An expression vector is a plasmid expression construct that is specifically used for the expression of one or more gene products. One or more expression constructs can be integrated into a host cell chromosome or maintained on an extrachromosomal polynucleotide, such as a plasmid or an artificial chromosome. In certain embodiments of the present invention, the expression construct is a pSOL expression vector (SEQ ID NO: 42).

[0055] Expression constructs may include certain polynucleotide elements, such as an origin of replication, a selectable marker, a promoter, such as a constitutive or inducible promoter (described further below), a ribosome binding site, and a multiple cloning site. Examples of these polynucleotide elements are well known in the art, and further descriptions thereof can be found in the following patent publications and application(s), all of which are expressly incorporated herein by reference: US9617335B2 and WO2014025663A1, "Inducible Coexpression System"; WO2016205570A1, "Vectors for Use in an Inducible Coexpression System"; and International Application PCT / US2016 / 067064, "Cytoplasmic Expression System."

[0056] Inducible promoters. As described further below, there are several different inducible promoters that can be included in an expression construct as part of the expression system of the present invention. Preferred inducible promoters share at least 80% polynucleotide sequence identity (more preferably, at least 90% identity, most preferably, at least 95% identity) over at least 30 (more preferably, at least 40, most preferably at least 50) consecutive bases of the promoter polynucleotide sequence as defined in Table 1 of WO2014025663A1, where the percent polynucleotide sequence identity is determined using the method of Example 11. Under "standard" induction conditions (see Example 10), preferred inducible promoters have at least 75% (more preferably at least 100%, most preferably at least 110%) of the strength of the corresponding "wild-type" inducible promoter of E. coli K-12 substrain MG1655, as determined using the quantitative PCR method of De Mey et al. "Promoter knock-in: a novel rational method for the fine tuning of gene", BMC Biotechnol 2010 Mar 24; 10: 26 (see WO2014025663A1, execution 8A). Within the expression construct, the inducible promoter is positioned 5' (or "upstream of") to the coding sequence for the gene product to be inducibly expressed, such that the presence of the inducible promoter induces transcription of the gene product coding sequence in the 5' to 3' direction relative to the coding strand of the polynucleotide encoding the gene product. The gene products expressed from inducible promoters in expression constructs are not the gene products naturally expressed from those inducible promoters; rather, they are heterologous gene products, with the consequence that expression constructs containing heterologous gene products expressed from inducible promoters are necessarily artificial constructs not found in nature.

[0057] Inducible promoters. Below is a description of inducible promoters that may be used in expression constructs for expression of gene products, along with some of the genetic modifications that may be made to host cells containing such expression constructs. These examples of inducible promoters and associated genes are derived from E. coli strain MG1655 (American Type Culture Collection deposit ATCC 700926), i.e., E. coli K-12 substrain (American Type Culture Collection deposit ATCC 10798), unless otherwise noted. Table 1 of International Application PCT / US13 / 53562 (published as WO2014025663A1) lists the genomic location in E. coli MG1655 of the nucleotide sequences for these examples of inducible promoters and associated genes, and WO2014025663A1 is incorporated herein by reference in its entirety. Nucleotides and other gene sequences referenced by genomic location as found in Table 1 of WO2014025663A1 are expressly incorporated herein by reference. Further information regarding the E. coli promoters, genes, and strains described herein can be found in many public sources, including the online EcoliWiki resource located at ecoliwiki.net.

[0058] Arabinose promoter. (As used herein, "arabinose" means L-arabinose.) Although several E. coli operons involved in arabinose utilization are inducible by arabinose (araBAD, araC, araE, and araFGH), the terms "arabinose promoter" and "ara promoter" are usually used to designate the araBAD promoter. ara , P ara B, P araBAD , and P BADSeveral additional terms, such as "ara promoter", "araC promoter", "araBAD ... BAD The AraC protein, which represses transcription from P. cerevisiae but changes its conformation upon binding of arabinose, is involved in the repression of transcription from P. cerevisiae in the presence of arabinose. BADThe araBAD operon encodes proteins that metabolize L-arabinose by converting it to D-xylulose-5-phosphate through the intermediates L-ribulose and L-ribulose phosphate. In order to maximize induction of expression from arabinose-inducible promoters, it is useful to eliminate or reduce the function of AraA, which catalyzes the conversion of L-arabinose to L-ribulose, and, optionally, to eliminate or reduce the function of at least one of AraB and AraD as well. By eliminating or reducing the ability of the cell to convert arabinose to other sugars, the host cell's ability to reduce the effective concentration of arabinose in the cell is eliminated, making more arabinose available for induction of arabinose-inducible promoters. Genes encoding transporters that move arabinose into the host cell are araE, which encodes a low-affinity L-arabinose proton symporter, and the araFGH operon, which encodes a subunit of the ABC superfamily high-affinity L-arabinose transporter. Other proteins capable of transporting L-arabinose into cells are certain mutants of the LacY lactose permease: LacY(A177C) and LacY(A177V) proteins, which have a cysteine ​​or valine amino acid, respectively, instead of alanine at position 177 (Morgan-Kiss et al., "Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity", Proc Natl Acad Sci USA 2002 May 28; 99(11):7373-7377). To achieve homologous induction of an arabinose-inducible promoter, it is useful to transport arabinose into cells independent of regulation by arabinose.This can be accomplished by eliminating or reducing the activity of the AraFGH transporter protein and altering the expression of araE so that araE is only transcribed from a constitutive promoter. Constitutive expression of araE can be accomplished by eliminating or reducing the function of the native araE gene and introducing into the cell an expression construct that includes the coding sequence for the AraE protein expressed from a constitutive promoter. Alternatively, in cells lacking AraFGH function, the promoter controlling expression of the host cell's chromosomal araE gene can be changed from an arabinose-inducible promoter to a constitutive promoter. In a similar manner, as a further alternative to homologous induction of an arabinose-inducible promoter, the host cell lacking AraE function can have any functional AraFGH coding sequence present in the cell expressed from a constitutive promoter. As another alternative, both the araE gene and the araFGH operon can be expressed from a constitutive promoter by replacing the native araE and araFGH promoters with constitutive promoters in the host chromosome. It is also possible to eliminate or reduce the activity of both the AraE and AraFGH arabinose transporters and use a mutation in the LacY lactose permease that allows this protein to transport arabinose in that situation. Because expression of the lacY gene is not normally regulated by arabinose, the use of a LacY mutant such as LacY(A177C) or LacY(A177V) does not result in an "all or nothing" induction phenomenon when an arabinose-inducible promoter is induced by the presence of arabinose. The use of a polynucleotide encoding the LacY(A177C) protein is preferred to the use of a polynucleotide encoding the LacY(A177V) protein, because the LacY(A177C) protein appears to be more effective at transporting arabinose into the cell.

[0059] Propionate promoter. The term "propionate promoter" or "prp promoter" refers to the promoter for the E. coli prpBCDE operon, P prpB Similar to the ara promoter, the prp promoter is part of a bidirectional promoter, controlling expression of the prpBCDE operon in one direction, and the prpR promoter controls expression of the prpR coding sequence in the other direction. The PrpR protein is a transcriptional regulator of the prp promoter, activating transcription from the prp promoter when the PrpR protein binds 2-methylcitric acid ("2-MC"). Propionate (also called propanoate) is the ion of propionic acid (or "propanoic acid"), CH3CH2COO - and share certain specific properties of this class of molecules: the property of producing an oily layer when salted out from water and the property of having a soap-like potassium salt. nIt is the smallest of the "fatty" acids that have a COOH. Commercially available propionate is generally sold as a monovalent cation salt of propionic acid, such as sodium propionate (CH3CH2COONa), or as a divalent cation salt, such as calcium propionate (Ca(CH3CH2COO)2). Propionate is membrane permeable and is metabolized to 2MC by conversion of propionate to propionyl-CoA by PrpE (propionyl-CoA synthetase), followed by conversion of propionyl-CoA to 2MC by PrpC (2-methylcitrate synthase). Other proteins encoded by the prpBCDE operon, PrpD (2-methylcitrate dehydratase) and PrpB (2-methylisocitrate lyase), are involved in the further metabolism of 2MC to smaller products such as pyruvate and succinate. Therefore, to maximize the induction of a propionate-inducible promoter by propionate added to the cell growth medium, it is desirable to have a host cell that has PrpC and PrpE activities to convert propionate to 2MC, but also has eliminated or reduced PrpD activity to prevent 2MC from being metabolized, and also optionally eliminated or reduced PrpB activity. Another operon that encodes proteins involved in 2MC biosynthesis is the scpA-argK-scpBC operon, also called the sbm-ygfDGH operon. These genes encode proteins required for the conversion of succinate to propionyl-CoA, which can then be converted to 2MC by PrpC. Elimination or reduction of the function of these proteins removes a parallel pathway for the production of 2-MC inducers, thus reducing the background level of expression of the propionate-inducible promoter and increasing the sensitivity of the propionate-inducible promoter to exogenously supplied propionate.The deletion of sbm-ygfD-ygfG-ygfH-ygfI introduced into E. coli BL21(DE3) to create strain JSB (Lee and Keasling, "A propionate-inducible expression system for enteric bacteria", Appl Environ Microbiol 2005 Nov; 71(11):6856-6862) served to reduce background expression in the absence of exogenously supplied inducer, but the deletion also reduced overall expression from the prp promoter in strain JSB. However, it should be noted that the deletion sbm-ygfD-ygfG-ygfH-ygfI also appears to affect ygfI, which encodes a putative LysR-family transcriptional regulator with unknown function. The genes sbm-ygfDGH are transcribed as one operon, and ygfI is transcribed from the opposite strand. The 3' ends of the ygfH and ygfI coding sequences overlap by several base pairs, so a deletion that removes all of the sbm-ygfDGH operon is likely to remove ygfI coding function as well. Eliminating or reducing the function of a subset of the sbm-ygfDGH gene products, such as YgfG (also called ScpB, methylmalonyl-CoA decarboxylase), or deleting most of the sbm-ygfDGH (or scpA-argK-scpBC) operon while leaving enough of the 3' end of the ygfH (or scpC) gene so as not to affect expression of ygfI, may be sufficient to reduce background expression from a propionate-inducible promoter without reducing the maximum level of induced expression.

[0060] Rhamnose promoter. (As used herein, "rhamnose" means L-rhamnose.) "Rhamnose promoter" or "rha promoter" or P rhaSRis the promoter for the E. coli rhaSR operon. Like the ara and prp promoters, the rha promoter is part of a bidirectional promoter, controlling expression of the rhaSR operon in one direction, and the rhaBAD promoter controls expression of the rhaBAD operon in the other direction. However, the rha promoter has two transcriptional regulators involved in regulating expression: RhaR and RhaS. The RhaR protein activates expression of the rhaSR operon in the presence of rhamnose, whereas the RhaS protein activates expression of the L-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively (Wickstrum et al., "The AraC / XylS family activator RhaS negatively autoregulates rhaSR expression by preventing cyclic AMP receptor protein activation", J Bacteriol 2010 Jan;192(1):225-232). The RhaS protein can also activate expression of the rhaSR operon, but in effect RhaS negatively autoregulates its expression by interfering with the ability of the cyclic AMP receptor protein (CRP) to coactivate expression with RhaR to higher levels. The rhaBAD operon encodes the rhamnose catabolic proteins RhaA (L-rhamnose isomerase), which converts L-rhamnulose to L-rhamnulose; RhaB (rhamnulokinase), which phosphorylates L-rhamnulose to form L-rhamnulose-1-P; and RhaD (rhamnulose-1-phosphate adrase), which converts L-rhamnulose-1-P to L-lactaldehyde and DHAP (dihydroxyacetone phosphate). To maximize the amount of rhamnose in the cell available for induction of expression from a rhamnose-inducible promoter, it is desirable to reduce the amount of rhamnose that is catalytically degraded by eliminating or reducing the function of RhaA, or, optionally, the function of at least one of RhaA, RhaB, and RhaD.E. coli cells can also synthesize L-rhamnose from alpha-D-glucose-1-P through the activity of the proteins RmlA, RmlB, RmlC, and RmlD (also called RfbA, RfbB, RfbC, and RfbD, respectively) encoded by the rmlBDACX (or rfbBDACX) operon. To reduce background expression from rhamnose-inducible promoters and to increase the sensitivity of induction of rhamnose-inducible promoters by exogenously supplied rhamnose, it may be useful to eliminate or reduce the function of one or more of the RmlA, RmlB, RmlC, and RmlD proteins. L-rhamnose is transported into the cell by RhaT, rhamnose permease, or L-rhamnose:proton symporter. As described above, expression of RhaT is activated by the transcriptional regulator RhaS. To effect expression of RhaT regardless of induction by rhamnose (which induces expression of RhaS), the host cell can be altered so that all functional RhaT coding sequences in the cell are expressed from a constitutive promoter. Additionally, the coding sequences for RhaS can be deleted or inactivated so that no functional RhaS is produced. By eliminating or reducing the function of RhaS in the cell, the level of expression from the rhaSR promoter is increased due to the absence of negative autoregulation by RhaS, and the level of expression of the rhamnose catalytic operon rhaBAD is decreased, further enhancing the ability of rhamnose to induce expression from the rha promoter.

[0061] Xylose promoter. (As used herein, "xylose" means D-xylose) "xylose promoter" or "xyl promoter" or P xylArefers to the promoter for the E. coli xylAB operon. The xylose promoter region is similar in organization to other inducible promoters in that both the xylAB and xylFGHR operons are expressed from adjacent xylose-inducible promoters in opposite orientations on the E. coli chromosome (Song and Park, "Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator", J Bacteriol. 1997 Nov;179(22):7025-7032). xylA and P xylFThe transcriptional regulator of both promoters is XylR, which activates expression of these promoters in the presence of xylose. The xylR gene is expressed as part of the xylFGHR operon or from its own weak promoter, which is not inducible by xylose and is located between the xylH and xylR protein coding sequences. D-xylose is catabolized by XylA (D-xylose isomerase), which converts D-xylose to D-xylulose, which is subsequently phosphorylated by XylB (xylulokinase) to form D-xylulose-5-P. To maximize the amount of xylose in the cell available for induction of expression from the xylose-inducible promoter, it is desirable to reduce the amount of xylose that is catalytically degraded by eliminating or reducing the function of at least XylA, or optionally both XylA and XylB. The xylFGHR operon encodes XylF, XylG, and XylH, subunits of the ABC superfamily high affinity D-xylose transporter. The xylE gene, which encodes the E. coli low affinity xylose proton symporter, represents an independent operon, the expression of which is also inducible by xylose. To effect expression of the xylose transporters independent of induction by xylose, the host cell can be altered so that all functional xylose transporters are expressed from a constitutive promoter. For example, the xylFGHR operon can be altered so that the xylFGH coding sequence is deleted, leaving XylR as the only active protein expressed from the xylose-inducible PxylF promoter, and the xylE coding sequence is expressed from a constitutive promoter rather than its native promoter. As another example, the xylR coding sequence can be expressed in an expression construct as PxylF. ylA Or P xylFThe xylFGH operon is either deleted so that xylE is constitutively expressed, or the xylFGH operon (which, while present in the expression construct, lacks the xylR coding sequence) is expressed from a constitutive promoter and the xylE coding sequence is deleted or altered so that it does not produce an active protein.

[0062] Lactose promoter. The term "lactose promoter" refers to the lactose-inducible promoter for the lacZYA operon, a promoter also called lacZp1, located at about 365603-365568 (minus strand, with an RNA polymerase binding ("-35") site at about 365603-365598, a Pribnow box ("-10") at 365579-365573, and a transcription start site at 365567) in the genomic sequence of E. coli K-12 substrain MG1655 (NCBI Reference Sequence NC_000913.2,11-JAN-2012). In some embodiments, the expression system of the invention may include a lactose-inducible promoter, such as the lacZYA promoter. In other embodiments, the expression system of the invention includes one or more inducible promoters that are not lactose-inducible promoters.

[0063] Alkaline phosphatase promoter. The terms "alkaline phosphatase promoter" and "phoA promoter" refer to the promoter for the phoApsiF operon, a promoter that is induced under conditions of phosphate starvation. The phoA promoter region is located at approximately 401647-401746 (plus strand, with a Pribnow box ("-10") at 401695-401701 (Kikuchi et al., "The nucleotide sequence of the promoter and the amino-terminal region of alkaline phosphatase structural gene (phoA) of Escherichia coli", Nucleic Acids Res 1981 Nov 11;9(21):5671-5678)) in the genomic sequence of E. coli K-12 substrain MG1655 (NCBI Reference Sequence NC_000913.3, 16-DEC-2014). The transcriptional activator for the phoA promoter is phoB, a transcriptional regulator that forms a two-component signal transduction system in E. coli together with the sensor protein PhoR. PhoB and PhoR are transcribed from the phoBR operon, located at approximately 417050-419300 (positive strand, with the PhoB coding sequence at 417,142-417,831 and PhoR at 417,889-419,184) in the genome sequence of E. coli K-12 substrain MG1655 (NCBI Reference Sequence NC_000913.3, 16-DEC-2014). The phoA promoter differs from the inducible promoters described above in that it is induced by the absence of a substance - intracellular phosphate - rather than by the addition of an inducer. For this reason, the phoA promoter generally induces the transcription of gene products to be produced at stages when the host is starved for phosphate, such as the later stages of fermentation. In some embodiments, the expression system of the present invention may include the phoA promoter. In other embodiments, the expression system of the invention comprises one or more inducible promoters that are not the phoA promoter.

[0064] III. Host cells. An expression construct encoding a gene product of interest is expressed in a host cell to produce the gene product of interest. The host cell can be any cell that contains and is capable of expressing such an expression construct. Particularly suitable host cells are capable of growing at high cell densities in fermentation cultures and producing gene products in the oxidizing host cell cytoplasm with highly controlled inducible gene expression. Host cells with these qualities are produced by combining some or all of the following characteristics: (1) The host cell is genetically engineered to have an oxidizing cytoplasm by increasing the expression or function of an oxidizing polypeptide in the cytoplasm and / or decreasing the expression or function of a reducing polypeptide in the cytoplasm. Specific examples of such genetic changes are provided herein. Optionally, the host cell can also be genetically engineered to express chaperones and / or cofactors that aid in the production of the desired gene product(s) and / or to glycosylate the polypeptide gene products. (2) The host cell comprises one or more expression constructs designed for expression of one or more genes of interest, and in certain embodiments, at least one expression construct comprises an inducible promoter and a polynucleotide encoding a gene product to be expressed from the inducible promoter, and (3) The host cell contains additional genetic modifications designed to improve certain aspects of gene product expression from the expression construct(s).In certain embodiments, the host cell has (A) an alteration in gene function of at least one gene encoding a transporter protein with respect to an inducer of at least one inducible promoter, where, as another example, the gene encoding the transporter protein is selected from the group consisting of araE, araF, araG, araH, rhaT, xylF, xylG, and xylH, or in particular araE, or the alteration in gene function is, more particularly, expression of araE from a constitutive promoter, and / or (B) an alteration in gene function of at least one gene encoding a protein that metabolizes an inducer of at least one inducible promoter. and / or (C) a reduced level of gene function of at least one gene encoding a protein involved in the biosynthesis of an inducer of at least one inducible promoter, which in a further embodiment is selected from the group consisting of scpA / sbm, argK / ygfD, scpB / ygfG, scpC / ygfH, rmlA, rmlB, rmlC, and rmlD.

[0065] Examples of host cells are provided that allow for efficient and cost-effective production of gene products, including multimeric products. Host cells can include isolated cells in culture as well as cells that are part of a multicellular organism or cells grown in different organisms or biological systems. In certain embodiments of the invention, the host cell is a microbial cell such as a yeast (Saccharomyces, Schizosaccharomyces, etc.) or a bacterium, or a Gram-positive or Gram-negative bacterium, or an E. coli, or an E. coli B strain, or an E. coli (B strain) EB0001 cell (also called an E. coli ASE (DGH) cell), or an E. coli (B strain) EB0002 cell. In growth experiments with E. coli host cells having an oxidizing cytoplasm, specifically E. coli B strains SHuffle® Express (NEB Catalog No. C3028H) and SHuffle® T7 Express (NEB Catalog No. C3029H) and E. coli K strain SHuffle® T7 (NEB Catalog No. C3026H), the inventors have determined that these E. coli B strains having an oxidizing cytoplasm are capable of growing to much higher cell densities than the most closely corresponding E. coli K strain.

[0066] Prokaryotic Host Cells In some embodiments of the invention, expression constructs designed for expression of gene products are provided in a host cell, such as a prokaryotic host cell. Prokaryotic host cells may include Archaea (e.g., Haloferax volcanii, Sulfolobus solfataricus), Gram-positive bacteria (e.g., Bacillus subtilis, Bacillus licheniformis, Brevibacillus choshinensis, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis, and Streptomyces lividans), or Gram-negative bacteria, including alphaproteobacteria (e.g., Agrobacterium tumefaciens). tumefaciens, Caulobacter crescentus, Rhodobacter sphaeroides, and Sinorhizobium meliloti), Betaproteobacteria (Alcaligenes eutrophus), and Gammaproteobacteria (Acinetobacter calcoaceticus, Azotobacter vinelandii, Escherichia coli, Pseudomonas aeruginosa, and Pseudomonas putida).Preferred host cells include gammaproteobacteria of the Enterobacteriaceae family, such as Enterobacter, Erwinia, Escherichia (including E. coli), Klebsiella, Proteus, Salmonella (including Salmonella typhimurium), Serratia (including Serratia marcescans), and Shigella.

[0067] Eukaryotic host cells. Yeasts such as Candida shehatae, Kluyveromyces lactis, Kluyveromyces fragilis, other Kluyveromyces species, Pichia pastoris, Saccharomyces cerevisiae, Saccharomyces pastorianus, also known as Saccharomyces carlsbergensis, Schizosaccharomyces pombe, Dekkera / Brettanomyces species, and Yarrowia lipolytica. lipolytica); other fungi (Aspergillus nidulans, Aspergillus niger, Neurospora crassa, Penicillium, Tolypocladium, Trichoderma reesia); insect cell lines (Drosophila melanogaster Schneider 2 cells and Spodoptera frugiperda Sf9 cells); and mammalian cell lines including immortalized cell lines (Chinese hamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), human embryonic kidney (HEK, 293, or HEK-293) cells, and human hepatocellular carcinoma cells (Hep Many additional types of host cells can be used in the expression system of the present invention, including eukaryotic cells such as G2), which are available from the American Type Culture Collection.

[0068] Changes to host cell gene functions. Certain changes can be made to gene functions of host cells containing an inducible expression construct to facilitate efficient and uniform induction of a host cell population by an inducer. Preferably, the combination of expression construct, host cell genotype, and induction conditions results in at least 75% (more preferably at least 85%, most preferably 95%) of the cells in the culture expressing a gene product from each of the induced promoters as measured by the method of Khlebnikov et al. "Regulatable arabinose-inducible gene expression system with consistent control in all cells of a culture", J Bacteriol 2000 Dec;182(24): 7029-7034, as described in Example 8B of WO2014025663A1. For host cells other than E. coli, these changes may involve the function of genes that are structurally similar to E. coli genes or that perform functions in the host cell similar to the function of E. coli genes. Changes to host cell gene function include eliminating or reducing gene function by deleting the gene protein coding sequence in its entirety or deleting a large enough portion of the gene, inserting sequences into the gene, or otherwise altering the gene sequence such that reduced levels of functional gene product are produced from that gene. Changes to host cell gene function also encompass enhancing gene function, for example, by altering a native promoter to create a stronger gene promoter that induces higher levels of transcription of the gene, or by introducing a missense mutation into the protein coding sequence that results in a more active gene product. Changes to host cell gene function include altering gene function in any way, for example, altering a naturally inducible promoter to create a constitutively activated promoter.In addition to changes in gene function related to inducer transport and metabolism, as described herein with respect to altering the expression of inducible promoters and / or chaperone proteins, it is also possible to alter the reducing-oxidizing environment of the host cell.

[0069] Host cell reducing-oxidizing environment. In bacterial cells such as E. coli, proteins that require disulfide bonds are normally exported to the periplasm where disulfide bond formation and isomerization is catalyzed by the Dsb system, including DsbABCD and DsbG. Increased expression of the cysteine ​​oxidase DsbA, the disulfide isomerase DsbC, or a combination of Dsb proteins, all normally transported to the periplasm, has been utilized in the expression of heterologous proteins that require disulfide bonds (Makino et al., "Strain engineering for improved expression of recombinant proteins in bacteria", Microb Cell Fact 2011 May 14;10: 32). It is also possible to express cytoplasmic forms of these Dsb proteins, such as cytoplasmic versions of DsbA and / or DsbC ("cDsbA" or "cDsbC") that lack the signal peptide and therefore are not transported to the periplasm. Cytoplasmic Dsb proteins such as cDsbA and / or cDsbC are useful for making the cytoplasm of a host cell more oxidizing and therefore more conducive to disulfide bond formation in heterologous proteins produced in the cytoplasm. The cytoplasm of a host cell can also be made less reducing and therefore more oxidizing by directly altering the thioredoxin and glutaredoxin / glutathione enzyme systems: mutant strains lacking glutathione reductase (gor) or glutathione synthetase (gshB), together with thioredoxin reductase (trxB), render the cell oxidizing. These strains are unable to reduce ribonucleotides and therefore cannot grow in the absence of exogenous reactants such as dithiothreitol (DTT). Suppressor mutations in the gene ahpC, which encodes the peroxiredoxin AhpC (e.g., ahpC * and ahpC Δ, Lobstein et al., "SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm", Microb Cell Fact 2012 May 8;11:56;doi:10.1186 / 1475-2859-11-56), converts it into a disulfide reductase that generates reduced glutathione, allowing channeling of electrons onto the enzyme ribonucleotide reductase, and allowing cells that are gor and trxB deficient or gshB and trxB deficient to grow in the absence of DTT. Different mutant forms of AhpC are capable of growing strains lacking gamma-glutamylcysteine ​​synthetase (gshA) activity and trxB in the absence of DTT: these include AhpC V164G, AhpC S71F, AhpC E173 / S71F, AhpC E171Ter, and AhpC dup162-169 (Faulkner et al., "Functional plasticity of a peroxidase allows evolution of diverse disulfide-reducing pathways", Proc Natl Acad Sci USA 2008 May 6;105(18):6735-6740, Epub 2008 May 2). In such strains with an oxidizing cytoplasm, exposed protein cysteines become readily oxidized, leading to the formation of disulfide bonds, in a process catalyzed by thioredoxin, reversing their physiological function.Other proteins that may be beneficial in reducing the effects of oxidative stress in oxidizing cytoplasmic host cells are the HPI (hydroperoxidase I) catalase-peroxidases encoded by E. coli katG and HPII (hydroperoxidase II) catalase-peroxidases encoded by E. coli katE, which disproportionate peroxides to water and O2 (Farr and Kogoma, "Oxidative stress responses in Escherichia coli and Salmonella typhimurium", Microbiol Rev. 1991 Dec;55(4):561-585; Review). Increasing the levels of KatG and / or KatE proteins in host cells, either by induced co-expression or by increasing the levels of constitutive expression, is an aspect of some embodiments of the invention.

[0070] Another change that can be made to the host cell is to express the sulfhydryl oxidase Erv1p from the inner membrane space of yeast mitochondria in the cytoplasm of the host cell, which has been shown to increase production of various complexes of disulfide-bonded proteins of eukaryotic origin in the cytoplasm of E. coli, even in the absence of mutations in gor or trxB (Nguyen et al., "Pre-expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E. coli" Microb Cell Fact 2011 Jan 7;10:1).

[0071] Host cells containing the expression constructs also preferably express cDsbA and / or cDsbC and / or Erv1p, are deficient in trxB gene function, are deficient in gor, gshB, and / or gshA gene function, optionally have increased levels of katG and / or katE genes, and optionally express an appropriate mutant form of AhpC, such that the host cells can be grown in the absence of DTT.

[0072] Glycosylation of polypeptide gene products. Host cells can have alterations in their ability to glycosylate polypeptides. For example, eukaryotic host cells can have eliminated or reduced gene functions in glycosyltransferases and / or oligosaccharyltransferases, impairing normal eukaryotic glycosylation of polypeptides to form glycoproteins. Prokaryotic host cells, such as E. coli, that do not normally glycosylate polypeptides, can be altered to express a set of eukaryotic or prokaryotic genes that provide glycosylation functions (DeLisa et al., "Glycosylated protein expression in prokaryotes", WO2009089154A2, 2009 Jul 16).

[0073] Available host cell lines with altered gene functions. To create preferred strains of host cells to be used in the expression systems and methods of the invention, it is useful to start with a strain that already contains the desired genetic alteration; examples of these are provided in Table 2.

[0074] [Table 3]

[0075] Methods for Altering Host Cell Gene Function. There are many methods known in the art for making changes to host cell genes to eliminate, reduce, or alter gene function. Methods for targeted disruption of genes in host cells such as E. coli and other prokaryotes have been described (Muyrers et al., "Rapid modification of bacterial artificial chromosomes by ET-recombination", Nucleic Acids Res 1999 Mar 15;27(6):1555-1557; Datsenko and Wanner, "One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products", Proc Natl Acad Sci USA 2000 Jun 6;97(12):6640-6645), and kits for using similar Red / ET recombination methods are commercially available (e.g., the Quick & Easy E. coli Gene Deletion Kit, Gene Bridges, Heidelberg, Germany). In one embodiment of the invention, the function of one or more genes of a host cell is eliminated or reduced by identifying a nucleotide sequence within the coding sequence of the gene to be disrupted, such as one of the E. coli K-12 substrain MG1655 coding sequences incorporated herein by reference to the genomic location of the sequence, more specifically by selecting two adjacent stretches of 50 nucleotides each within the coding sequence. A polynucleotide construct containing a selectable marker is then inserted between the selected adjacent stretches of the coding sequence using The Quick & Easy E. coli Gene Deletion Kit according to the manufacturer's instructions to eliminate or reduce the normal function of the gene.Also, using the Red / ET recombination method, promoter sequences can be replaced with sequences of different promoters, for example constitutive promoters or artificial promoters predicted to promote a certain level of transcription (De Mey et al., "Promoter knock-in: a novel rational method for the fine tuning of genes", BMC Biotechnol 2010 Mar 24;10:26). The function of host cell genes can also be eliminated or reduced by RNA silencing methods (Man et al., "Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria", Nucleic Acids Res 2011 Apr;39(8):e50, Epub 2011 Feb 3). Furthermore, known mutations that alter host cell gene function can be introduced into host cells by traditional genetic methods.

[0076] IV. Methods for Propagating Host Cells. Small volume growth. The host cells used to carry out the methods of the invention can be grown in small volumes, such as for the purpose of testing growth or induction conditions, or for the production of multiple different gene products. The nature of the experiment to be performed determines the volume in which the host cells are grown, such as 1 mL up to 1 liter, or 5 mL to 500 mL, or any convenient volume. In certain embodiments, the vessel in which the host cells are grown is moved repeatedly to agitate the growth medium and thus provide oxygen to the host cells. The host cells are grown in a medium containing appropriate nutrients and any antibiotics required to select for retention by the host cells of the expression construct providing antibiotic resistance. An example of small volume growth of host cells is provided in Example 1. To determine the amount of inducer suitable to be used to induce expression of the inducible expression construct present in the cells, experiments such as those described in Example 10 can be suitably carried out with the host cells grown in small volumes, for example in multi-well plates.

[0077] Fermentation. Fermentation processes involved in the production of recombinant proteins use modes of operation that fall into one of the following categories: (1) discontinuous (batch process) operation, (2) continuous operation, and (3) semi-continuous (fed-batch) operation. Batch processes are characterized by inoculating a sterile culture medium (batch medium) with microorganisms at the beginning of the process and culturing for a specific reaction period. During the cultivation, cell concentration, substrate concentration (carbon source, nutrient salts, vitamins, etc.), and product concentration vary. Good mixing ensures that there are no significant local differences in the composition or temperature of the reaction mixture. The reaction is non-stationary and cells are grown until the growth-limiting substrate (generally the carbon source) is consumed.

[0078] Continuous operation is characterized by the continuous addition of fresh culture medium (feed medium) to the fermenter and the continuous withdrawal of spent medium and cells from the fermenter at the same rate. In continuous operation, the growth rate is determined by the rate of medium addition and the growth yield is determined by the concentration of the growth-limiting substrate (i.e., carbon source). All reaction variables and control parameters remain constant in time, thus a time-constant state is established in the fermenter and subsequently at constant productivity and output.

[0079] Semi-continuous operation may be viewed as a combination of batch and continuous operation. The fermentation is undertaken as a batch process and a continuous feed medium containing glucose and minerals is added in a specified manner when the growth limiting substrate is consumed (fed-batch). In other words, the operation uses both batch and feed medium to achieve cell growth and efficient production of the desired protein. Cells are neither added nor removed during the culture period, thus the fermenter operates in batch mode as far as the microorganism is concerned. While the present invention may be utilized in a variety of processes, including those described above, certain applications are in conjunction with fed-batch processes.

[0080] In each of the above processes, cell growth and product accumulation can be monitored indirectly by exploiting the correlation between metabolite formation and several other variables such as medium pH, optical density, color, and titratable acidity. For example, optical density provides an indication of the accumulation of insoluble cell particles and can be monitored on-the-fly using a micro-OD unit connected to a display device or recorder, or off-line by sampling. Optical density readings at 600 nanometers (OD600) are used as a means of determining dry cell weight.

[0081] High cell density fermentations generally produce yields of at least 30 g cell dry weight / liter (OD >60). 600), and in certain embodiments, yields of greater than 40 g / liter cell dry weight (OD >80 600 ) are described as processes that result in high cell density fermentation. All high cell density fermentation processes use a concentrated nutrient medium that is gradually metered into the fermentor in a "fed-batch" process. A concentrated nutrient feed medium is required for high cell density processes to minimize dilution of the fermentor contents during feeding. The fed-batch process is required because it allows the operator to control the carbon source feeding, which is important because cells produce so much of the inhibitory by-product acetate that their growth stops when exposed to carbon source concentrations high enough to produce high cell densities (Majewski and Domach, "Simple constrained-optimization view of acetate overflow in E. coli", Biotechnol Bioeng 1990 Mar 25;35(7):732-738).

[0082] Both acetic acid and its deprotonated ion, acetate, represent one of the main inhibitory by-products of bacterial growth and recombinant protein production in bioreactors. At pH 7, acetate is the most common form of acetate. Any excess carbon energy source can be converted to acetate when the amount of carbon energy source greatly exceeds the processing capacity of the bacteria. Studies have found that saturation of the tricarboxylic acid cycle and / or electron transport chain is the most likely cause of acetate accumulation. The choice of growth medium can affect the level of acetate inhibition, and cells grown in defined media can be more affected by acetate than cells grown in complex media. Replacement of glucose with glycerol can also greatly reduce the amount of acetate produced. It is believed that glycerol produces less acetate than glucose because its rate of transport into the cell is much slower than that of glucose. However, glycerol is more expensive than glucose and can cause the bacteria to grow more slowly. The use of reduced growth temperature can also reduce the rate of carbon source uptake and growth rate, thus reducing acetate production. Bacteria not only produce acetate in the presence of excess carbon energy sources or during fast growth, but also under anaerobic conditions. When bacteria such as E. coli are grown at very high rates, they can exceed the oxygen delivery capacity of the bioreactor system, which can lead to anaerobic growth conditions. To prevent this from happening, a slower, constant growth rate can be maintained by nutrient limitation. Other methods to reduce acetate accumulation include genetic modification to prevent acetate production, addition of acetate utilization genes, and selection of acetate-reduced strains. E. coli BL21(DE3) is one strain that has been found to produce lower levels of acetate due to its ability to use acetate in its glyoxylate shunt cycle.

[0083] A variety of larger scale fed-batch fermenters are available for recombinant protein production. Larger fermenters have a capacity (i.e., working volume) of at least 1000 liters, preferably between about 1000 liters and 100,000 liters, leaving sufficient room for headspace. These fermenters use agitator impellers or other suitable means for distributing oxygen and nutrients, especially glucose (the preferred carbon / energy source). Small scale fermentation generally refers to fermentation in fermenters with a volumetric capacity of approximately 100 liters or less, and in some specific embodiments, approximately 10 liters or less.

[0084] Standard reaction conditions for fermentation processes used to produce recombinant proteins generally involve, for microbial host cells such as E. coli, maintaining a pH of about 5.0 to 8.0 and a culture temperature in the range of 20° C. to 50° C. In one embodiment of the invention utilizing E. coli as a host system, fermentation is carried out at an optimum pH of about 7.0 and an optimum culture temperature of about 30° C.

[0085] Standard nutrient medium components in these fermentation processes generally include energy, carbon, nitrogen, phosphorus, magnesium, and trace amounts of iron and calcium. In addition, the medium may contain growth factors (e.g., vitamins and amino acids), inorganic salts, and any other precursors necessary for product formation. The medium may contain transportable organic phosphates such as glycerophosphate, e.g., alpha-glycerophosphate and / or beta-glycerophosphate, and more specifically, glycerol-2-phosphate and / or glycerol-3-phosphate. The elemental composition of the host cells in culture can be used to calculate the ratio of each of the components required to support cell growth. The component concentrations vary depending on whether the process is a low cell density process or a high cell density process. For example, glucose concentrations in low cell density batch fermentation processes range from 1 g / L to 5 g / L, whereas high cell density batch processes use glucose concentrations in the range of 45 g / L to 75 g / L. Additionally, the growth medium may contain appropriate concentrations (e.g., in the range of 0.1 mM to 5 mM, or 0.25 mM, 0.5 mM, 1 mM, 1.5 mM, or 2 mM) of protective osmolytes such as betaine, dimethylsulfonioisopropionate, and / or chlorine.

[0086] One or more inducers can be introduced into the growth medium to induce expression of the gene product(s) of interest. Induction can begin during the exponential growth phase, for example toward the end of the exponential growth phase but before the culture reaches maximum cell density, or earlier or later during fermentation. If the gene product(s) of interest are expressed from one or more promoters that are inducible by depletion of a nutrient, such as phosphate, induction occurs when the nutrient has been sufficiently depleted from the growth medium without the addition of an exogenous inducer.

[0087] During exponential growth of host cells, metabolic rate is directly proportional to the availability of oxygen and carbon / energy sources, and thus reducing the levels of available oxygen or carbon / energy sources, or both, reduces the metabolic rate. The available oxygen level can be adjusted and the host cell metabolic rate reduced by manipulating fermenter operating parameters such as agitation rate or back pressure, or by reducing O2 pressure. Reducing the concentration or delivery rate of the carbon / energy source(s), or both, has a similar effect. Furthermore, depending on the nature of the expression system, induction of expression can cause a decrease in the host cell metabolic rate. Eventually, once maximum cell density is reached, the growth rate stops or is dramatically reduced. A reduction in the host cell metabolic rate can result in a more controlled expression of the gene product(s) of interest, including the process of protein folding and assembly. The host cell metabolic rate can be assessed by measuring the cell growth rate, either specific growth rate or instantaneous growth rate (by measuring optical density (OD), such as OD600, and / or optionally converting OD to biomass). Calculate the approximate biomass (cell dry weight) at each assayed time point: Approximate biomass (g) = (OD 600 ÷ 2) × volume (L). The desired growth rate, in certain embodiments of the invention, is in the range of 0.01 to 0.7, or in the range of 0.05 to 0.3, or in the range of 0.1 to 0.2, or approximately 0.15 (0.15 + or - 10%), or is 0.15.

[0088] Fermentation Equipment. The following is an example of equipment that can be used to grow the host cells; many other configurations of fermentation systems are commercially available. The host cells can be grown in a New Brunswick BioFlo / CelliGen 115 water jacketed fermenter (Eppendorf North America, Hauppauge, NY) with a 1 L vessel size equipped with a 2X Rushton impeller and a BioFlo / CelliGen 115 fermenter / bioreactor controller, with temperature, pH and dissolved oxygen (DO) monitored. The host cells can also be grown in a 4-fold configurable DSAGIP system (Eppendorf North America, Hauppauge, NY) containing four 60-250 ml DASbox fermentation vessels, each equipped with a 2X Rushton impeller, a DASbox exhaust cooler, and a DASbox feed and monitoring module, which includes temperature, pH / redox and dissolved oxygen sensors. Suitable fermentation equipment also includes an NLF 22 30 L lab fermenter (Bioengineering, Somerville, Massachusetts) with 30 L capacity and 20 L maximum working volume in a stainless steel vessel, two Rushton impellers sparged with air only, and a control system running BioSCADA software allowing tracking and control of all relevant parameters including pH, DO, exhaust O2, exhaust CO2, temperature, and pressure.

[0089] V. Solubilization and Purification Methods. The gene products expressed by the methods described herein can be purified using a variety of purification methods. When gene products are expressed to produce solubilizable complexes as described herein, particularly as seen in the specific embodiments described in Examples 1 and 2, highly suitable purification methods can be used to efficiently produce properly folded and active gene products without the need for additional refolding steps. Example 3 describes a further "direct solubilization" method of purifying gene products expressed as solubilizable complexes, including gene products that form disulfide bonds, without the need for centrifugation after lysis to separate soluble and insoluble fractions, and without the use of reducing agents. The methods of purifying solubilizable gene acid product complexes using these methods are outlined diagrammatically in FIG. 1 and described in more detail below.

[0090] Harvesting the host cells by centrifugation. The host cells containing the expression construct are grown and expression of the gene product of interest is induced as further described herein, resulting in the production of a solubilizable complex of the gene product of interest within the host cells. After the growth and induction period is complete, the host cells are harvested, for example, by centrifugation at 4,000 x g for 10 minutes at 4°C. The host cells can be frozen at this point and stored for later purification.

[0091] Lysis of host cells. The resulting pellet of intact host cells is lysed using one of several alternative methods. The host cell pellet is resuspended in a non-denaturing lysis buffer, such as phosphate buffered saline (PBS) or Tris buffered saline (TBS) supplemented with 0 mM-300 mM NaCl or 2.5 mM L-cysteine, pH 9.5. After resuspension in the lysis buffer, the host cells can be lysed by methods including enzymatic or chemical lysis, mechanical lysis, and / or freeze-thaw methods. For enzymatic lysis, lysis can be achieved by adding recombinant lysozyme, benzonase, and octylglucoside to the lysis buffer. For mechanical lysis, the resuspended host cells are passed one or more times through a microfluidizer, such as a Microfluidics Model LV1 Microfluidizer for volumes up to 60 ml, or a Microfluidics Model M-110Y1 Microfluidizer (Microfluidics International, Westwood, Mass.) for volumes greater than 60 mL, or through a PandaPLUS 2000 benchtop homogenizer or GEO Niro (GEA North America, Columbia, Md.) For the freeze-thaw method, the cell suspension is frozen at -80°C and thawed at a temperature between 25°C and 37°C.

[0092] After lysis, the lysed cell mixture is optionally centrifuged to pellet the solubilizable gene product complexes. The speed and time of this centrifugation step can vary from 3,300 to 20,000×g and 30 to 60 minutes. Using a higher speed can result in a pellet of solubilizable gene product complexes that are more difficult to resuspend. The lower the speed used in this centrifugation step, the longer the duration of centrifugation required to complete separation of the solubilizable gene product complexes from the supernatant. The salt concentration and / or pH of the cell lysate can be altered to change the centrifugation or other conditions required to separate the solubilizable gene product complexes from other components in the cell lysate.

[0093] One important advantage of harvesting the gene product of interest in this manner is that the majority of potentially contaminating host cell proteins and other molecules remain in the supernatant and are removed from the pelleted solubilized gene product complex, which is therefore a preparation highly enriched in the gene product of interest. Alternatively, if the supernatant remaining after pelleting of the solubilizable complex retains sufficient gene product, the gene product in the supernatant can be solubilized and / or further purified as described for the direct solubilization method. If analysis of the pelleted material indicates that a significant number of cells survive without lysis and are spun down together with the solubilizable gene product complex, it is possible to use a thick and / or viscous solution, such as a high concentration sucrose solution, as a "cushion" in the centrifugation procedure to separate the intact cells from the solubilizable gene product complex. If mechanical lysis is used, the lysed cell mixture can be passed multiple times (e.g., 4 or 5 times) through a microfluidizer. As described below, where the centrifugation step is omitted in direct solubilization methods, the cell lysate is mixed with reagents to create conditions for solubilization of the solubilizable gene product complexes.

[0094] The gene product is released from the solubilizable complex by placing it in a solubilization solution, resulting in a solubilized gene product. The gene product, either in the pellet resulting from centrifugation or in the cell lysate, is solubilized as described below. The solubilization solution preferably contains one or more chaotropic agents, such as one or more of n-butanol, ethanol, guanidinium chloride, guanidinium hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, or urea. Exemplary solubilization solutions can contain 7M-8M urea in PBS or TRIS at pH 9.5, optionally with 2.5 mM L-cysteine, or 6M guanidine hydrochloride in PBS at pH 7.5, and successful solubilization experiments have used solubilization buffers at pH values ​​ranging from 6.5 to 11.0. Experiments to determine alternative buffer compositions to be used for solubilization are described in Example 5. The pellet may be optionally washed prior to resuspension. After addition of solubilization buffer to the pellet, it is more efficient to mechanically agitate the tube containing the pellet for at least 10 minutes using, for example, a plate vortexer, using a pipette tip rather than manually resuspending the pellet. Reversible chemical modifications of gene products, such as citraconylation of free lysine residues and primary amino groups, can alter the solubility of the gene product.

[0095] Optional clarification of solubilized gene products by centrifugation. As shown in Figure 1, the solubilized gene product solution can optionally be clarified by centrifugation, for example, at 7,000 x g for 1 hour at 16°C. The supernatant containing the solubilized gene products is retained after centrifugation. The clarification procedure can be performed before and / or after placing the solubilized gene products in a solution that is less concentrated than the solubilization solution, as described below.

[0096] Placing the solubilized gene product in a solution with a lower concentration than the solubilization solution. For samples of protein gene products to be analyzed by peptide mapping, since 7M-8M urea or 6M guanidine hydrochloride inhibit the cleavage efficiency of many proteases, the solubilized gene product is usually placed in a solution with a 2-fold to 10-fold reduced concentration of denaturant using methods such as dialysis, dilution, or diafiltration. For example, after solubilization in 7M-8M urea in PBS or TRIS, pH 6.5-9.5, optionally with 2.5 mM L-cysteine, the sample containing the gene product can be placed in 2M-4M urea in PBS or TRIS, pH 6.5-9.5, optionally with 2.5 mM L-cysteine, and incubated, for example, at 16° C. with shaking for 10 to 120 hours.

[0097] Optional formation of a solution with a higher concentration of gene product. For purposes such as storage, further purification, or characterization, the solubilized gene product solution can be reconstituted to produce a solution with a higher concentration of gene product. This can be accomplished by running the solution through a chromatography column and eluting in a desired buffer, as described below, or by spin desalting or diafiltration, as described below, or by other known methods. Another alternative is the use of precipitation methods, such as ammonium sulfate precipitation, to precipitate the gene product, which can optionally be washed before resuspending the pellet in a desired buffer at the desired concentration.

[0098] Gene products having cleavage sequences can optionally be cleaved by chemical or enzymatic treatment. For example, gene products containing sequences that are cleaved by enzymes such as trypsin and / or sequences such as the "DP" (Asp-Pro) chemical cleavage sequence described above and in Example 2 can be cleaved by appropriate enzymatic or chemical treatment prior to use or further purification.

[0099] The solubilized gene product can be optionally further purified. For example, a gene product containing a 6xHis tag can be purified by immobilized metal affinity chromatography (IMAC), such as using a nickel-nitrilotriacetic acid (Ni-NTA) column that specifically retains the 6xHis-tagged gene product of interest while passing other molecules through. IMAC utilizes the interaction between histidine residues and divalent metal ions, most commonly Ni2+, and other metal ions, including Cu2+, Co2+, Fe2+, and Zn2+, have also been found to have affinity for His residues. Metal ions are usually immobilized on matrices by various metal-chelator systems, including iminodiacetic acid (IDA) and the more commonly used nitrilotriacetic acid (NTA). A wide variety of matrices are commercially available, such as nickel-nitrilotriacetic acid (Ni-NTA), Ni-Sepharose, and copper-carboxymethylaspartic acid (CO-CMA). The column may be equilibrated with a buffer such as 50 mM Tris, 3 M urea, 0.5 M NaCl, 25 mM imidazole, pH 8.0. After binding of the 6xHis tagged gene product, a wash step with a buffer containing a low concentration of imidazole (0 mM, or 10 mM to 50 mM), or a buffer having a higher or lower pH than the pH of the binding buffer, may be included to remove non-specific proteins that are weakly bound to the column during sample loading. For example, a wash buffer of 50 mM Tris, 100 mM NaCl, pH 10 may be used. The 6xHis tagged gene product may be eluted from the matrix using a buffer containing imidazole at a concentration of at least 100 mM imidazole, or 250 mM to 500 mM imidazole, or 500 mM imidazole. It is also possible to elute the gene product of interest by lowering the buffer pH and / or including a chelating agent such as EDTA (at a concentration of 50 mM to 200 mM, or 100 mM) in the elution buffer. For example, an elution buffer of 50 mM Tris, 100 mM NaCl, 100 mM imidazole, pH 10 can be used.Purification methods for gene products containing polyhistidine tags are further described in Bornhorst and Falke, "Purification of proteins using polyhistidine affinity tags," Methods Enzymol 2000;326:245-254, which is incorporated herein by reference. In the purification of 6xHis-tagged CPBpro proinsulin protein from solubilizable complexes using Ni-NTA Superflow (QIAgen, Germantown, MD) or HisTrap HP Ni Sepharose columns (GE Healthcare, Pittsburgh, PA), this method allowed purification of the proinsulin gene product to greater than 90% purity.

[0100] For samples lacking a 6xHis tag, or for procedures where the use of such a tag is not necessary, cation or anion exchange chromatography, such as using DEAE resin, and / or reverse phase or high performance liquid chromatography (PRLC or HPLC) can be used to further separate the gene product of interest from other contaminants or from undesired product(s) of chemical or enzymatic treatments.

[0101] Chemical or enzymatic procedures can optionally be performed on the gene product retained by a solid substrate such as a column: for example, trypsin cleavage of the proinsulin gene product for preparative or analytical purposes, also referred to as transversion of proinsulin to mature insulin, as described below in Example 3C.

[0102] Also, chromatographic means such as IMAC can be used to elute the solubilized complex in a buffer other than that used to solubilize the complex, for example, 250 mM up to 500 mM imidazole in PBS pH 7.5, followed by spin desalting to exchange the elution buffer for a more preferred buffer, as described in Example 2D. Methods for removing undesirable buffer components such as salts include dialysis, diafiltration (e.g., using centrifugal concentrators or tangential flow filtration), and gel filtration using other chromatographic resins, such as, for example, polyacrylamide beads (Bio-Rad, Hercules, CA), Sephadex resin (GE Healthcare, Pittsburgh, PA), and / or size exclusion resins (Zeba™ Spin Desalting Columns, ThermoFisher Scientific, Waltham, MA).

[0103] The solubilized gene products can be characterized chemically and / or structurally. For protein gene products that contain disulfide bonds, proper folding of the protein produced by the methods of the invention can be inferred from the presence of correctly formed disulfide bonds. Identification and characterization of disulfide bonds can be achieved using peptide mapping methods that use chemical or enzymatic treatment of the protein to produce peptide fragments. Separation and identification of these fragments is accomplished by liquid chromatography-mass spectrometry (LC-MS) analysis, and peptide mapping and LC-MS methods are further described in Example 8 below. Peptide mapping and LC-MS analysis can also identify differences in protein primary structure, such as point mutations and post-translational modifications (PTMs).

[0104] The number and presence of oxidized disulfide bonds can be verified on intact protein samples. Protein gene products can be treated with reducing agents such as dithiothreitol (DTT) and / or sulfhydryl-reactive reagents such as iodoacetamide (IAA). LC-MS analysis of reduced and / or alkylated samples results in a mass increase of 2 Da per disulfide bond reduction and a mass increase of 57 Da per alkylation of each free thiol. Protein gene products can be characterized not only for the formation of the correct number of disulfides, but also for the correct bridge configuration or "disulfide structure". This procedure consists of proteolysis, separation of the resulting peptides by high performance liquid chromatography (HPLC), and mass spectrometry (MS) analysis of the peptides represented by the HPLC peaks. To generate proteolytic peptide products, the protein gene product can be fragmented by chemical agents such as cyanogen bromide and / or enzymatic agents such as trypsin, pepsin, lysyl endopeptidase (Lys-C), glutamyl endopeptidase (Glu-C), and peptidyl-Asp metalloendopeptidase (Asp-N). For the protein gene product proinsulin, sequential proteolytic reactions can be carried out using Glu-C and trypsin, where the order of addition of proteases can be interchanged (i.e., Glu-C followed by trypsin, or trypsin followed by Glu-C). Protease digestion reactions can be carried out for 4-16 hours at temperatures ranging from 25-37° C., with substrate to enzyme ratios ranging from 12-200 micrograms of proinsulin per microgram of protease. Proteolytic efficiency and specificity can be improved by the addition of commercially available detergents such as ProteaseMax™ (Promega, Madison, WI) and RapiGest SF (Waters, Milford, MA), and / or low concentrations of organic solvents such as 10%-20% acetonitrile.

[0105] As further described in Example 2C and shown in Figure 5, LC-MS analysis demonstrated that approximately 93% of the solubilized protein gene products had properly formed disulfide bonds without further refolding or purification steps after solubilization. Other methods that may be used to characterize the solubilized gene products include gel electrophoresis, activity assays, and analytical reversed-phase high performance liquid chromatography (HPLC) separation or size exclusion chromatography (SEC). EXAMPLES

[0106] Example 1 Use of the CPBpro mutant propeptide in the production of lispro proinsulin A. Preparation of expression construct for CPBpro_lispro proinsulin In these experiments, certain CPBpro variants were used as propeptides in small-scale expression of lisproproinsulin polypeptides. Expression constructs containing polynucleotides encoding the CPBproproinsulin polypeptides shown in Table 3 and optimized for expression in E. coli were synthesized by ATUM (Newark, Calif.). The first column of Table 3 provides the protein number (PN) and SEQ ID NO for each complete CPBproproinsulin polypeptide amino acid sequence. The polynucleotides encoding each of the presented CPBproproinsulin polypeptides from the RBS sequence to the stop codon have SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, SEQ ID NO: 50, SEQ ID NO: 52, and SEQ ID NO: 54, respectively. The second to fifth columns of Table 3 show the amino acid sequence of each portion of each CPBproproinsulin polypeptide: the N-terminal CPBpro propeptide sequence, followed by the lisproinsulin B chain (as shown in Table 1), the C-peptide, and the lisproinsulin A chain (as shown in Table 1), in the following order from N to C:

[0107] [Table 4]

[0108] Polynucleotides encoding each of the CPBpro proinsulin polypeptides were positioned downstream of the araBAD promoter in the pSOL expression vector (SEQ ID NO: 42). Each of these expression constructs also contained a coding sequence for protein disulfide isomerase (PDI, SEQ ID NO: 41) downstream of the prpBCDE promoter in the pSOL expression vector.

[0109] B. Host cell transformation and expression of CPBpro_lispro proinsulin pSOL:CPBpro-Lispro / PDI expression constructs were transformed into EB0001 cells as described below; the genotype of EB0001 cells is shown in Table 2. Chemically competent (CaCl2-treated) EB0001 cells were thawed on ice for 10 minutes. DNA (1 microliter from each expression construct DNA stock) was added to cold sterile Eppendorf tubes. EB0001 cells (100 microliters) were added to each tube of DNA and the mixture was incubated on ice for 30 minutes. The tubes were heat shocked at 42°C for 20 seconds and allowed to sit on ice for 5 minutes. The transformed cells were allowed to recover in 900 microliters of SOC growth medium (NEW England Biolabs Cat# B9020S) at 37°C for 1 hour with shaking at 275 RPM. After the recovery period, cells were pelleted at 3.8k×g for 2 minutes and resuspended in approximately 100 microliters of remaining recovery medium from the supernatant before plating on agar plates containing 50 micrograms / mL kanamycin. The transformed plated cells were grown at 37° C. for 18 hours. For each transformation, three colonies were picked from the plate and cultured in LB medium with 50 micrograms / mL kanamycin overnight at 30° C. with shaking at 275 RPM until stationary phase (OD600 >2.0) was reached. Glycerol stocks were made by adding 750 microliters of 40% glycerol to 750 microliters of the overnight culture. Glycerol stocks were stored at −80° C.

[0110] Host cell cultures for expression of CPBpro_lisproproinsulin were initiated by stabbing the glycerol stock and inoculating 0.1 L of LB medium containing 50 micrograms / mL kanamycin in a 0.5 L unbaffled flask. Cells were grown overnight at 30°C with shaking at 275 RPM until an OD600 of 2 was reached. Host cell cultures were diluted into LB medium containing 50 micrograms / mL kanamycin to an OD600 of 0.2 and grown in a 0.5 L baffled flask in a total volume of 0.1 L at 30°C with shaking at 275 RPM until an OD600 of 0.6-0.8 was reached. At this point, an appropriate volume was pelleted (3800 x g, 10 min) so that resuspension in M9 minimal medium containing 50 micrograms / mL kanamycin gave an OD600 of 0.7-0.75.

[0111] M9 minimal medium. Autoclave in a volume of 1.2 L: Sodium phosphate, dibasic, heptahydrate 15.36g Potassium phosphate, monobasic 3.6g Sodium chloride 0.6g Ammonium chloride 1.2g Casamino acids 2.4g Adjust pH to 7.2 with KOH, autoclave at 121° C. for 45 minutes, and allow to cool to room temperature; this creates incomplete M9 minimal medium. To complete the medium, add the following volumes of filter-sterilized salts for each 10 mL of incomplete medium: 20 microliters 1 M MgSO4, 1 microliter 1 M CaCl2, 1 microliter 5 mg / mL FeSO4.

[0112] Cultures were transferred to 24-well deep well plates. For each of the induction conditions below, 3 mL samples of host cell cultures were added to each well: 6 wells for each expression construct with 15 micromolar arabinose, and 6 wells for each construct with 45 micromolar arabinose. Host cells were induced at 27° C. or 30° C. for 6 hours with shaking at 275 RPM. Host cell optical density was measured after the induction period, OD600 was 1.0-1.2 in all wells. Replicate samples (3×1 mL, 2×5 mL pellets) for each induction condition for each expression construct were collected by centrifugation at 3800×g for 7 minutes at room temperature.

[0113] Successful induction of PN2.5, PN2.7, and PN2.9 expression constructs was confirmed using SDS-PAGE with Coomassie Blue staining. 5 mL pellets for each induction condition for each expression construct were thawed on ice for 10 min. Host cells were lysed in GLB-OG lysis buffer, pH 7.4 (50 mM Tris pH 7.4, 200 mM NaCl with 1% octylglucoside, 1X protease inhibitors, 2 U benzonase (EMD#70746) per mL culture and 2.25 kU rLysozyme (EMD#71110) per mL culture) at 6-fold higher than culture concentration at harvest. Lysis was allowed to proceed by incubating on ice for 10 min. After lysis, samples were split into two pools, one of which contained the total lysate preparation and the other contained the soluble lysate preparation. For total lysate preparations, after lysis, 8M urea in 50mM Tris pH 7.4, 200mM NaCl was added to each sample in a 1:1 ratio and incubated at room temperature for 20 minutes before the samples were ready to be run on the gel. For soluble lysate preparations, samples were centrifuged at 20k×g for 30 minutes at 4° C., the supernatant (soluble fraction) was removed and added to 8M urea in 50mM Tris pH 7.4, 200mM NaCl in a 1:1 ratio and incubated at room temperature for 20 minutes before the samples were ready to be run on the gel. Polyacrylamide gel electrophoresis (PAGE) was performed on samples on a reducing 12% Bis-Tris gel in SDS-MES buffer and the gel was stained with Coomassie blue stain. In the lanes with total lysate preparations, significant bands of the expected size were seen only for the PN2.5 (SEQ ID NO: 43), PN2.7 (SEQ ID NO: 47), and PN2.9 (SEQ ID NO: 51) samples: the CPBpro polypeptides in these samples all have a 6xHis sequence immediately following the N-terminal methionine residue. However, bands for PN2.5, PN2.7, and PN2.9 were not observed in the soluble lysate preparations, indicating that significant amounts of the proteins produced from the corresponding expression constructs were produced in an insoluble (and solubilizable) form. No expression was observed for PN2.6, PN2.8, and PN2.10 expression in any preparation, and no expression was detected from the PN2.6 expression construct in follow-up experiments.Although the cause of the absence of expression from the expression constructs encoding PN2.6, PN2.8, and PN2.10 has not been determined, these expression constructs share a common nucleotide sequence around the translation start site that differs from that in the expression constructs encoding PN2.5, PN2.7, and PN2.9, and it is possible that the messages transcribed from the PN2.6, PN2.8, and PN2.10 expression constructs are not translated efficiently.

[0114] C. Solubilization and characterization of CPBpro_lispro proinsulin Solubilization with 2M-6M urea. To determine the conditions for solubilization of PN2.5 CPBpro_lisproproinsulin (SEQ ID NO: 43), a 5 mL pellet of host cells containing PN2.5 CPBpro_lisproproinsulin produced in Example 1.B was thawed on ice for 10 minutes. The host cells were lysed in GLB-OG lysis buffer pH 7.4 at 2x the culture concentration at harvest for 10 minutes on ice. The lysate was then split into 12 samples and processed as follows; all samples except the total lysate sample were centrifuged at 20k×g for 30 minutes at 4° C.: Total melt: no spin, no solubilizing additives Untreated: no solubilizing additives 6M urea: Addition of 8M urea in GLB 4M urea: Addition of 5.3M urea in GLB 2M urea: Addition of 2.66M urea in GLB 1M urea: Addition of 1.33M urea in GLB 0.5M urea: Addition of 0.66M urea in GLB 4% Triton-X100: 5% Triton-X100 in GLB 2% Triton-X100: 2.5% Triton-X100 in GLB 1% Triton-X100: 1.25% Triton-X100 in GLB 0.5% Triton-X100: 0.625% Triton-X100 in GLB 0.25% Triton-X100: 0.3125% Triton-X100 in GLB The amount of PN2.5 CPBpro_lispro proinsulin present in each sample was determined by automated capillary electrophoresis "Western blot" using a WES instrument (ProteinSimple, San Jose, CA) following the manufacturer's protocol and generally as described in Example 6. In preparation for analysis under reducing conditions, total lysate, untreated and solubilized samples as indicated above were diluted 1:300 in 0.1x WES buffer (ProteinSimple) supplemented with DTT (48mM) to give samples a final concentration of 0.0033X. PN2.5 CPBpro_lispro proinsulin was detected by capillary electrophoresis on the WES instrument using a mouse anti-lispro primary antibody and a HRP-conjugated goat anti-mouse secondary antibody at exposures of 5, 15, 30, 60, 120, 240 and 480 seconds (only 5 seconds of exposure was used for quantification). Triton-X solubilization alone was generally unsuccessful in this experiment, solubilizing only approximately 10% or less of the total PN2.5 CPBpro_lisproproinsulin present in the sample, as indicated by the amount of PN2.5 CPBpro_lisproproinsulin detected in the total lysate. Solubilization with urea at a concentration of at least 2M was more successful: the amount of solubilized PN2.5 CPBpro_lisproproinsulin increased with increasing urea concentration, with 6M urea solubilizing approximately 70% of the PN2.5 CPBpro_lisproproinsulin present in the sample.

[0115] Characterization of the size of solubilizable CPBpro_lispro proinsulin complex. Host cells containing solubilizable PN2.5 CPBpro_lispro proinsulin complex were lysed as follows. To create a control "guanidine lysis" sample representing the total amount of protein produced by host cell lysis, a 1 mL pellet of host cells containing PN2.5 CPBpro_lispro proinsulin produced in Example 1.B was thawed on ice for 10 minutes and resuspended in 500 microliters of 6M guanidine HCl buffer, pH 8 (6M guanidine HCl, 100 mM NaPO4, 10 mM Tris base, 10 mM imidazole, adjusted to pH 8 with 5M NaOH). Host cells were lysed by freezing at -80°C for 1 hour and then thawed at room temperature for 30 minutes or until completely thawed. Also as described above, host cells were lysed in GLB-OG lysis buffer and the lysate was centrifuged at 900×g for 15 minutes at 4° C. to create pellet fraction ("P1") and supernatant ("S1") fractions. A portion of the S1 supernatant fraction was retained and the remainder was centrifuged at 7000×g for 30 minutes at 4° C. to create pellet fraction ("P2") and supernatant ("S2") fractions. A portion of the S2 supernatant fraction was retained and the remainder was centrifuged at 20K×g for 30 minutes at 4° C. to create pellet fraction ("P3") and supernatant ("S3") fractions. The P1 pellet was solubilized in 6M guanidine HCl buffer, pH 8. The guanidine lysed, solubilized P1, S1, S2, and S3 samples were analyzed by capillary electrophoresis on a WES instrument under reducing conditions as described above. After a 900×g spin, the amount of PN2.5 CPBpro_lispro proinsulin detected in the S1 soluble fraction was approximately 42% of the "total dissolved" amount detected in the guanidine-dissolved sample. The amount of PN2.5 CPBpro_lispro proinsulin detected in the solubilized P1 pellet was approximately 35% of the "total dissolved" amount in the guanidine-dissolved sample instead of the expected 58%, suggesting that losses of potentially recoverable PN2.5 CPBpro_lispro proinsulin occur at several steps in the GLB-OG dissolution, centrifugation, and solubilization procedures.After faster spins at 7000×g and 20K×g, only small amounts (about 7%) of PN2.5 CPBpro_lisproproinsulin were detected in the S2 and S3 soluble fractions, with the majority of the protein in the P2 and P3 pellets. These results are consistent with a significant portion of N2.5 CPBpro_lisproproinsulin, probably about half of that present in the host cells, being present in complexes large enough to be spun down at a relatively low centrifugation speed of 900×g (see Cube Biotech, "Screening detergents for optimal solubilization and purification of membrane proteins", 2013, retrieved from www.cube-biotech.com / files / protocols / Screening_Detergents.pdf, March 29, 2017), which can pellet cell debris from lysed cells but not soluble proteins. Of the PN2.5 CPBpro_lispro proinsulin present in the host cells that remained soluble during the 900×g spin, the majority was pelleted at the intermediate centrifugation speed of 7000×g, consistent with the remaining CPBpro_lispro proinsulin also being present in the host in the form of large solubilizable complexes.

[0116] Solubilization in 8M urea and 3.5M urea / 5% Triton-X. To further evaluate the solubilization conditions for the preparation of PN2.5 CPBpro_lispro proinsulin (SEQ ID NO: 43), EB0001 host cells containing the PN2.5 pSOL:CPBpro-lispro / PDI expression construct were grown and induced with 15 micromolar arabinose as described in Example 1B. After induction, 1 mL samples of host cell culture were collected by centrifugation at 3800×g for 10 minutes at 4° C. For lysis, the host cell pellet was redissolved in GLB-OG lysis buffer pH 7.4 and lysis was allowed to proceed on ice for 15 minutes. Host cell lysates were centrifuged at 20K×g for 15 min at 4° C. and the resulting pellets were resuspended in either 8 M urea in 1× Tris-buffered saline (TBS) pH 8.0 or 3.5 M urea / 5% triton-X in 1× TBS pH 8.0. Samples of solubilized protein were prepared for PAGE by removing triton-X from the 3.5 M urea / 5% triton-X samples using a Pierce™ SDS PAGE Prep kit (Thermo Fisher Scientific, Waltham, Mass.) and by preparing both non-reduced (in LDS sample buffer, Thermo Fisher Scientific) and reduced samples (in LDS sample buffer+100 mM DTT) and analyzed by PAGE for each solubilization condition. The amount of solubilized PN2.5 CPBpro_lispro proinsulin in each sample was assessed by PAGE on a 12% Bis-Tris gel in SDS-MES buffer and the gel was stained with Coomassie Blue stain. The band migration as expected for the molecular weight of PN2.5 CPBpro_lispro proinsulin (12.25 kD) was clearly seen, with the sample solubilized in 8M urea yielding a much more intense band than the sample solubilized in 3.5M urea / 5% triton-X. This result indicates that under these conditions, 8M urea is more effective at solubilizing the proinsulin complex than 3.5M urea / 5% triton-X.

[0117] CPBpro_lisproproinsulin from the solubilizable complex has disulfide bonds and is highly purified. To determine whether PN2.5 CPBpro_lisproproinsulin from the solubilizable complex has disulfide bonds or free thiol residues, samples solubilized in 8M urea and prepared as described above were analyzed by PAGE on a 12% Bis-Tris gel in SDS-MES buffer, with non-reduced and reduced pairs of samples run in adjacent lanes. The gel was stained with Coomassie blue and is shown in FIG. 2. Upon treatment with DTT, the reduced samples (lanes 4 and 6) ran slightly slower than the corresponding non-reduced samples (lanes 3 and 5), indicating the presence of disulfide bonds in non-reduced PN2.5 CPBpro_lisproproinsulin. The difference in migration rates between reduced and non-reduced CPBpro_lisproproinsulin was confirmed by analytical reversed-phase chromatography. FIG. 2 also shows that the solubilized PN2.5 CPBpro_lispro proinsulin (lanes 3 to 6) was highly purified as a result of removal of soluble proteins in the total host cell lysate (lanes 1 and 2) from the solubilizable CPBpro_lispro proinsulin pellet.

[0118] Solubilizable CPBpro_lispro proinsulin complex forms throughout induction. To further characterize the formation of solubilizable CPBpro_lispro proinsulin complexes, a time-course induction was performed using EB0001 host cells containing the PN2.5 pSOL:CPBpro-lispro / PDI expression construct, which were grown and induced generally as described in Example 1B. In this experiment, induction was performed on host cell cultures in three volumes of 200 mL each in 1 L baffled flasks, with 15 micromolar arabinose as inducer, and 1 mL samples were taken at 0, 2, 4, and 6 hours after the start of induction. Host cells were harvested, lysis was performed, and the solubilizable pellets were resuspended in 8M urea in 1× Tris-buffered saline (TBS) pH 8.0 and analyzed by PAGE as described above. Solubilizable CPBpro_lispro proinsulin complexes were present in the host cells at the 2, 4, and 6 hour time points, respectively, as indicated by bands in each lane at the expected positions. This result indicates that solubilizable complexes were formed in the host cells throughout the induction period, and that sufficient CPBpro_lispro proinsulin was present in the host cells to form solubilizable complexes after only 2 hours of induction.

[0119] Example 2 Use of CPBpro variant propeptides in the production of glargine proinsulin A. Host cells for expression of CPBpro_glargine proinsulin In these experiments, the CPBpro propeptide ("His-CPB1") having the amino acid sequence of SEQ ID NO:27 was used in the fermentation-scale expression of the glargine proinsulin polypeptide. A polynucleotide encoding the His-CPB1 propeptide and optimized for expression in E. coli has been previously synthesized (Example 1A). The sequence encoding the His-CPB1 propeptide was cloned into an existing expression construct containing a polynucleotide sequence encoding glargine proinsulin, also optimized for expression in E. coli and also synthesized by ATUM, Inc. (Newark, Calif.). The PN3.13 CPBpro_glargine proinsulin polypeptide (SEQ ID NO:55) has at its N-terminus the His-CPB1 propeptide (SEQ ID NO:27), followed by the glargine insulin B chain (SEQ ID NO:7), the C-peptide (SEQ ID NO:11) corresponding to RRYPGDVKR, except that the initial arginine (RR) of the C-peptide is shown in this case as present at the end of the B chain of SEQ ID NO:7, and in the glargine insulin A chain (SEQ ID NO:6). The structure of the PN3.13 CPBpro_glargine proinsulin polypeptide, including the disulfide bonds found in insulin glargine, is shown diagrammatically in Figure 3. The polynucleotide encoding the PN3.13 CPBpro_glargine proinsulin polypeptide (SEQ ID NO: 56) was inserted downstream of the araBAD promoter in the pSOL expression vector (SEQ ID NO: 42). This expression construct was also optimized for expression in E. coli (SEQ ID NO: 57) and contained Erv1p (SEQ ID NO: 38) downstream of the prpBCDE promoter in the pSOL expression vector. Using the method described in Example 1B, the pSOL:PN3.13-CPBpro_glargine / Erv1p expression construct was transformed into E. coli EB0001 cells, and glycerol stocks of transformed host cells were prepared and stored at -80°C.

[0120] B. CPBpro_Glargine Proinsulin Induces Host Cell Proliferation and Its Expression EB0001 (pSOL:PN3.13-CPBpro_glargine / Erv1p) host cells were grown in a DSAGIP fermentation system (Eppendorf North America, Hauppauge, NY) in one of the 250 ml DASbox fermentation vessels, Bioreactor 1 ("BR1") (see "Fermentation Equipment" above). The bioreactor was calibrated as follows: pH offset 0.80 pH, pH slope 104.15%, DO offset 0.01 nA; DO slope 66.72 nA.

[0121] Fermentation medium. Fermentation medium, and growth and induction feeds, total volume of 100 mL, were prepared as follows: Fermentation medium; pre-sterilized components, concentrations in g / L per 90 mL volume added to each bioreactor Potassium phosphate (monobasic) 14.8 Potassium citrate tribasic (monohydrate) 3.3 Ammonium sulfate 4.4 Sodium chloride 2.2 ·Yeast extract 11.1 Modified Korz trace metals (100x stock); Combine and filter the following components (where final concentrations are given in g / L): ·CoCl2·6H2O 0.25 MnCl2 4H2O 1.5 CuSO4 5H2O 0.22 H3BO30.3 ·Na2MoO4·2H2O 0.25 ZnSO4 7H2O 1.7 Fermentation medium; post-sterilization components (sterile stock concentrations), amounts in mL added to reach a total volume of approximately 100 mL in the bioreactor: Glucose (700g / L) 1.4 EDTA (100x stock, 0.84g / L) 1.0 Modified Korz Trace Metals (100x Stock) 1.0 Ferrous ammonium sulfate (40g / L) 0.8 Magnesium sulfate heptahydrate (500 g / L) diluted 1:5 1.3 Sterile Antiform 204 (Sigma-Aldrich, St. Louis, MO) 10% dissolved in 70% ethanol / 30% H2O 0.3 Kanamycin (50g / L) diluted 1:10 1.0 Calcium chloride (200g / L) 1.0 Growth feed; components (sterile stock concentrations), amounts in mL that can be prepared for one bioreactor: Glucose (700g / L) 80 EDTA (100x stock, 0.84g / L) 1.36 Modified Korz Trace Metals (100x Stock) 1.44 Ferrous ammonium sulfate (40g / L) 1.40 Magnesium sulfate heptahydrate (500g / L) 4.0 Kanamycin (50g / L) 0.08 ·Yeast extract (250g / L) 2.8 Induction feed; components (sterile stock concentrations), amounts in mL that can be prepared for one bioreactor: Glycerol (700g / L) 80 EDTA (100x stock, 0.84g / L) 1.36 Modified Korz Trace Metals (100x Stock) 1.44 Ferrous ammonium sulfate (40g / L) 1.40 Magnesium sulfate heptahydrate (500g / L) 4.0 Kanamycin (50g / L) 0.08 Arabinose (500g / L) 0.97 10 x Tremendous Bros. ("10 x TB"): To 90 mL ddH2O add: 12 g soytone, 24 g yeast extract. Adjust to 100 mL with ddH2O. Sterilize by autoclaving. Allow to cool to room temperature.

[0122] Fermentation procedure. A feeder culture of EB0001 (pSOL:PN3.13-CPBpro_glargine / Erv1p) host cells was grown generally according to the method described in Example 1B, except that it was grown overnight until the OD600 reached approximately 3, and a larger day 2 inoculum was used into LB medium with 1% glucose to reach a final cell density OD600 of 2.40 after 5.5 hours of growth. This feeder culture was used to inoculate the fermentation medium in the bioreactor, with a 4.2 mL aliquot being added to approximately 100 mL of medium, resulting in an initial optical density reading (OD600) of approximately 0.1.

[0123] Cells were grown for 29 hours under growth phase conditions (growth feed containing 70% glucose at 30.0° C., DO 30%, pH 7.0, initial feed rate of 0.6 mL / hour for a set growth rate of 0.15 / hour with a maximum feed rate of 3.2 mL per hour). Just prior to the start of induction, 5 mL of 10× Tremendous Bro was added to the bioreactor. Induction was started and fermentation conditions were set to induction phase conditions: 30.0° C., DO 30%, pH 7.0, and induction feed containing 70% glycerol with an induction feed factor of 2.1 mL per hour. The induction feed also contained the inducer L-arabinose at a concentration calculated from the total volume of components added to make the induction feed as follows: [L-arabinose] in induction feed: (0.97mL x 500g / L) / 89.25mL = 5.4g / L The host cells in the bioreactor were sampled at several time points during fermentation and induction and the optical density of the growth broth at these time points expressed in terms of elapsed fermentation time (EFT(hrs)) and elapsed induction time (EIT(hrs)) are shown below.

[0124] [Table 5]

[0125] At 9 hours or more after induction, host cells in 2 mL samples taken for optical density measurements and 125 microliters of host cells diluted 1:20 in PBS buffer were harvested by centrifugation at 4300 RPM for 7 minutes at 4°C and stored at -80°C as a dry lyophilized pellet.

[0126] C. Solubilization and characterization of CPBpro glargine proinsulin To investigate whether the disulfide bond present in the solubilizable CPBpro_glargine proinsulin complex is formed between the correct residues, the solubilized CPBpro_glargine proinsulin complex was analyzed by liquid chromatography-mass spectrometry (LC-MS). The pellet of host cells harvested from 1 mL of culture, grown and induced as described in Example 2B, was resuspended in 15 mL of GLB-OG lysis buffer pH 7.4, and lysis was allowed to proceed for 15 minutes on ice. The host cell lysate was centrifuged at 20K×g for 30 minutes at 4° C., and the resulting pellet was resuspended in 5 mL of 8 M urea in 1× phosphate buffered saline (PBS) pH 7.5.

[0127] The following procedure was performed in the enzymatic digestion and non-reduced disulfide mapping of CPBpro_glargine proinsulin and is shown diagrammatically in Figure 4. The starting sample contained solubilized CPBpro_glargine proinsulin at a concentration of 0.63 mg / mL in 8 M urea, PBS, pH 7.5. To prepare the samples for enzymatic digestion, 25 microliters of 1 M Tris pH 7.5, 165 microliters of deionized water, and 60 microliters of CPBpro_glargine proinsulin sample were added to a 1.5 mL Eppendorf tube for each sample to produce a final concentration of CPBpro_glargine proinsulin sample of 0.15 mg / mL (and a total of 37.5 micrograms of CPBpro_glargine proinsulin) and the concentration of urea was reduced to approximately 1.9 M. Sequencing grade trypsin (Promega, Madison, WI) was reconstituted at 0.1 mg / mL in 50 mM acetic acid. Ten microliter volumes (or 1 microgram) of trypsin were added to each sample tube and incubated at 37° C. for 4 hours with shaking at 275 RPM. Pierce™ Glutamyl Endopeptidase ("Glu-C") MS Grade (Thermo Fisher Scientific, Waltham, MA) was reconstituted at 0.04 mg / mL in deionized water and 5 microliters (or 0.2 micrograms) of Glu-C were added to each sample tube. Samples were incubated at 37° C. for 16 hours with shaking at 275 RPM. A 5 microliter volume of 10% acetic acid was added to each tube to inactivate the protease. At this point, samples were optionally frozen at −80° C. prior to analysis. Samples can also optionally be analyzed by SDS-PAGE or reverse phase LC to determine that digestion has occurred. After enzymatic digestion, samples were centrifuged at 14×g for 5 minutes at 4° C. and 20 microliters of supernatant was transferred to an appropriate autosampler vial for use in the MS analysis described below.

[0128] Nano-LC MS / MS analysis was performed using a 60 min method on a recently calibrated Orbitrap Fusion™ Tribrid™ mass spectrometer and a Dionex UltiMate™ 3000R SLC nano System (Thermo Fisher Scientific). An Acclaim™ Pepmap™ 100 C18 75 micrometer x 25 cm x 2 micrometer analytical column was used with an Acclaim™ Pepmap™ 100 C18 100 micrometer x 2 cm x 5 micrometer trapping column (Thermo Fisher Scientific). Buffer A consisted of 0.1% formic acid in LC-MS grade water and buffer B consisted of 0.1% formic acid in LC-MS grade acetonitrile. 200 ng of sample was injected onto the trap. The gradient was run as follows: 0 min to 5 min 2% Buffer B; 5 min to 5.1 min 2% to 7.5% Buffer B; 5.1 min to 35 min 7.5% to 30% Buffer B; 36 min to 41 min 30% to 98% Buffer B, and 42 min to 60 min 2% Buffer B. Samples were analyzed in positive ion mode using an EASY-Spray™ source (Thermo Fisher Scientific) at 2400 V with an ion transfer tube temperature of 275° C. MS1 scans were acquired from 400 m / z to 1600 m / z with an AGC (automatic gain control) of 400,000 at a resolution of 120 K and a maximum injection time of 50 ms. Targeted MS / MS was performed at 742.8330 m / z (z=4) and represented the following sequences QCCTSICSLYQLE (SEQ ID NO:58) and FVNQHLGSHLVE (SEQ ID NO:59) with one interchain and one intrachain disulfide. Masses were calculated to four decimal places for calculations (e.g., H+=1.0073). Targeted MS / MS settings included 3 m / z quadrupole isolation, HCD activation at 30%, and detection on an Orbitrap mass spectrometer at resolution 15K from 100 m / z to 2000 m / z with a maximum injection time of 250 ms and an AGC target of 50,000. Further data-dependent or targeted MS / MS events can be optionally scheduled as desired, except that MS1 survey scans are performed at least every 2 seconds.The results of this liquid chromatography-mass spectrometry (LC-MS) analysis are shown in FIG.

[0129] D. Further purification and solubility of CPBpro_glargine proinsulin Further variants CPBpro_glargine proinsulin were prepared (PN3.15, PN3.16, and PN3.17), each having a propeptide portion corresponding to SEQ ID NO: 33, SEQ ID NO: 34, and SEQ ID NO: 35, respectively, in which one or more acid-cleavable DP(Asp-Pro) sequences were inserted before the arginine present at the C-terminus of the propeptide. Modifications were made to the expression construct (SEQ ID NO: 56) encoding PN3.13 CPBpro_glargine proinsulin (SEQ ID NO: 55) to produce expression constructs encoding PN3.15 CPBpro_glargine proinsulin (SEQ ID NO: 62), PN3.16 CPBpro_glargine proinsulin (SEQ ID NO: 64), and PN3.17 CPBpro_glargine proinsulin (SEQ ID NO: 66) (SEQ ID NO: 63, SEQ ID NO: 65, and SEQ ID NO: 67, respectively, +18 bp downstream nucleotide sequence shown from the ribosome binding site (RBS) to the stop codon), each expression construct having a polynucleotide encoding CPBpro_glargine proinsulin located downstream of the araBAD promoter in the pSOL expression vector, and a polynucleotide encoding Erv1p (SEQ ID NO: 38) downstream of the prpBCDE promoter (SEQ ID NO: 57), as described in Example 2A. These pSOL:PN3.15-CPBpro_glargine / Erv1P, pSOL:PN3.16-CPBpro_glargine / Erv1P, and pSOL:PN3.17-CPBpro_glargine / Erv1P expression constructs were transformed into E. coli EB0001 cells. PN3.15, PN3.16, and PN3.17CPBpro_glargine proinsulin were produced by fermentation essentially as described in Example 2B, with lysis of the host cells followed by centrifugation at 20K×g for 30 min at 4° C. The resulting pellet, containing the solubilizable complex of CPBpro_glargine proinsulin, was solubilized in 8 M urea in 1× phosphate buffered saline (PBS) pH 7.5.

[0130] A portion of PN3.15 CPBpro_glargine proinsulin (SEQ ID NO: 62) prepared from the solubilizable complex was loaded onto a 5 mL Ni-NTA column washed with 8 M urea and 10 mM imidazole in 1×PBS pH 7.5, and subsequently eluted in 500 mM imidazole in 1×PBS pH 7.5. PN3.15 CPBpro_glargine proinsulin was stable and soluble under the non-denaturing conditions of Ni-NTA column purification and elution into 500 mM imidazole in 1×PBS at neutral pH 7.5. After elution from the Ni-NTA column, PN3.15, PN3.16, and PN3.17 CPBpro_glargine proinsulin samples in 500 mM imidazole in 1×PBS pH 7.5 were each adjusted to pH 6 with formic acid, purified CPBpro_glargine proinsulin was precipitated at 16K×g for 10 min at 4° C., and the pellet was resuspended in 0.1 M acetic acid at pH 2 and incubated at 65° C. for 12 h to cleave each propeptide at the DP(Asp-Pro) sequence present in the PN3.15, PN3.16, and PN3.17 propeptides, respectively. After incubation, the samples were neutralized with 2 M NH4HCO3 (ammonium bicarbonate) to a final pH of 7.0-8.0. Cleavage of the propeptides was monitored by polyacrylamide gel electrophoresis.

[0131] Separation of the cleaved N-terminal portion of the PN3.17 propeptide from the remainder of PN3.17 CPBpro_glargine proinsulin (SEQ ID NO: 66) was achieved by cation exchange chromatography ("CEX") using Capto S medium (GE Healthcare, Pittsburgh, PA). The cleavage reaction in which PN3.17 CPBpro_glargine proinsulin was treated with 0.1 M acetic acid at pH 2 and incubated at 60° C. for 24 hours with shaking at 275 RPM was adjusted to pH 4 with 1 M hydrochloric acid and loaded onto a cation exchange column, subsequently equilibrated with 8 M urea in 20 mM NaOAc pH 6.5 and eluted with increasing salt concentrations from 0 M to 0.35 M NaCl in 8 M urea in 20 mM NaOAc pH 6.5. LC-MS analysis of the CEX-purified glargine proinsulin fragments confirmed that the masses of the fragments were as expected for glargine proinsulin with all of its double bonds intact. Trypsin digestion of the CEX-purified glargine proinsulin produced a mature glargine insulin molecule with intact disulfide bonds as shown by LC-MS analysis.

[0132] To further investigate the precipitation of PN3.15 CPBpro_glargine proinsulin (SEQ ID NO: 62) by acidic conditions, samples of PN3.15 CPBpro_glargine proinsulin prepared by solubilization of the solubilizable complex and elution from a Ni-NTA column with 500 mM imidazole in 1×PBS pH 7.8 as described above were adjusted to the following pH values ​​using 10% formic acid: pH 7.5, 7.2, 7.0, 6.7, 6.5, 6.0, 5.5, and 5.0. The samples were then centrifuged at 14K×g for 15 min at 4° C., and the supernatant was removed and dried by centrifugal evaporation. The pellet and dried supernatant were resuspended in 8 M urea in 1×PBS pH 7.5 and analyzed by polyacrylamide gel electrophoresis under denaturing conditions. At pH values ​​between 7.8 and 7.2, the majority of PN3.15 CPBpro_glargine proinsulin remained soluble. At pH 7.0, approximately equal amounts of PN3.15 CPBpro_glargine proinsulin were observed in the supernatant and pellet. As the pH decreased, an increasing portion of PN3.15 CPBpro_glargine proinsulin was present in the pellet until nearly all precipitated at pH 5.0. The ability to precipitate proteins by changing the pH of the protein solution is useful, for example, for resuspension of proteins in smaller, more concentrated volumes and / or in different buffers. This effect of pH on solubility was also observed for PN3.17 CPBpro_glargine proinsulin and is likely characteristic of other polypeptide gene products that form solubilizable complexes.

[0133] To obtain purified PN3.17 CPBpro_glargine proinsulin (SEQ ID NO: 66) for further analysis, PN3.17 CPBpro_glargine proinsulin was produced by fermentation essentially as described in Example 2B, with lysis of host cells followed by centrifugation at 20K×g for 30 min at 4° C. The pelleted material was solubilized in 8 M urea in 1× phosphate buffered saline (PBS) pH 7.5 with 10 mL of resuspension buffer per gram wet cell weight of harvested host cell weight, vortexed, incubated at room temperature for 20-30 min, followed by a clarifying spin at 4000×g for 5 min at 4° C. The solubilized PN3.17 CPBpro_glargine proinsulin was loaded onto a 5 mL Ni-NTA column. The column was equilibrated using 5 column volumes (CV) of 8M urea in PBS pH 7.5, the sample was loaded, and washed 1 (5CV of 8M urea and 20mM imidazole in PBS pH 7.5), washed 2 (1.25CV of 20mM imidazole in PBS pH 7.5), elution (5CV of 500mM imidazole in PBS pH 7.5), and cleaning (1.25CV 0.2N NaOH, 6CV 20% EtOH). To study the solubility of PN3.17 CPBpro_glargine proinsulin in various buffers, a sample of Ni-NTA purified PN3.17 CPBpro_glargine proinsulin corresponding to 0.5g wet cell weight of harvested host cells was run on a 5mL Zeba™ spin desalting column with 7K molecular weight cut-off (MWCO) (Thermo Fisher Scientific, Waltham, MA). The buffers into which the samples were spun were 500 mM imidazole in PBS pH 7.5, 200 mM imidazole in PBS pH 7.5, PBS pH 7.5, 50 mM EDTA in PBS pH 7.5, and 25 mM L-arginine in 10 mM K phosphate pH 7.5.The Bradford protein assay was used to measure the protein concentration of PN3.17 CPBpro_glargine proinsulin solutions when eluted in 500 mM imidazole in PBS pH 7.5 and after spin desalting into various buffers. The yield of PN3.17 CPBpro_glargine proinsulin in experiments where the starting buffer, 500 mM imidazole in PBS pH 7.5, was replaced with the same buffer, representing the efficiency of the spin desalting procedure, was approximately 80%. The yields of the other samples transferred by spin desalting into various buffers, respectively, ranged from 77% to 91%, which was not significantly different from the yield expected from the spin desalting procedure itself, suggesting that no further loss of PN3.17 CPBpro_glargine proinsulin from precipitation was observed when transferred to different buffers. The desalted PN3.17 CPBpro_glargine proinsulin sample was also analyzed by polyacrylamide gel electrophoresis under reducing and non-reducing conditions, and the shift in electrophoretic mobility of the PN3.17 CPBpro_glargine proinsulin band upon exposure to reducing conditions indicates that PN3.17 CPBpro_glargine proinsulin when eluted from the Ni-NTA column and desalted contained disulfide bonds.

[0134] Example 3 Use of variant propeptides and C-peptides in the production of glargine proinsulin A. Preparation of Glargine Proinsulin Additional glargine proinsulins were prepared (PN3.62, PN3.116, PN3.165, PN3.172, and PN3.185) each having a variant propeptide portion corresponding to either SEQ ID NO:36 or SEQ ID NO:37, and a variant C-peptide corresponding to one of SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 (see Table 4).

[0135] [Table 6]

[0136] A polynucleotide encoding each of the glargine proinsulin polypeptides was inserted downstream of the araBAD promoter in the pSOL expression vector (SEQ ID NO: 42). Each of these expression constructs contained a coding sequence for protein disulfide isomerase (PDI, SEQ ID NO: 41) downstream of the prpBCDE promoter in the pSOL expression vector.

[0137] Each of the expression vectors encoding the glargine proinsulin polypeptides was used to transform E. Coli EB0001 host cells to form: EB0001 (pSOL:PN3.62 proglargin / PDI), EB0001 (pSOL:PN3.116 proglargin / PDI), EB0001 (pSOL:PN3.165 proglargine / PDI), EB0001 (pSOL:PN3.172 proglargine / PDI), and EB0001 (pSOL:PN3.185 proglargine / PDI).

[0138] Each of the five types of host cells was grown in fermentation culture, induced for protein expression, and then harvested, generally as described in Example 2B. Two factors that varied between samples during the fermentation process were whether the fermentation was carried out in a DASbox device or an NLF device, and whether MnCl2 was added or not to the fermentation as a component of Korz trace metals (see Example 2B). These factors are shown for each glargine proinsulin sample that was purified and analyzed, as described in Example 3B.

[0139] B. Purification of Glargine Proinsulin by Direct Solubilization As described in Example 3C, to prepare a highly purified sample of properly folded glargine proinsulin with correctly placed disulfide bonds for the purpose of transversion to mature glargine insulin, the host cells harvested above were subjected to a direct solubilization process after lysis without an initial centrifugation step to separate soluble and insoluble fractions to collect the glargine proinsulin in the form of a solubilizable complex.

[0140] Purified harvest host cell samples are referred to as shown in the list below, noting the fermentor and whether MnCl2 was added (+) or not (-) during fermentation.

[0141] PN3.62A DASbox MnCl2 added (+) PN3.62B NLF MnCl2 absent (-) PN3.62C NLF MnCl2 absent (-) PN3.116 DASbox MnCl2 absent (-) PN3.165A DASbox MnCl2 absent (-) PN3.165B NLF MnCl2 added (+) PN3.165C NLF MnCl2 added (+) PN3.172 DASbox MnCl2 absent (-) PN3.185A DASbox MnCl2 added (+) PN3.185B NLF MnCl2 added (+) PN3.185C NLF MnCl2 added (+) For lysis, the "main sample group" includes PN3.62A, PN3.62B, and PN3.62C; PN3.116; PN3.165A; PN3.172; and PN3.185A. For the main sample group, harvested host cells were resuspended in 7M urea, 50 mM Tris pH 8 at a 10-fold dilution relative to the fermentation culture volume. Additional samples (PN3.165B and PN3.165C, and PN3.185B and PN3.185C) were resuspended in 7M urea, 2.5 mM L-Cys, 50 mM Tris pH 9.5 at a 2-fold dilution relative to the fermentation culture volume. All samples were homogenized at 8,000 psi a total of five times to lyse the cells. Lysates were diluted 3.5-fold into 50 mM Tris pH 8 (main samples) or 2.5 mL L-Cys, 50 mM Tris pH 9.5 (PN3.165B and PN3.165C, and PN3.185B and PN3.185C) so that all samples were in 2 M urea solution. All samples except PN3.165C and PN3.185C were incubated at 16° C. with shaking at 120 RPM for 48-72 hours, or 24 hours for PN3.165C and PN3.185C.

[0142] After incubation, all lysate samples were clarified by centrifugation at 3300×g, the soluble lysate was filtered through a 0.45 microliter polyethersulfone (PES) membrane, collected, and imidazole was added to the lysate for a final concentration of 10 mM for purification using immobilized metal affinity chromatography (IMAC). The centrifugation step in the clarification can also be performed at 7000-20,000×g for 30-60 minutes, and the soluble lysate can also be filtered by glass fiber filtration (retention of 0.7 micrometer particles in the liquid). Additives to the clarified lysate to prevent non-specific binding during IMAC can include 10 mM-20 mM imidazole and / or 0 mM-300 mM NaCl.

[0143] The IMAC column was equilibrated with 2-4 column volumes (CV) of 7 M urea, 0.3 M NaCl, 10 mM imidazole, 25 mM Tris pH 8, followed by washing with 2-4 CV of 0.1 M NaCl, 40 mM Tris pH 10. For one set of samples (PN3.62A and PN3.62B, PN3.165A, PN3.172, and PN3.185A), each sample was loaded onto a Ni Sepharose Fast Flow column (GE Healthcare Life Sciences, Pittsburgh, PA). For the second set of samples (PN3.62C, PN3.116, PN3.165B and PN3.165C, and PN3.185B and PN3.185C), each sample was loaded onto a Ni HisTrap High Performance column (GE Healthcare Life Sciences, Pittsburgh, PA). Samples were loaded at the equivalent of 0.5 mL to 1 mL of fermentation culture volume per mL of resin. All samples were eluted using 2-4 CVs of 0.5 M imidazole, 40 mM Tris, 0.1 M NaCl pH 10. The column was cleaned in place with 2 CVs of 0.5 M NaOH and stripped with 7 M urea, 0.3 M NaCl, 0.5 M imidazole, and 25 mM Tris pH 8.

[0144] After IMAC, samples were concentrated and desalted. For one set of samples (PN3.62A, PN3.165A, PN3.172, and PN3.185A), each sample was concentrated using a 3 kDa molecular weight cut-off (MWCO) Amicon® centrifugal concentrator (Sigma-Aldrich, St. Louis, MO), and distilled water was added to return each sample to its starting volume and exchanged two to three times using the same centrifugal concentrator. For the other sample groups (PN3.62B and PN3.62C, PN3.116, PN3.165B and PN3.165C, and PN3.185B and PN3.185C), each sample was concentrated by tangential flow filtration and discontinuous diafiltration in a 3-kDa MWCO Vivaflow 50 tangential concentrator (Sartorius, Gottingen, Germany) until the volume in the feed reservoir was approximately one-tenth of the starting volume, followed by the addition of distilled water to return each sample to its starting volume, and the process was repeated two to three times.

[0145] C. Transversion of glargine proinsulin to mature glargine insulin Experiments were carried out to identify optimal conditions for the transversion of various forms of glargine proinsulin to mature glargine insulin ("B32 glargine") by digestion with trypsin. Following the processing protocol, the results were analyzed by solid phase extraction mass spectrometry (SPE-MS).

[0146] The SPE-MS parameters were as follows:

[0147] [Table 7]

[0148] According to the SPE-MS results, the conditions for each glargine proinsulin that produce optimal B32 glargine were selected, and the corresponding samples were run by quadrupole time-of-flight mass spectrometry (QTOF) liquid chromatography mass spectrometry (LCMS). The authentic standard of USP glargine was used for external standard curve to quantify the percentage of material that underwent transversion. The percentage of transversion (transversion%) is equal to the concentration of B32 glargine as determined by A280 LC integration against USP glargine standard divided by the starting concentration of glargine proinsulin as determined by A280 LC integration against amino acid analysis standard multiplied by 100%. The integration at A214 against the same glargine external standard curve and by the extracted ion chromatogram was consistent with the integration at A280.

[0149] The QTOF-LCMS parameters were as follows:

[0150] [Table 8]

[0151] The following gradient table was used:

[0152] [Table 9]

[0153] In experiment 1, 1.5 microliters of each glargine proinsulin sample was mixed with 5 microliters of NiCl2 solutions of various concentrations and 5 microliters of trypsin solution (4.5 g / L trypsin in 120 mM Tris 300 mM NaCl pH 9, 15 mM CaCl2). The combined volumes were spun at 500×g for 1 minute and then incubated at room temperature with shaking at 100 RPM for variable amounts of time, followed by the addition of 8 M urea with 1% formic acid (pH 3-3.5) to stop the reaction.

[0154] In experiment 2, various concentrations of NiCl2, FeCl2, NaCl, and CaCl2 were added to the trypsin reaction mixture and the reactions were carried out for various amounts of time but were otherwise generally as described above.

[0155] In experiment 3, various concentrations of NaCl or Tris buffer were added to a trypsin reaction mixture that had a fixed amount of 1.5 g / L trypsin, 50 mM CaCl2, and 7 micromolar NiCl2, and the trypsin mixture was run for various amounts of time but was otherwise generally as described in experiment 1.

[0156] In experiment 4, various concentrations of NaCl and reaction times were tested in a trypsin reaction mixture containing 1.5 g / L trypsin, 50 mM CaCl2, 120 mM Tris, 300 mM NiCl2.

[0157] In experiment 5, two trypsin reaction conditions containing 1.5 g / L trypsin were tested at various lengths of reaction time. Each of the conditions produced comparable maximum results, as shown in the table below.

[0158] In experiment 6, various concentrations of NaCl and pH were tested for various amounts of time in a trypsin reaction mixture containing 1.5 g / L trypsin and 7 micromolar NiCl2.

[0159] For each experiment, the best reaction conditions for each glargine proinsulin of interest tested are shown below along with the percentage of transversion.

[0160] [Table 10]

[0161] These results demonstrate that high transversion frequencies can be obtained for the variant glargine proinsulin polypeptides of the present invention using the methods disclosed herein.

[0162] Example 4 Determining the solubility of expression products: a method to detect inclusion bodies When the methods of the invention are used to express a gene product in the cytoplasm of a host cell, the following procedure can be used to determine the extent to which the gene product is produced in the cell in a soluble form.

[0163] The most straightforward approach is to lyse the cells using any effective method, such as enzymatic lysis with lysozyme or by cell disruption using a microfluidizer, as described in more detail in Example 1. A sample of the cell lysate can be retained as a measure of the total soluble and insoluble gene products produced by the host cells. The lysed cells are then centrifuged at 20,000×g for 15 minutes at room temperature to separate and remove the insoluble fraction as a pellet, and the soluble fraction (supernatant) is collected. The amount of total gene product present in the cell lysate minus the amount of soluble gene expression recovered in the supernatant represents the total amount of insoluble gene product present in the pellet. Using the methods for solubilization described herein, it can be determined what portion of the insoluble fraction in the pellet is solubilizable. Any method that can be used to detect the gene product, preferably to specifically and quantifiably detect the gene product in each fraction, such as ELISA or capillary electrophoresis Western blot, is used to compare the amounts present in the soluble and insoluble fractions. To test the validity of this approach, endogenous host cell proteins that are known to be soluble and present only in the cytoplasm of the host cell are detected in both the soluble and insoluble fractions to determine whether the lysis and fractionation method captures detectable amounts of soluble cytoplasmic products in the insoluble fraction.

[0164] It is also possible to directly assess whether cells contain inclusion bodies. Inclusion bodies can be collected by centrifugation of lysed host cells, stained with dyes such as Congo Red, and visualized using bright field or cross-polarized light microscopy at medium (10x) magnification (Wang et al., "Bacterial inclusion bodies contain amyloid-like structure", PLoS Biol 2008 Aug 5;6(8):e195; doi:10.1371 / journal.pbio.0060195). Such inclusion bodies can also be resolubilized (Singh and Panda, "Solubilization and refolding of bacterial inclusion body proteins", J Biosci Bioeng 2005 Apr;99(4):303-310; Review) and specific binding assays or other methods of protein identification can be used to determine, for example, whether they contain specific gene products. Inclusion bodies can be distinguished from the solubilizable complexes described herein in that the majority of the gene product recovered from inclusion bodies by solubilization is not active or in a properly folded form, and at least one further refolding step is required to obtain the majority of the gene product that is active and / or properly folded.

[0165] Example 5 Determination of further methods for solubilization of solubilizable gene product complexes The buffer used to solubilize the gene product complex produced by the method of the present invention may contain several different types of components, as described below. To optimize the solubilization of any gene product of interest, experiments can be undertaken to identify the most effective combination of solubilization buffer components. Initial experiments are performed to identify which combinations of buffer components can be easily prepared in the laboratory using commercially available compounds. Once a test buffer is prepared, it can be used in solubilization experiments with the gene product complex of interest and, optionally, with a control gene product complex that is known to be solubilizable to different degrees in a reference solubilization buffer. Examples of solubilization protocols for use with gene product complexes, such as those described in Examples 1 and 2, are provided herein.

[0166] Solubilization buffer contains: The following description of the buffer components outlined in Table 5 is intended to provide examples of different types of components that can be used in combination in a solubilization buffer, without limitation on possible buffer components or combinations thereof. For example, chaotropic agents include n-butanol, ethanol, guanidinium chloride, guanidinium hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and / or urea. One or more compounds of each type of buffer component can be used in combination with one or more compounds of any or all of the other component types in preparing a solubilization buffer to be tested for effectiveness in solubilizing gene product complexes. The buffer concentrations shown in Table 5 include ranges of concentrations that can be tested for effectiveness, as well as specific examples of those concentrations.

[0167] In preparations of the gene product of interest that preserve the conformation of the gene product properly folded, that preserve properly formed disulfide bonds, and / or that preserve protein activity, reducing agents are not included in the solubilization buffer. However, certain analytical assays, such as capillary electrophoresis Western blots (see Example 6), are preferably performed with solubilized gene product samples in the reduced state. To prepare samples for such assays, reducing agents (e.g., DTE (dithioerythritol), DTT (dithiothreitol), and / or TCEP (tris(2-carboxyethyl)phosphine)) can be included in the buffer, for example at a concentration of 10 mM, or up to a concentration of 100 mM.

[0168] [Table 11]

[0169] Example 6 Characterization by capillary electrophoresis Western blot As described below, using soluble or solubilized protein gene products as examples, gene products can be detected and quantified by capillary electrophoresis Western blot on the WES system (ProteinSimple, San Jose, Calif.) according to the manufacturer's instructions. Soluble protein extracts are loaded into a set of capillaries and proteins are electrophoretically separated by size. Proteins of interest in the sample are detected with a primary antibody specific for that protein and incubated with an HRP-conjugated secondary antibody, such as a goat anti-human or anti-mouse secondary antibody, that recognizes the heavy and / or light chains of the primary antibody. Detection of the presence of the HRP-conjugated secondary antibody is achieved by addition of a chemiluminescent substrate to the capillary and direct capture of the light emitted during the enzyme-catalyzed reaction. Molecular weight estimates are calculated using a standard curve generated using six biotinylated proteins ranging from 12k to 230kDa for each run. Fluorescence standards are included in the sample loading buffer to provide each sample with an internal standard that is used to align each sample with the molecular weight standards.

[0170] To determine the amount of protein present at a given molecular weight, a known amount of a protein standard preparation of interest is run through some of the capillaries and detected using the same primary and secondary antibodies as for the experimental samples. A serial dilution of a standard of a protein of interest with a known concentration, such as a commercial protein standard, is prepared, for example, starting from 10 micrograms / mL and diluting down to 1.0 nanograms / mL. Approximately five WES system capillaries are used to run the serial dilution. For each protein band in both the experimental capillary and the serial dilution capillary, a curve is generated by the WES system software that represents the chemiluminescence intensity of the protein band, the area under each curve is evaluated, and a standard curve of these areas is plotted for the protein band in the serial dilution capillary. To determine the concentration of the experimental sample, the area under each curve that represents the chemiluminescence intensity of the experimental sample can be compared to the standard curve generated for a sample of known concentration.

[0171] Example 7 Determination of yield and recovery of gene products produced using the solubilization and purification methods of the invention The methods described below can be used to calculate the amount of gene product recovered at various stages of the solubilization and purification process when compared to the total amount present in the cell lysate.

[0172] A standard sample of the gene product is required. This can be a commercially available sample of the gene product with known concentration, or an amino acid analyzed (AAA) fully purified sample of the gene product. Cell lysates from host cell cultures, such as fermentation broth, are prepared from the host cell cultures at known levels of dilution. SDS-PAGE gels, such as 4%-12% gels, are prepared and a set of serial dilutions of samples of both the cell lysates and standards of gene products are run on the SDS-PAGE gel under reducing conditions and then stained with SimplyBlue SafeStain (Thermo Fisher Scientific, Waltham, MA). The use of reducing conditions is necessary to be able to measure the total amount of gene product in the cell lysates. Densitometric measurements of the gene product bands on the SDS-PAGE gel are performed for each of the samples and curves based on the densitometric data are plotted as follows:

[0173] For gene product standards, the band intensity of each standard's gene product band on the gel is plotted on the y-axis, and sample volume (in microliters) is plotted on the x-axis. The volume of standard solution present in the least diluted sample (e.g., 6 microliters) is plotted against sample volume. For each serially diluted standard, the volume is plotted as the volume of standard in the least diluted sample (e.g., 6 microliters) divided by each dilution factor (e.g., 2). For these values, the sample volumes (in microliters) are 6, 3, 1.5, 0.75, etc. A best-fit linear standard curve is generated based on the plotted data, which can be represented by the equation y=m(standard)x+k, where m is the slope of the standard curve and k is the y-intercept.

[0174] To determine the yield (or titer, in g / L) of gene product present in the cell lysate, the band intensity of the gene product band for each cell lysate sample is plotted on the y-axis against the sample volume on the x-axis in the same manner as for the standard samples described above. A best-fit linear curve for the cell lysate samples is also generated in the form y=m(experimental)x+k. To calculate the yield of gene product in the cell lysate, the slope for the cell lysate sample is divided by the slope for the standard samples, then multiplied by the concentration of the standard sample solution and by the degree to which the cell lysate sample was diluted into the host cell culture fluid (e.g., 100 for a 100-fold dilution).

[0175] To illustrate the use of this method, the following example is the determination of the total gene product yield of PN3.172 proglargin from a fermentation process. A highly purified and amino acid analyzed (AAA) standard sample of PN3.172 was prepared, which has a concentration of 0.266 micrograms / microliter, which is equivalent to 0.266 g / L. The PN3.172 proglargin polypeptide was expressed and dissolved in a host cell fermentation broth, generally according to the method described in Example 2B and Example 3A above. The analyzed cell lysate was diluted 80 times into the host cell fermentation broth, so the dilution factor is 80. Both the AAA PN3.172 standard and PN3.172 cell lysate samples were prepared as a set of 1.25-fold serial dilutions with volumes of 6.0, 4.8, 3.8, 3.1, and 2.5 microliters, and these samples were run on a 4%-12% SDS-PAGE gel under reducing conditions and then stained with SimplyBlue SafeStain (Thermo Fisher Scientific, Waltham, MA). Band densitometry was performed for the AAA PN3.172 standard and the PN3.172 cell lysate samples, respectively, and a best-fit linear curve was plotted. For the AAA PN3.172 standard, the curve was y=93,899x-129,917, and the slope or m(standard) was equal to 93,899. For PN3.172 cell lysate, the curve was y=72,614x-228,763 and the slope or m(experimental) was equal to 72,614. The calculation of the yield of PN3.172 in the cell lysate was: (m(experimental) / m(standard)) × dilution factor × concentration of standard = (72,614 / 93,899) × 80 × 0.266 g / L = 16.5 g / L. In further experiments, the yield of proinsulin gene product in cell lysates ranged from 5 g / L to 20 g / L.

[0176] The optical density (e.g., OD) of the host cell growth culture 600 ) is measured upon lysis, it is also possible to calculate the yield of gene product as g / L / OD by dividing the yield in g / L as calculated above by the optical density.

[0177] This method of calculating yield can also be used at later steps in the solubilization and purification process. For example, an SDS-PAGE gel can be run with a standard sample and an experimental sample solubilized by one of the methods described herein to determine the yield after solubilization of the experimental sample. This yield calculation method can also be used to determine the yield of gene products after purification by column chromatography, such as Ni-IMAC purification, preferably using RP-UPLC analysis of the standard and experimental sample peaks. When using RP-UPLC analysis, the calculated area under the chromatogram peak(s) at the expected retention time (in seconds) for the desired gene product is used in much the same way as the band density in the yield calculation method described above. Serial dilutions of the standard sample are made and samples with known amounts of gene product are run on the chromatography column one at a time, and the area under the gene product peak is calculated and a standard curve plotted. For an experimental sample, the calculated area under the chromatogram peak(s) at the expected retention time (seconds) from any single run through RP-UPLC can be compared to a standard curve calculated from serial dilutions of the standard sample to obtain the quantity of gene product in the experimental sample.

[0178] The percentage of gene product recovered between successive process steps can be determined by dividing the yield at the later step by the yield at the earlier process step and multiplying by 100%. A purification process was performed on PN3.172 Proglargin, where the yield at the cell lysis step was determined using the method described above, and PN3.172 Proglargin was solubilized by centrifuging the solubilizable complex to form a pellet and then solubilizing PN3.172 Proglargin from the pellet (as in Examples 1 and 2) or by a direct solubilization method (as in Example 3). The yield of soluble PN3.172 Proglargin was determined using the method described above, and the percent recovery of soluble PN3.172 Proglargin was calculated for each solubilization method. The "pellet and solubilize" method of Example 1 and Example 2 resulted in PN3.172 proglargine with a recovery of 84.7%, with the recovered material being 75.3% pure PN3.172 proglargine protein as determined by RP-UPLC analysis using a BEH 300A 1.7 μm 2.1×150 mm C4 protein column (product number 186006549, Waters, Milford, Massachusetts). The "direct solubilize" method of Example 3 resulted in a comparable recovery of 81.4% PN3.172 proglargine, with the recovered material being 30.4% pure PN3.172 proglargine protein as determined by RP-UPLC analysis. Subsequent purification of PN3.172 proglargin prepared by each solubilization method using a Ni-IMAC column and a buffer exchange step resulted in a total recovery of 70.8% for the "pelleted and solubilized" PN3.172 proglargin with a purity of 98.2% and a total recovery of 71.0% for the "directly solubilized" PN3.172 proglargin with a purity of 94.7%. This experiment demonstrated that the "pelleted and solubilized" method of Example 1 and Example 2 recovers as much gene product as the direct solubilization method of Example 3 and produces higher purity material both before and after the subsequent chromatography step.

[0179] Example 8 Characterization of disulfide bonds present in the expression products The number and location of disulfide bonds in protein expression products can be determined by digesting the protein with a protease such as trypsin under non-reducing conditions and subjecting the resulting peptide fragments to mass spectrometry (MS) combining a series of electron transfer dissociation (ETD) and collision-induced dissociation (CID) MS steps (MS2, MS3) (Nili et al., "Defining the disulfide bonds of insulin-like growth factor-binding protein-5 by tandem mass spectrometry with electron transfer dissociation and collision-induced dissociation", J Biol Chem 2012 Jan 6;287(2):1510-1519;Epub 2011 Nov 22).

[0180] Digestion of the expressed proteins. To prevent rearrangement of disulfide bonds, any free cysteine ​​residues are first blocked by alkylation: the expressed proteins are incubated in a buffer with 4M urea with the alkylating agent iodoacetamide (5 mM) for 30 minutes at 20° C. with shaking, protected from light. Alternatively, and preferably, NEM is used as the alkylating agent and trypsin proteolysis is performed under denaturing conditions (6M GuaHCl) in combination with reduction / alkylation. After alkylation, the expressed proteins are separated by non-reducing SDS-PAGE using precast gels. Alternatively, the expressed proteins are incubated in the gel after electrophoresis with iodoacetamide or NEM or without it as a control. The protein bands are stained, destained with double deionized water, excised and incubated twice in 500 microliters of 50 mM ammonium bicarbonate, 50% (v / v) acetonitrile with shaking, for 30 minutes at 20° C. Protein samples are dehydrated in 100% acetonitrile for 2 min, dried by vacuum centrifugation, and rehydrated with 10 mg / ml trypsin or chymotrypsin in a buffer containing 50 mM ammonium bicarbonate and 5 mM calcium chloride for 15 min on ice. Excess buffer is removed and replaced with 50 microliters of the same buffer without enzyme, followed by incubation with shaking for 16 h at 37° C. or 20° C. for trypsin and chymotrypsin, respectively. Digestion is stopped by the addition of 3 microliters of 88% formic acid, and after brief vortexing, the supernatant is removed and stored at −20° C. until analysis. In cases where trypsinolysis provides insufficient sequence coverage (less than 75%), alternative protein fragmentation methods (LysC, Glu-C, or CNBr) are used. The use of the reducing agent TCEP (tris(2-carboxyethyl)phosphine) in the presence of NEM under acidic conditions offers the opportunity to obtain fragments with partially intact disulfide bonds. The disulfide intact digestion maps are compared with the reduced (DTT or TCEP) digestion maps.

[0181] Disulfide bond localization by mass spectrometry. Peptides are injected onto a 1 mm x 8 mm trap column (Michrom BioResources, Auburn, CA) at 20 microliters / min in a mobile phase containing 0.1% formic acid. The trap cartridge is then placed alongside a 0.5 mm x 250 mm column containing 5 mm of Zorbax SB-C18 stationary phase (Agilent Technologies, Santa Clara, CA) and the peptides are separated using a 2% to 30% acetonitrile gradient over 90 min at 10 microliters / min using an 1100 series capillary HPLC (Agilent Technologies) or using a C18 column suitable for UPLC. Peptides are analyzed using an LTQ Velos linear ion trap with an ETD source (Thermo Fisher Scientific, Waltham, MA). Electrospray ionization is performed using a Captive Spray source (Michrom Bioresources) or preferably a 3.0 kV uncoated drawn fused silica emitter (New Objective, Woburn, Massachusetts). Alternatively, analysis of medium-sized proteolytic fragments is performed using a Thermo LTQ-FT MS (7 Tesla) instrument or a Synapt G2-Si quadrupole traveling wave ion mobility time-of-flight (ToF) mass spectrometer (WATERS, Milford, Massachusetts). Preferably, peptides are analyzed using an Orbitrap Fusion™ Tribrid™ mass spectrometer (Thermo Fisher Scientific). Disulfide-bonded peptides have a charge state of +4 or higher after trypsinization due to the presence of two basic residues (arginine or lysine) at the two N- and carboxy-termini. These disulfide-bonded peptides are preferentially isolated by the Orbitrap Fusion™ instrument so that the disulfide bonds can be disrupted using ETD fragmentation.After the survey MS scan, seven data-dependent scans consisting of CID and ETD MS2 scans are performed on the most intense ions in the survey scan, followed by five MS3 CID scans on the first through fifth most intense ions in the ETD MS2 scan. The CID scan uses a normalized collision energy of 35, and the ETD scan uses an activation time of 100 ms with additional activation enabled. The minimum signal to initiate the MS2 CID and ETD scans is 10,000, the minimum signal to initiate the MS3 CID scan is 1000, and the isolation width for all MS2 and MS3 scans is 3.0 m / z. The dynamic exclusion feature of the software is enabled with an iteration count of 1, an exclusion list size of 100, and an exclusion duration of 30 seconds. An inclusion list for target-specific crosslinked species for the collection of ETD MS2 scans is used. Separation data files for the MS2 and MS3 scans are generated by Bioworks 3.3 (Thermo Fisher Scientific) using ZSA charge state analysis. Matching of MS2 and MS3 scans to peptide sequences is performed by Sequest (V27, Rev 12, Thermo Fisher Scientific). Analysis is performed with no enzyme specificity, parent ion mass tolerance of 2.5, fragment mass tolerance of 1.0, and a drift mass of +16 for oxidized methionine residues. Results are then analyzed using the program Scaffold (V3_00_08, Proteome Software, Portland, OR) using minimum peptide and protein probabilities of 95% and 99%. Data interpretation software tools also include Proteome Discoverer™ 2.0 with Disulfinator node (Thermo Fisher Scientific). Peptides from the MS3 results are sorted by scan number, and cysteine ​​containing peptides are identified from the group of MS3 scans generated from the five most intense ions observed in the ETD MS2 scan.The identity of the cysteine ​​peptides involved in the disulfide bonded species is further confirmed by manual inspection of the parent ion masses observed in the survey and ETD MS2 scans.

[0182] Example 9 Solubilization and purification of expression products from bacterial cell periplasm, from spheroplasts, and from whole cells The solubilization and purification methods of the present invention can be used in the production of gene products that accumulate in different compartments of a cell, such as the cytoplasm or periplasm. Host cells, such as E. coli or S. cerevisiae, have an outer cell membrane or wall and can form spheroplasts when the outer membrane or wall is removed. Expressed proteins made in such hosts can be purified specifically from the periplasm, or from spheroplasts, or from whole cells using the methods described below (Schoenfeld, "Convenient, rapid enrichment of periplasmic and spheroplasmic protein fractions using the newPeriPreps™ Periplasting Kit", Epicentre Forum 1998 5(1):5; available at epibio.com / docs / default-source / forum-archive / forum-05-1---convenient-rapid-enrichment-of-periplasmic-and-spheroplasmic-protein-fractions-using-the-new-peripreps-periplasting-kit.pdf). The methods are designed for E. coli and other Gram-negative bacteria, but the general approach can be modified for other host cells, such as S. cerevisiae.

[0183] 1. Bacterial host cell cultures are grown only to late log phase because older cell cultures in stationary phase usually show some resistance to lysozyme treatment. If recombinant protein expression is in excess, the cells may lyse early, and therefore the cell cultures are not grown in rich media or at higher growth temperatures that may induce excess protein synthesis. Protein expression is then induced and the cells should be in log phase or early stationary phase.

[0184] 2. Pellet the cell culture by centrifugation at a minimum of 1,000 x g for 10 minutes at room temperature. Note: Cells must be fresh and not frozen. Determine the wet weight of the cell pellet to calculate the amounts of reagents needed for this protocol.

[0185] 3. Thoroughly resuspend cells in a minimum of 2 ml of PeriPreps Periplasting Buffer (200 mM Tris-HCl pH 7.5, 20% sucrose, 1 mM EDTA, and 30 U / microliter Ready-Lyse Lysozyme) per gram of cells by vortex mixing or pipetting until the cell suspension is homogenous. Note: Excessive agitation may cause premature lysis of spheroplasts, resulting in contamination of the periplasmic fraction with cytoplasmic proteins.

[0186] 4. Incubate at room temperature for 5 minutes. Ready-Lyse Lysozyme is optimally activated at room temperature. Lysis at lower temperatures (0°C to 4°C) requires additional incubation time, which is extended by 2-4 times.

[0187] 5. Add 3 ml of purified water at 4° C. per gram of original cell pellet weight (step 2) and mix by inversion. 6. Incubate on ice for 10 minutes.

[0188] 7. Pellet the lysed cells by centrifugation at a minimum of 4,000 x g for 15 minutes at room temperature. 8. Transfer the supernatant containing the periplasmic fraction to a clean tube.

[0189] 9. OmniCleave Endonuclease is optionally added to the PeriPreps Lysis Buffer to degrade contaminating nucleic acids. Although the inclusion of nuclease generally improves protein yield and ease of handling of the lysate, the addition of nuclease may be undesirable in some cases, e.g., the use of nuclease should be avoided if residual nuclease activity or transient exposure to magnesium cofactor interferes with subsequent assays or use of the purified protein. The addition of EDTA to the lysate to inactivate OmniCleave Endonuclease may similarly interfere with subsequent assays or use of the purified protein. If nuclease is to be added, dilute 2 microliters of OmniCleave Endonuclease and 10 microliters of 1.0 M MgCl2 in PeriPreps Lysis Buffer (10 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM EDTA, and 0.1% deoxycholate) up to 1 ml per milliliter of Lysis Buffer required in step 10.

[0190] 10. Resuspend the pellet in 5 ml of PeriPreps Lysis Buffer per gram of original cell pellet weight. 11. Incubate the pellet at room temperature for 10 minutes (continue incubation until OmniCleave Endonuclease activity, if present, has caused a significant reduction in viscosity and the cell suspension has the consistency of water).

[0191] 12. Pellet cell debris by centrifugation at a minimum of 4,000 x g for 15 minutes at 4°C. 13. Transfer the supernatant containing the spheroplast fraction to a clean tube.

[0192] 14. When OmniCleave Endonuclease is added to the PeriPreps Lysis Buffer, 20 microliters of 500 mM EDTA is added per milliliter of the resulting spheroplast fraction to chelate magnesium (final concentration of EDTA in the lysate is 10 mM). After hydrolysis of nucleic acids with OmniCleave Endonuclease, the lysate may contain significant amounts of mono- or oligonucleotides. The presence of these degradation products may affect the further processing of the lysate: for example, nucleotides may reduce the binding capacity of anion exchange resins by interacting with the resin.

[0193] The above protocol can be used to prepare total cellular protein with the following modifications: the cells pelleted in step 2 can be fresh or frozen, in step 4, the cells are incubated for 15 minutes, steps 5-8 are omitted, and in step 10, 3 ml of PeriPreps Lysis Buffer is added per gram of original cell pellet weight.

[0194] Following preparation of periplasmic, or spheroplast, or whole cell protein samples, the samples may be analyzed by any of a number of protein characterization and / or quantification methods. In one example, successful fractionation of periplasmic and spheroplast proteins is confirmed by analyzing aliquots of both the periplasmic and spheroplast fractions by SDS-PAGE (2 microliters of each fraction is generally sufficient for visualization by staining with Coomassie Brilliant Blue). The presence of a unique protein or enrichment of a particular protein in a given fraction indicates successful fractionation. For example, if the host cell contains a high copy number plasmid with an ampicillin resistance marker, the presence of β-lactamase (31.5 kDa) primarily in the periplasmic fraction indicates successful fractionation. Other E. coli proteins found in the periplasmic space include alkaline phosphatase (50 kDa) and elongation factor Tu (43 kDa). The amount of protein found in a given fraction can be quantified using any of a number of methods (e.g., SDS-PAGE and densitometric analysis of stained or labeled protein bands, scintillation counting of radiolabeled proteins, enzyme-linked immunosorbent assay (ELISA), or scintillation proximity assay, among others). Comparing the amount of protein found in the periplasmic fraction as compared to the spheroplast fraction indicates the extent to which the protein has been exported from the cytoplasm into the periplasm.

[0195] Example 10 Titration of expression by varying inducer concentration To optimize production of gene products using the expression systems of the present invention, the concentration of inducer can be independently adjusted or titrated. Host cells containing an expression construct including an inducible promoter (such as an L-arabinose-inducible, propionate-inducible, L-rhamnose-inducible, or D-xylose-inducible promoter) are grown in small volume titrations in M9 minimal medium containing the appropriate antibiotic to a desired density (e.g., OD 600After growth to approximately OD 0.5), the cells are aliquoted into small volumes of M9 minimal medium, optionally prepared without a carbon source such as glycerol, with the appropriate antibiotic and various concentrations of each inducer. Small volume titrations can be performed in 200-500 ml shake flasks. The concentration of L-arabinose, L-rhamnose, or D-xylose required to induce expression is typically less than 0.02% (and often much less) per OD unit of cells. In the titration experiments, the tested concentrations of L-arabinose were 2% to 1.5%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.005%, 0.002%, 0.001%, 0.0005%, 0.0002%, 0.0001%, and 0.0002% per unit of cell OD. The concentration of 66.61 micromolar L-arabinose corresponds to 0.001% L-arabinose. An alternative titration experiment for L-arabinose, L-rhamnose, or D-xylose is to test the following concentrations, expressed in terms of molar concentrations: 250 mM, 100 mM, 50 mM, 25 mM, 10 mM, 5 mM, 2.5 mM, 1.0 mM, 500 micromolar, 250 micromolar, 100 micromolar, 75 micromolar, 50 micromolar, 25 micromolar, 10 micromolar, 5.0 micromolar, 2.5 micromolar, 1.0 micromolar, 500 nM, 250 nM, 100 nM, 50 nM, 25 nM, 10 nM, 5.0 nM, 2.5 nM, 1.0 nM, 500 pM, 250 pM, 100 pM, 50 pM, 25 pM, 10 pM, 5.0 pM, 2.5 pM, and 1.0 pM, all per OD unit of cells.For propionate, concentrations to be tested can range from 1M to 750 mM, 500 mM, 250 mM, 100 mM, 75 mM, 50 mM, 25 mM, 10 mM, 5 mM, 1 mM, 750 micromolar, 500 micromolar, 250 micromolar, 100 micromolar, 50 micromolar, 25 micromolar, 10 micromolar, 5.0 micromolar, 2.5 micromolar, 1.0 micromolar, 500 nM, 250 nM, 100 nM, 50 nM, 25 nM, 10 nM, 5.0 nM, 2.5 nM, and 1.0 nM, all per OD unit of cells.

[0196] For each concentration "x" of L-arabinose (or L-rhamnose or D-xylose) to be tested, the concentration of a different inducer, such as propionate, added to each tube containing the first inducer at concentration "x" is varied in each series of samples. Alternatively, a titration experiment can start with a "standard" combination of inducer concentrations, which for host cells with reduced levels of at least one gene function encoding a protein that metabolizes the inducer, is 0.0015% (100 micromoles) of either L-arabinose, L-rhamnose, or D-xylose per OD unit of cells, and / or 100 micromoles of propionate per OD unit of cells. For host cells in which the inducer-metabolizing proteins are functional, the "standard" combination of inducer concentrations is 0.0033% (220 micromolar) of either L-arabinose, L-rhamnose, or D-xylose per OD unit of cells, and / or 83 mM propionate per OD unit of cells. Further combinations of inducer concentrations different from the "standard" combination can be tested in a series of titration experiments, and the results from earlier experiments can be used to "fine-tune" the inducer concentrations used in later experiments. Similar titration experiments can be performed with any combination of inducers used in the expression system of the present invention, including but not limited to L-arabinose, propionate, L-rhamnose, and D-xylose. After 6 hours of growth in the presence of inducer, the cells are pelleted, the desired product is extracted from the cells, and the yield of product per mass value of cells is determined by a quantitative immunological assay such as ELISA, or by purification of the product and quantification by UV absorbance at 280 nm.

[0197] It is also possible to titrate inducer concentrations using high-throughput assays, where the protein to be expressed is engineered to contain a fluorescent protein moiety, such as that provided by mKate2 red fluorescent protein (Evrogen, Moscow, Russia) or enhanced green fluorescent protein from Aequorea victoria and Bacillus cereus. Another approach to determine the amount and activity of gene products produced by different concentrations of inducer in high-throughput titration experiments is to use sensors capable of measuring binding interactions of biomolecules, such as sensors that detect surface plasmon resonance or bio-layer interferometry (BLI) (e.g., Octet® QK system, from forteBIO, Menlo Park, CA). If an antibody is available that binds with sufficient specificity to the expressed gene product, the gene product can be detected and quantified using capillary electrophoresis Western blots, such as those implemented in a WES system as described in Example 6.

[0198] Example 11 Determining Polynucleotide or Amino Acid Sequence Similarity Percentage of polynucleotide or amino acid sequence identity is defined as the number of aligned symbols, i.e., nucleotides or amino acids, that are identical in both aligned sequences, divided by the total number of symbols in the alignment of the two sequences, including gaps. The degree of similarity (percentage identity) between two sequences can be determined by aligning the sequences using the Needleman and Wunsch global alignment method (J. Mol. Biol. 48:443, 1970), as implemented by the National Center for Biotechnology Information (NCBI) in the Needleman-Wunsch global sequence alignment tool, available through the website blast.ncbi.nlm.nih.gov / Blast.cgi. In one embodiment, the Needleman and Wunsch alignment parameters are set to default values ​​(match / mismatch score 2 and -3, respectively, and gap cost 5 and 2 for Existence and Extension, respectively). Sequences may be aligned using the Basic Local Alignment Search Tool, as implemented by NCBI using the default parameter settings described at the blast.ncbi.nlm.nih.gov / Blast.cgi website, or other programs used by those skilled in the art of sequence comparison, such as the BLAST (registered trademark) program (Altschul et al., "Basic local alignment search tool", J Mol Biol 1990 Oct 5;215(3):403-410).The BLAST algorithm has multiple optional parameters, including two that can be used as follows: (A) the inclusion of a filter to mask segments of the query sequence that have low compositional complexity or segments that are preferably not utilized or consist of short periodic internal repeats that are set to "off", and (B) a statistical significance threshold for reporting matches against database sequences, called the "Expect" or E-score (the expected probability of a match being found simply by chance; if the statistical significance ascribed to a match is greater than this E-score threshold, the match will not be reported). If this "Expect" or E-score value is adjusted from the default value (10), the preferred threshold is 0.5, or increasing preference in the order of 0.25, 0.1, 0.05, 0.01, 0.001, 0.0001, 0.00001, and 0.000001.

[0199] In carrying out the present invention, many conventional techniques in molecular biology, microbiology, and recombinant DNA technology are optionally used. Such conventional techniques relate to vectors, host cells, and recombinant methods. These techniques are well known and are described, for example, in Berger and Kimmel, Guide to Molecular Cloning Techniques, Methods in Enzymology volume 152 Academic Press, Mc, San Diego, CA; Sambrook et al., Molecular Cloning-A Laboratory Manual (3rd Ed.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 2000, and Current Protocols in Molecular Biology, FMAusubel et al., eds., Current Protocols, a joint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (supplemented throughout 2006).For example, other useful references regarding cell isolation and culture, and subsequent nucleic acid or protein isolation, include Freshney (1994) Culture of Animal Cells, a Manual of Basic Technique, third edition, Wiley-Liss, New York and references cited therein; Payne et al. (1992) Plant Cell and Tissue Culture in Liquid Systems John Wiley & Sons, Inc. New York, NY; Gamborg and Phillips (Eds.) (1995) Plant Cell, Tissue and Organ Culture; Fundamental Methods Springer Lab Manual, Springer-Verlag (Berlin Heidelberg New York); and Atlas and Parks (Eds.) The Handbook of Microbiological Media (1993) CRC Press, Boca Raton, FL. Methods for making nucleic acids (e.g., by in vitro amplification, purification from cells, or chemical synthesis), methods for manipulating nucleic acids (e.g., by site-directed mutagenesis, restriction enzyme digestion, ligation, etc.), and various vectors, cell lines, etc. useful in manipulating and making nucleic acids are described in the above references. Additionally, essentially any polynucleotide (including labeled or biotinylated polynucleotides) can be custom ordered or standard ordered from any of a variety of commercially available sources.

[0200] The invention has been described in terms of specific embodiments found or proposed to include certain particular modes for carrying out the invention. In light of this disclosure, those skilled in the art will recognize that numerous modifications and variations can be made in the specific embodiments exemplified without departing from the intended scope of the invention.

[0201] All cited references, including patent publications, are incorporated herein by reference in their entirety. Nucleotide and other gene sequences referred to by published gene location or other description are also expressly incorporated herein by reference.

[0202] Sequences presented in the sequence listing

[0203] [Table 12-1]

[0204] [Table 12-2]

[0205] [Table 12-3]

[0206] [Table 12-4] In one aspect, the present invention may be as follows. [Aspect 1] A method for producing one or more gene products, comprising: providing a first solution comprising at least one gene product expressed in a host cell, wherein at least some of the at least one gene product in the first solution can be sedimented by centrifugation at 7000×g to form a solubilizable pellet; placing at least some of said at least one gene product in a solubilization solution; recovering at least some of the at least one gene product from the solubilization solution, wherein the amount of the at least one gene product recovered from the solubilization solution is at least 50% of the total amount of the at least one gene product present in the first solution; wherein the at least one gene product is not contacted with a reducing agent. [Aspect 2] A step of subjecting the first solution to centrifugation, whereby the first solution is separated into a soluble fraction and a pellet; Recovering at least some of the at least one gene product from the pellet, wherein at least some of the at least one gene product is disposed in the solubilization solution. 3. The method of embodiment 2, further comprising: [Aspect 3] A method for producing one or more gene products, comprising: Providing a first solution comprising at least one gene product expressed in a host cell; subjecting the first solution to centrifugation, whereby the first solution is separated into a soluble fraction and a pellet; recovering at least some of the at least one gene product from the pellet; placing at least some of the at least one gene product recovered from the pellet in a solubilization solution; recovering at least some of the at least one gene product from the solubilization solution, wherein the amount of the at least one gene product recovered from the solubilization solution is at least 50% of the total amount of the at least one gene product present in the first solution; wherein the at least one gene product is not contacted with a reducing agent. [Aspect 4] The method according to any one of aspects 1 to 3, wherein the at least one gene product is a polypeptide that forms at least one disulfide bond. [Aspect 5] The method according to any one of aspects 1 to 4, wherein the at least one gene product is a polypeptide lacking a signal peptide. [Aspect 6] The method according to any one of Aspects 1 to 5, wherein the at least one gene product comprises a polypeptide selected from the group consisting of (a) a polypeptide comprising the amino acid sequence of the mature chain of leptin, metreleptin, growth hormone, human growth hormone, or insulin, and (b) a fragment of any of the polypeptides of (a). [Aspect 7] The method described in any one of aspects 1 to 6, wherein the first solution is a lysate of the host cells. [Aspect 8] The method described in aspect 7, wherein the host cell lysate is produced by contacting the host cell with lysozyme. [Aspect 9] The method of aspect 7, wherein the host cell lysate is produced by mechanical lysis. [Aspect 10] The method according to any one of aspects 1 to 9, wherein the host cell is a prokaryotic cell. [Aspect 11] The method according to aspect 10, wherein the host cell is an Escherichia coli cell. [Aspect 12] The method according to any one of aspects 1 to 11, wherein the host cell has been modified to have a more oxidized cytoplasm. [Aspect 13] The method according to aspect 12, wherein the modification to the host cell results in the loss of expression of at least one gene selected from the group consisting of trxB, gor, gshA, and gshB. [Aspect 14] The method described in aspect 13, wherein the host cell further comprises a mutation in the ahpC gene. [Aspect 15] A method according to any one of aspects 1 to 14, wherein the host cell comprises one or more expression constructs. [Aspect 16] The method described in aspect 15, wherein at least one of the expression constructs comprises at least one inducible promoter. [Aspect 17] The method described in aspect 16, wherein the host cell has a reduced level of gene function of at least one gene encoding a protein that metabolizes at least one inducer of the at least one inducible promoter. [Aspect 18] A method described in any of aspects 2 to 17, wherein centrifugation is performed at a force of 900 x g to 25,000 x g. [Aspect 19] The method described in any one of aspects 1 to 18, wherein the solubilization solution contains at least one chaotropic agent. [Aspect 20] The method according to any one of aspects 1 to 19, wherein the at least one chaotropic agent is selected from the group consisting of n-butanol, ethanol, guanidinium chloride, guanidine hydrochloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. [Aspect 21] The method according to aspect 20, wherein the at least one chaotropic agent is selected from the group consisting of urea at a concentration of 2 M to 10 M and guanidine hydrochloride at a concentration of 2 M to 8 M. [Aspect 22] The method according to any one of aspects 1 to 21, further comprising a step of reducing the concentration of the at least one chaotropic agent in the solubilization solution. [Aspect 23] The method of aspect 22, wherein the step of diluting the solubilization solution reduces the concentration of the at least one chaotropic agent to 50% or less of its initial concentration in the solubilization solution. [Aspect 24] The method described in aspect 22 or 23, further comprising a step of incubating the solubilization solution containing a reduced concentration of the at least one chaotropic agent for at least 1 hour. [Aspect 25] A method according to any one of aspects 1 to 24, wherein at least some of the at least one gene product recovered from the solubilized solution have a characteristic selected from the group consisting of properly formed disulfide bonds and gene product activity. [Aspect 26] The method described in aspect 25, wherein at least 50% of the at least one gene product recovered from the solubilized solution has properly formed disulfide bonds. [Aspect 27] The method according to any one of aspects 1 to 26, further comprising chromatographic purification of the at least one gene product. [Embodiment 28] The method according to embodiment 27, wherein the chromatographic purification is immobilized metal affinity chromatography (IMAC). [Aspect 29] The method described in aspect 28, wherein the chromatographic purification utilizes a Ni-NTA column.

Claims

1. 1. A method for producing one or more gene products, comprising: providing a first solution comprising at least one gene product expressed in a host cell, wherein at least some of the at least one gene product in the first solution can be sedimented by centrifugation at 7000×g to form a solubilizable pellet; placing at least some of said at least one gene product in a solubilization solution; recovering at least some of the at least one gene product from the solubilization solution, wherein the amount of the at least one gene product recovered from the solubilization solution is at least 50% of the total amount of the at least one gene product present in the first solution; wherein the at least one gene product is not contacted with a reducing agent; the gene product comprises a proinsulin polypeptide, the host cell is Escherichia coli; The solubilization solution contains 7M or more urea. The above method.

2. subjecting the first solution to centrifugation, whereby the first solution is separated into a soluble fraction and a pellet; Recovering at least some of the at least one gene product from the pellet, wherein at least some of the at least one gene product is disposed in the solubilization solution. The method of claim 1 further comprising:

3. 1. A method for producing one or more gene products, comprising: Providing a first solution comprising at least one gene product expressed in a host cell; Centrifugation of the first solution at 7000×g, wherein the first solution is separated into a soluble fraction and a pellet; recovering at least some of the at least one gene product from the pellet; placing at least some of the at least one gene product recovered from the pellet in a solubilization solution; recovering at least some of the at least one gene product from the solubilization solution, wherein the amount of the at least one gene product recovered from the solubilization solution is at least 50% of the total amount of the at least one gene product present in the first solution; wherein the at least one gene product is not contacted with a reducing agent; the gene product comprises a proinsulin polypeptide, the host cell is Escherichia coli; The solubilization solution contains 7M or more urea. The above method.

4. The method according to any one of claims 1 to 3, wherein the first solution is a lysate of said host cells.

5. The method of claim 4 , wherein the host cell lysate is produced by contacting a host cell with lysozyme.

6. The method of claim 4 , wherein the host cell lysate is produced by mechanical lysis.

7. The method of any one of claims 1 to 6, wherein the host cell has been modified to have a more oxidized cytoplasm.

8. 8. The method of claim 7, wherein the modification to the host cell results in the deficiency of expression of at least one gene selected from the group consisting of trxB, gor, gshA, and gshB.

9. The method of claim 8 , wherein the host cell further comprises a mutation in the ahpC gene.

10. The method of any one of claims 1 to 9, wherein the host cell comprises one or more expression constructs.

11. The method of claim 10 , wherein at least one of the expression constructs comprises at least one inducible promoter.

12. 12. The method of claim 11, wherein the host cell has a reduced level of gene function of at least one gene encoding a protein that metabolizes at least one inducer of the at least one inducible promoter.

13. The method of any one of claims 1 to 12, further comprising the step of reducing the concentration of urea in the solubilization solution.

14. 14. The method of claim 13, wherein diluting the solubilization solution reduces the concentration of urea to 50% or less of its initial concentration in the solubilization solution.

15. 15. The method of claim 13 or 14, further comprising incubating the solubilization solution containing the reduced concentration of urea for at least 1 hour.

16. 16. The method of any one of claims 1 to 15, wherein at least some of the at least one gene product recovered from the solubilization solution have a property selected from the group consisting of properly formed disulfide bonds and a gene product activity.

17. 17. The method of claim 16, wherein at least 50% of the at least one gene product recovered from the solubilization solution has properly formed disulfide bonds.

18. The method of any one of claims 1 to 17, further comprising chromatographic purification of said at least one gene product.

19. 19. The method of claim 18, wherein the chromatographic purification is immobilized metal affinity chromatography (IMAC).

20. The method of claim 19, wherein the chromatographic purification utilizes a Ni-NTA column.

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