FolA selection assay to identify strains with increased soluble target protein expression

Abstract

The present disclosure provides compositions and methods for identifying cells or strains that produce soluble proteins. The present disclosure further provides a growth / no growth selection strategy for identifying E. coli strains in a mixed population (library) that produce more soluble proteins.

Description

[Background technology]

[0001] Incorporation by Reference of Electronically Submitted Materials The sequence listing, which is part of this disclosure, is submitted as a text file concurrently herewith. The name of the text file containing the sequence listing is "57387_Seqlisting.txt", created on November 15, 2022, and is 1,893 bytes in size. The subject matter of the sequence listing is incorporated herein by reference in its entirety.

[0002] Antibiotic resistance genes are commonly used as fusion partners for selection assays. A commonly used selectable fusion partner is an antibiotic resistance gene. However, these methods suffer from a limited linear response range, where expression of only a small amount of the antibiotic resistance gene confers nearly the maximum practically achievable concentration of the antibiotic. Protocols that aim to overcome this limitation use indirect selection schemes, such as translational coupling. To date, no assay has been provided that allows for a direct correlation between the expression of antibiotic resistance genes and the expression of soluble proteins. The present disclosure addresses this unmet need. Summary of the Invention

[0003] In one embodiment, the disclosure provides a method for identifying host cells capable of producing a soluble protein of interest, the method comprising: (a) preparing a population of host cells, wherein each host cell of the population comprises an expression construct capable of expressing a fusion protein comprising (i) a protein of interest and (ii) a selection protein; (b) incubating the host cells of (a) under conditions that allow expression of the fusion protein, wherein the conditions comprise a growth substance comprising at least two synergistic selection agents; and (c) visualizing the host cells that are able to grow, thereby identifying the host cells that are able to produce the soluble protein of interest.

[0004] In another embodiment, a method is provided for identifying a host cell that produces the greatest amount of a soluble protein of interest in a population of host cells, the method comprising: (a) preparing a population of host cells, wherein each host cell of the population comprises an expression construct capable of expressing a fusion protein comprising (i) a protein of interest and (ii) a selection protein; (b) incubating the host cells of (a) under conditions that allow expression of the fusion protein, the conditions comprising a growth substance comprising at least two synergistic selection agents; and (c) visualizing the host cells that are able to grow, thereby identifying a host cell that produces the greatest amount of the soluble protein of interest in the population of host cells.

[0005] In some embodiments, the aforementioned method is provided, further comprising binning the host cells based on the amount of soluble protein produced. In some embodiments, the selection protein is a target of an antibiotic or an antibiotic resistance protein. In one embodiment, the selection protein is FolA. In another embodiment, FolA is from E. coli. In yet another embodiment, FolA is set forth in SEQ ID NO: 1.

[0006] In yet another embodiment, the aforementioned method is provided, comprising using two synergistic selection agents, the synergistic selection agents being selected from the group consisting of antibiotics, sugars, chemical agents, enzyme substrate analogs, enzyme inhibitors, agents that sequester biomolecules, chelating agents, and agents that damage cell walls or cell membranes. In one embodiment, the two synergistic selection agents are trimethoprim and sulfamethoxazole. In some embodiments, the aforementioned method is provided, wherein the protein of interest is a heterologous protein. In another embodiment, the heterologous protein is selected from the group consisting of antibodies, Fabs, scFvs, nanobodies, T-cell receptors, and chimeric antigen receptors, growth factors, cytokines, hormones, enzymes, or functional fragments thereof.

[0007] In some embodiments, the aforementioned method is provided, wherein the population of host cells comprises a library of host cells, the library being comprised of cells having unique genotypes and / or unique genetic engineering, In one embodiment, the library comprises about 1,000 to about 1 billion host cells.

[0008] In some embodiments, the aforementioned method is provided, wherein the host cell is selected from the group consisting of a eukaryotic cell, a prokaryotic cell, a bacterial cell, a mammalian cell, and an insect cell. In another embodiment, the bacterial cell is an E. coli cell. In yet another embodiment, the E. coli cell comprises (a) an altered gene function of at least one gene encoding a transporter protein for the inducer of the at least one inducible promoter, (b) a reduced level of gene function of at least one gene encoding a protein that metabolizes the inducer of the at least one inducible promoter, (c) a reduced level of gene function of at least one gene encoding a protein involved in the biosynthesis of the inducer of the at least one inducible promoter, (d) an altered gene function of a gene that affects the reducing / oxidizing environment of the cytoplasm of the host cell, (e) a reduced level of gene function of a gene encoding a reductase, (f) at least one expression construct encoding at least one disulfide bond isomerase protein, (g) at least one polynucleotide encoding a form of DsbC lacking a signal peptide, and / or (h) at least one polynucleotide encoding Ervlp.

[0009] In yet another embodiment, the aforementioned method is provided, wherein the expression construct is an extrachromosomal construct selected from the group consisting of a polynucleotide, a plasmid, and an artificial chromosome. In one embodiment, the expression construct comprises an inducible promoter. In another embodiment, the expression construct comprises two or more inducible promoters. In yet another embodiment, at least one inducible promoter is a propionate inducible promoter and at least one other inducible promoter is an L-arabinose inducible promoter.

[0010] In yet another embodiment, the aforementioned method is provided, wherein the growth substrate is selected from the group consisting of selective media. In some embodiments, the aforementioned method is provided, wherein the growth substrate comprises a matrix of two or more synergistic selective agents. In one embodiment, the two synergistic selective agents are trimethoprim and sulfamethoxazole, which are present in the growth medium at a concentration range of 1 ug / ml to 1000 ug / ml and 1 ug / ml to 1000 ug / ml, respectively.

[0011] In some embodiments, the aforementioned method is provided, wherein the conditions allowing expression of the fusion protein comprise the presence of one or more inducers of expression of the fusion protein. In some embodiments, the aforementioned method is provided, wherein the host cells are identified using a technique selected from the group consisting of visual inspection, chemiluminescence, radiography, fluorescence, and colorimetry. In yet another embodiment, the aforementioned method is provided, wherein visualizing the host cells in step (c) comprises detecting growth on an agar plate.

[0012] In some embodiments, the disclosure provides the aforementioned method, further comprising a solid phase assay for detecting soluble protein expression. For example, in one embodiment, there is provided a method as described above, further comprising the steps of: (a) plating host cells on a growth substrate and incubating the host cells under conditions that allow for host cell growth on the growth substrate; (b) optionally preparing at least one replicate plate and incubating the replicate plate under conditions that allow for host cell growth and production of the protein of interest; (c) transferring the host cells from the growth substrate or the at least one replicate plate of (b) to a membrane; (d) preparing the host cells transferred to the membrane in (c) for probing, comprising: (i) optionally fixing the host cells under conditions that allow for immobilization of cellular components; (ii) blocking the host cells; and (iii) optionally lysing the host cells under conditions that allow for permeabilization of the host cells; (e) contacting the permeabilized host cells with a probe solution comprising at least one probe under conditions that allow for binding of the at least one probe to the protein of interest, thereby forming a probe-protein of interest complex; and (f) imaging the host cells under conditions that allow for identifying host cells producing the protein of interest.

[0013] Additional embodiments are provided below in which the protein of interest is encoded by a gene product of interest. In one embodiment, the disclosure provides a method of identifying host cells that produce a gene product of interest, the method comprising the steps of: (a) plating a population of host cells comprising an expression construct encoding one or more gene products onto a growth substrate and incubating the host cells under conditions that permit host cell growth on the growth substrate; (b) preparing at least one replicate plate and incubating the replicate plate under conditions that permit host cell growth and production of the gene product; (c) transferring the host cells from the at least one replicate plate of (b) to a membrane; and (d) preparing the host cells transferred to the membrane in (c) for probing. The method further comprises the steps of: (i) optionally preparing the host cells by fixing the host cells under conditions that allow for immobilization of cellular components, (ii) blocking the host cells, and (iii) optionally lysing the host cells under conditions that allow for permeabilization of the host cells, (e) contacting the permeabilized host cells with a probe solution that includes at least one probe under conditions that allow for binding of the at least one probe to a gene product of interest, thereby forming a probe-gene product complex, and (f) imaging the host cells under conditions that allow for identification of host cells that produce the gene product of interest. In another embodiment, step (c) further comprises marking the at least one replicate plate and membrane to allow for spatial alignment of the at least one replicate plate and membrane.

[0014] In some embodiments, the aforementioned methods are provided, wherein the host cell is selected from the group consisting of eukaryotic cells, prokaryotic cells, bacterial cells, mammalian cells, and insect cells. In yet other embodiments, the aforementioned methods are provided, wherein the gene product of interest is selected from the group consisting of therapeutic proteins, antibodies, Fabs, scFvs, nanobodies, T-cell receptors, and chimeric antigen receptors, or fragments thereof. In one embodiment, the gene product of interest is an intracellular protein.

[0015] In still other embodiments, the aforementioned method is provided, wherein the population of host cells comprises approximately 1,000 to approximately 1 billion host cells. In other embodiments, the population of host cells is modified to produce a gene product from an expression construct. In one embodiment, the expression construct comprises two or more inducible promoters. In yet another embodiment, at least one inducible promoter is a propionate-inducible promoter and at least one other inducible promoter is an L-arabinose-inducible promoter.

[0016] The disclosure also provides, in some embodiments, the aforementioned methods, wherein the host cell has been genetically modified to include one or more of: (a) altered gene function of at least one gene encoding a transporter protein for the inducer of the at least one inducible promoter; (b) a reduced level of gene function of at least one gene encoding a protein that metabolizes the inducer of the at least one inducible promoter; (c) a reduced level of gene function of at least one gene encoding a protein involved in the biosynthesis of the inducer of the at least one inducible promoter; (d) an altered gene function of a gene that affects the reducing / oxidizing environment of the cytoplasm of the host cell; (e) a reduced level of gene function of a gene encoding a reductase; (f) at least one expression construct encoding at least one disulfide bond isomerase protein; (g) at least one polynucleotide encoding a form of DsbC lacking a signal peptide; and / or (h) at least one polynucleotide encoding Ervlp.

[0017] In yet another embodiment, the aforementioned method is provided, wherein two, three, four, five or more replicate plates are prepared. In one embodiment, the replicate plate comprises an antibiotic and at least one inducer of expression of the gene product of interest. In yet another embodiment, the membrane is selected from the group consisting of nitrocellulose membrane and PVDF (polyvinylidene fluoride) membrane. In another embodiment, the replicate plate and membrane of step (c) are contacted with a colored substance. In one embodiment, the colored substance is selected from the group consisting of acrylic paints and dyes. In another embodiment, the colored substance is contacted with the plate and membrane with an instrument selected from the group consisting of a needle and a pen.

[0018] In another embodiment, the aforementioned method is provided, wherein the conditions that allow for fixation of cellular components include contacting the membrane with a composition comprising one or more of glutaraldehyde and paraformaldehyde, hi another embodiment, the conditions that allow for permeabilization of host cells include contacting the membrane with a composition comprising one or more of lysozyme and EDTA.

[0019] In yet another embodiment, the aforementioned method is provided, wherein at least one probe is selected from the group consisting of an antibody or functional fragment thereof, a nucleic acid binding protein or functional fragment thereof, a receptor or functional fragment thereof, a ligand or functional fragment thereof, and an antigen or functional fragment thereof, and a peptide or polypeptide capable of being bound by a gene product of interest. In one embodiment, the probe comprises a reporter moiety selected from the group consisting of biotin, a histidine tag, an Fc tag, a spy tag, a Strp tag, and an Avi tag.

[0020] The present disclosure also provides, in some embodiments, the aforementioned method, where when two, three, four, five or more replicate plates are prepared, each membrane prepared from the replicate plates is contacted with a different concentration of the probe solution. In yet another embodiment, the aforementioned method is provided, where the imaging comprises contacting the membrane with a composition comprising an activator of the reporter moiety. In another embodiment, the contacting of the membrane with the composition comprising an activator of the reporter moiety is repeated once, twice, or three or more times. In yet another embodiment, the activator is selected from the group consisting of alkaline phosphatase, streptavidin alkaline phosphatase, and streptavidin alkaline phosphatase dextran polymer.

[0021] In yet other embodiments, the aforementioned methods are provided, wherein imaging includes a method selected from the group consisting of chemiluminescence, radiography, fluorescence, and colorimetry. In yet other embodiments, the aforementioned are provided, further comprising the step of replating one or more host cells identified as capable of producing the gene product of interest. In another embodiment, the replated host cells are subjected to the method of claim 1 to confirm the ability of the host cells to produce the gene product of interest and / or to isolate the host cell line from other host cell lines identified as capable of producing the gene product of interest. In some embodiments, the method may further comprise determining the affinity of the probe-gene product complex.

[0022] The disclosure also provides, in one embodiment, a method of screening host cells from a population of host cells that produce a gene product of interest, the method comprising the steps of: (a) plating a population of host cells comprising an expression construct encoding one or more gene products onto a growth substrate and incubating the host cells under conditions that permit host cell growth on the growth substrate; (b) preparing at least one replicate plate and incubating the replicate plate under conditions that permit host cell growth and production of the gene product; (c) transferring the host cells from the at least one replicate plate of (b) to a membrane; and (d) probing the host cells transferred to the membrane in (c). the method includes the steps of: (i) preparing the host cells under conditions that allow for immobilization of cellular components, (ii) blocking the host cells, and (iii) optionally lysing the host cells under conditions that allow for permeabilization of the host cells; (e) contacting the permeabilized host cells with a probe solution comprising at least one probe under conditions that allow for binding of the at least one probe to a gene product of interest, thereby forming a probe-gene product complex; and (f) imaging the host cells under conditions that allow for identification of host cells that produce the gene product of interest.

[0023] In yet another embodiment, the disclosure provides a method for determining probe-gene product of interest relative affinity, the method comprising the steps of: (a) plating a population of host cells comprising an expression construct encoding one or more gene products onto a growth substrate and incubating the host cells under conditions that permit host cell growth on the growth substrate; (b) preparing at least three replicate plates and incubating the replicate plates under conditions that permit host cell growth and production of the gene products; (c) transferring the host cells from the at least three replicate plates of (b) onto separate membranes; and (d) preparing the host cells transferred to the membranes in (c) for probing, the steps comprising: (i) plating the host cells onto a growth substrate; and (ii) incubating the host cells under conditions that permit host cell growth on the growth substrate; (iii) transferring the host cells onto a growth substrate; The method includes preparing the host cells, the method including (a) fixing the host cells under conditions that allow for immobilization of cellular components; (ii) blocking the host cells; and (iii) optionally lysing the host cells under conditions that allow for permeabilization of the host cells; (e) contacting the permeabilized host cells on at least three replicate membranes with a probe solution comprising at least one probe under conditions that allow for binding of the at least one probe to a gene product of interest, thereby forming a probe-gene product complex, the probe solutions comprising different probe concentrations; and (f) imaging the host cells under conditions that allow for determining the relative affinity of the probe to the gene product of interest. [Brief description of the drawings]

[0024] [Figure 1] Figure 2 shows that cells harboring the mCherry-FolA fusion are highly resistant to trimethoprim. [Diagram 2] 1 shows that synergy with sulfamethoxazole improves trimethoprim dynamic range. [Diagram 3] 1 shows a trimethoprim-sulfamethoxazole matrix. [Figure 4] An exemplary plasmid construct is shown. [Diagram 5] A plate with colonies after one week of incubation is shown. [Figure 6] Images from round 1 results of the solid phase assay are shown. [Figure 7] The results of round 2 are shown below. [Figure 8] An image of the treated membrane is shown. [Figure 9] An exemplary plasmid construct is shown. [Figure 10] Additional results from a solid phase assay are shown. [Figure 11] 4 shows the results of liquid performer compared to agar plates. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] Embodiments of the present disclosure provide compositions and methods for identifying cells or lines from a library of cells or lines capable of producing high amounts of a soluble protein of interest. In some embodiments, the protein of interest is genetically fused to a selection protein, such as an antibiotic resistance gene or a protein that is the target of an antibiotic. As described herein, in some embodiments, the selection process includes using two (or more) selection agents that act synergistically to improve the dynamic range of the assay.

[0026] Considerations for the selection-enabling fusion partner (i.e., selection protein) include, but are not limited to, (1) allowing growth / no growth selection, (2) allowing identification of rankings of expressing strains from low to very high by modifying the selection conditions, and (3) being clean (low background noise). A selectable assay should allow testing of large diversity libraries up to billions of variants on a single 10 cm agar plate.

[0027] As described herein, each 10 cm plate may contain up to 10 billion cells (eg, 10 billion for a lethal combination of drugs, 1 billion for a static combination of drugs).

[0028] Identifying host cells capable of producing a soluble protein of interest In one embodiment of the present disclosure, a growth / no growth selection strategy is provided to identify E. coli strains in a mixed population (library) that yield higher soluble protein expression (i.e., relative to other strains in the library). As described herein, the synergistic effect of selection agents increases the dynamic range of the assay. As an example, a protein of interest is engineered and fused to a reporter gene (folA) that is the target of an antibiotic (trimethoprim). In this exemplary embodiment, selection of the reporter gene (folA) / antibiotic (trimethoprim) pair allows for synergistic action with a second antibiotic agent, improving the efficacy of the first antibiotic (trimethoprim). Thus, the benefits of a successful growth / no growth selection strategy can be used to identify strains of E. coli with higher soluble protein expression (i.e., relative to other strains in the library). As described herein, the synergistic effect of selection agents increases the dynamic range of the assay. As an example, a protein of interest is genetically engineered and fused to a reporter gene (folA) that is the target of an antibiotic (trimethoprim). In this exemplary embodiment, selection of the reporter gene (folA) / antibiotic (trimethoprim) pair allows for synergistic action with a second antibiotic agent, improving the efficacy of the first antibiotic (trimethoprim). Thus, the benefits of a successful growth / no growth selection strategy can be used to identify strains of E. coli with higher soluble protein expression (i.e., relative to other strains in the library). 9 ) This allows for parallel testing of libraries.

[0029] The fusion proteins described herein can be directed against any antibiotic target or antibiotic resistance gene, so long as a second synergist can be used to increase the useful dynamic range of the assay.

[0030] Fusion proteins Protein of interest As described herein, a protein of interest (and in some embodiments described herein, a gene of interest encoding the protein of interest) can be used with the methods provided herein. As used herein, a "soluble protein" or a "soluble protein of interest" refers, in one embodiment, to a protein that remains stable in solution and does not precipitate over time or remains in solution when centrifugal force (16,000 x g) is applied for 10 minutes or more. Protein solubility is, in some embodiments, a thermodynamic parameter defined as the concentration of a protein in a saturated solution that is in equilibrium with a solid phase, either crystalline or amorphous, under a given set of conditions. Solubility can be affected by many extrinsic and intrinsic factors, including pH, ionic strength, temperature, and the presence of various solvent additives (Kramer, RM, et al., Biphys J., 2012, 102(8):1907-1915). In one embodiment, a soluble protein is one that has a solubility of greater than 70% and is insoluble with a solubility of less than 30% (Chan, P., et al., Scientific Reports, 2013, 3, 3333).

[0031] Proteins may include biologically active derivatives or variants or fragments. As used herein, a "biologically active derivative" or "biologically active variant" includes any derivative or variant of a molecule having substantially the same functional and / or biological properties of the molecule, such as binding properties, and / or the same structural basis, such as a peptide backbone or basic polymer unit.

[0032] An "analog," such as a "variant" or "derivative," is a compound that is substantially similar in structure and has the same biological activity as a naturally occurring molecule, albeit to a different extent in certain instances. For example, a polypeptide variant refers to a polypeptide that shares a substantially similar structure and has the same biological activity as a reference polypeptide. Variants or analogs differ in the composition of their amino acid sequence compared to the naturally occurring polypeptide from which they are derived based on one or more mutations, including (i) a deletion of one or more amino acid residues at one or more termini of the polypeptide and / or at one or more internal regions (e.g., fragments) of the naturally occurring polypeptide sequence, (ii) an insertion or addition of one or more amino acids at one or more termini of the polypeptide (typically an "addition" or "fusion") and / or at one or more internal regions (typically an "insertion") of the naturally occurring polypeptide sequence, or (iii) a substitution of one or more amino acids for other amino acids in the naturally occurring polypeptide sequence. As an example, a "derivative" refers to a polypeptide that is a type of analog and shares the same or substantially similar structure as a reference polypeptide that has been, for example, chemically modified.

[0033] Variant polypeptides are a type of analog polypeptide, including insertion variants, in which one or more amino acid residues are added to the biomolecule amino acid sequence of the disclosure. The insertion may be located at either or both termini of the protein, and / or may be located within an internal region of the therapeutic protein amino acid sequence. Insertion variants with additional residues at either or both termini include, for example, fusion proteins and proteins that include amino acid tags or other amino acid labels. In one aspect, the biomolecule optionally includes an N-terminal Met, especially when the molecule is recombinantly expressed in bacterial cells such as E. coli. In another aspect, the biomolecule includes a histidine tag (His tag).

[0034] In deletion variants, one or more amino acid residues are removed in the biomolecule polypeptides described herein. Deletions can occur at one or both termini of the protein polypeptide and / or by removal of one or more residues within the therapeutic protein amino acid sequence. Thus, deletion variants include fragments of the protein polypeptide sequence.

[0035] In substitution variants, one or more amino acid residues of a biomolecule are removed and replaced with alternative residues. In one aspect, these substitutions are conservative in nature, and this type of conservative substitution is well known in the art. Alternatively, the present disclosure encompasses substitutions that are also non-conservative. Exemplary conservative substitutions are described in Lehninger, [Biochemistry, 2nd Edition; Worth Publishers, Inc., New York (1975), pp.71-77] and are shown immediately below.

[0036] Proteins contemplated herein include full-length proteins, precursors of full-length proteins, biologically active subunits or fragments of full-length proteins, and biologically active derivatives and variants of any of these forms of therapeutic proteins. Thus, proteins include those that (1) have an amino acid sequence that has greater than about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% or more amino acid sequence identity with a polypeptide encoded by a reference nucleic acid or amino acid sequence described herein over a region of at least about 25, about 50, about 100, about 200, about 300, about 400, or more amino acids. In accordance with the present disclosure, the term "recombinant protein" includes any protein obtained via recombinant DNA technology. In certain embodiments, the term encompasses the proteins described herein.

[0037] In some embodiments, the protein is a therapeutic protein, such as a monoclonal or polyclonal antibody, or other glycoprotein, a biosimilar, an Fc fusion, an enzyme, a vaccine, a hormone, a cytokine, or a growth factor. In yet other embodiments, the gene product is an anticoagulant, a blood factor, a bone morphogenetic protein, an interleukin, an interferon, a thrombolytic agent, or any protein produced by recombinant means. In other embodiments, the method provides that the method can be used with any biomolecule or chemical entity, including small molecules, that is produced by the cells described herein and that has a probe that can be used for the binding step.

[0038] The term "antibody" as used herein refers to a whole antibody that interacts (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) with an epitope on a target antigen. A naturally occurring "antibody" is a glycoprotein that comprises at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain: CL. The VH and VL regions can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant region of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single chain Fvs (scFvs), disulfide-linked Fvs (sdFvs), Fab fragments, F(ab') fragments, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the invention), as well as epitope-binding fragments of any of the above. The antibody can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. The antibody or epitope-binding fragment may be, or be a component of, a multispecific molecule, including a bispecific antibody.

[0039] Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light (VL) and heavy (VH) chain portions determine antigen recognition and specificity. Conversely, the constant domains of the light (CL) and heavy (CH1, CH2, or CH3) chains confer important biological properties such as secretion, transplacental transfer, Fc receptor binding, and complement binding. By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding site or amino-terminus of the antibody. The N-terminus is the variable region and the constant region is at the C-terminus. The CH3 and CL domains actually comprise the carboxy termini of the heavy and light chains, respectively.

[0040] The phrase "antibody fragment" as used herein refers to one or more portions of an antibody that retain the ability to specifically interact with a target epitope (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution). Examples of binding fragments include, but are not limited to, Fab fragments, which are monovalent fragments consisting of VL, VH, CL, and CH1; F(ab)2, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments consisting of the VH domain (Ward et al., (1989) Nature 341:544-546); and isolated complementarity determining regions (CDRs). Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker that allows the VL and VH regions to pair into a single protein chain forming a monovalent molecule (known as single-chain Fv (scFv), see, e.g., Bird et al., (1988) Science 242:423-426, and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed within the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies.

[0041] In other embodiments, the protein of interest is a therapeutic protein, a T cell receptor, a chimeric antigen receptor, a growth factor, a cytokine, a hormone, an enzyme, or a functional fragment thereof, hi one embodiment, the protein is insulin.

[0042] The present disclosure also provides fusion proteins that, in some embodiments, include an optional linker, e.g., a flexible linker sequence, between two protein components. In some embodiments, the linker is at least 5, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, or at least 50 amino acids long. In other embodiments, the linker is less than 5, less than 10, less than 15, less than 20, less than 25, less than 30, less than 35, less than 40, less than 45, or less than 50 amino acids long. In one embodiment, the linker is 23 amino acids long. The linker may be flexible, rigid, or cleavable (e.g., disulfide, protease-sensitive sequence) in various embodiments (Chen, X., et al., Advanced Drug Delivery Reviews, 65(1):1357-1369 (2013)). Linkers may be derived from naturally occurring multidomain proteins or may be empirical (see Argos, P., J. Mol. Biol., 211:943-958 (1990), and Heringa, GR, Protein Eng., 15:871-879 (2002), which compare several properties of natural linkers independently, such as length, hydrophobicity, amino acid residues, and secondary structure). Threonine (Thr), serine (Ser), proline (Pro), glycine (Gly), aspartic acid (Asp), lysine (Lys), glutamine (Gln), asparagine (Asn), and alanine (Ala) are preferred linker components, as are, in some embodiments, arginine (Arg), phenylalanine (Phe), and glutamic acid (Glu). In general, preferred amino acids are polar uncharged or charged residues. Natural linkers adopt a variety of secondary structures, including helical, beta-stranded, coil / bend, and twist, in order to perform their functions.

[0043] Selection proteins and selection agents The selection proteins provided herein are, in some embodiments, proteins, polypeptides, or analogs, such as variants or derivatives of the above. Any selection agent can be used in the methods provided by the present disclosure, so long as the agents act synergistically to improve the dynamic range. A "synergistic selection agent" refers to any combination of two or more agents where the effect of the combination of agents is greater than the sum of the individual agents alone. Synergistic selection agents include, but are not limited to, antibiotics, sugars, chemical agents, enzyme substrates and analogs, enzyme inhibitors, agents that sequester biomolecules, chelating agents, and agents that damage cell walls or cell membranes. The selection agent can be part of the composition that constitutes the growth substrate, or can be provided separately.

[0044] In one example, the selection protein is dihydrofolate reductase (dhfr) and the synergistic selection agents are trimethoprim and sulfamethoxazole. In certain embodiments, the dhfr is from a bacterial source. Typically, the bacterial dhfr is referred to as FolA. In a preferred embodiment, FolA is from E. coli. In one embodiment, endogenous FolA is knocked out or otherwise inactivated in the cells or strains used in accordance with the methods described herein. In one embodiment, FolA is from E. coli as follows (www.uniprot.org / uniprot / P0ABQ4.fasta): [Table 1]

[0045] In other embodiments, the selection protein and synergistic selection agent include, but are not limited to, synergistic combinations of trimethoprim / sulfamethoxazole, b-lactam / b-lactamase inhibitor, cell wall active agent / aminoglycoside, and fosfomycin / b-lactam.

[0046] cell Cells, including cells from a population of cells or cells that are part of a library of cells, are contemplated by the present disclosure. As used herein, a "library" with respect to cells refers to any collection of genetically distinct variants of cells, either deliberately engineered or naturally occurring. For example, cells with unique genotypes and / or uniquely engineered are contemplated. Exemplary genetic modifications are described herein, including modifications that allow the cells to express and produce recombinant heterologous proteins of interest. Unique phenotypes or uniquely engineered cells, in some embodiments, refer to cell populations that differ from each other in genetic aspects, such as the presence of mutations, plasmids, genes within plasmids, etc.

[0047] Cells comprising one or more of the expression constructs described herein are contemplated in various embodiments of the present disclosure. The cells of the present disclosure comprise an outer membrane (e.g., composed of proteins and lipids) within which the fusion proteins described herein, when expressed, may interact with or otherwise reside.

[0048] Prokaryotic host cells. In some embodiments of the present disclosure, expression constructs designed for expression of gene products, including the fusion proteins described herein, are provided in host cells, such as prokaryotic host cells. Prokaryotic host cells include those derived from 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 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.

[0049] Eukaryotic Host Cells. Many additional types of host cells can be used in the expression systems of the present disclosure, including eukaryotic cells, such as yeast (Candida shehatae, Kluyveromyces lactis, Kluyveromyces fragilis, other Kluyveromyces species, Pichia pastoris, Saccharomyces cerevisiae, Saccharomyces pastorianus, also known as Saccharomyces carlsbergensis, Schizosaccharomyces pombe, and the like). pombe, Dekkera / Brettanomyces, and Yarrowia 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, 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 G2)). The above host cells are available from the American Type Culture Collection.

[0050] As described in WO / 2017 / 106583, which is incorporated herein by reference in its entirety, the production of gene products such as therapeutic proteins at commercial scale and in soluble form is addressed by providing suitable host cells that can grow at high cell densities in fermentation cultures and produce soluble gene products in an oxidizing host cell cytoplasm through highly controlled inducible gene expression. Host cells of the present disclosure with these qualities are produced by combining some or all of the following features: (1) The host cell is genetically modified to have an oxidizing cytoplasm through increasing the expression or function of an oxidizing polypeptide in the cytoplasm and / or by decreasing the expression or function of a reducing polypeptide in the cytoplasm. Specific examples of such genetic modifications are provided herein. Optionally, the host cell can also be genetically modified to express chaperones and / or cofactors that assist in the production of the desired gene product and / or to glycosylate the polypeptide gene product. (2) The host cell comprises one or more expression constructs designed for expression of one or more gene products of interest, and in certain embodiments, at least one expression construct comprises an inducible promoter and a polynucleotide encoding a gene product expressed from the inducible promoter, and (3) the host cell comprises additional genetic modifications designed to improve certain aspects of gene product expression from the expression constructs.In a particular embodiment, the host cell has (A) a modification of the gene function of at least one gene encoding a transporter protein for an inducer of at least one inducible promoter, and as another example, the gene encoding the transporter protein is selected from the group consisting of araE, araE, araG, araH, rhaT, xylF, xylG, and xylH, or in particular araE, or more specifically, the modification of the gene function is expression of araE from a constitutive promoter, and / or (B) a modification of the gene function of at least one gene encoding a protein that metabolizes the inducer of at least one of the inducible promoters. and, as a further example, a gene encoding a protein that metabolizes the inducer of the at least one inducible promoter is selected from the group consisting of araA, araB, araD, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB; and / or (C) a reduced level of gene function of at least one gene encoding a protein involved in the biosynthesis of the inducer of the 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.

[0051] A host cell with an oxidized cytoplasm. The expression system of the present disclosure is designed to express a gene product, and in certain embodiments of the present disclosure, the gene product is expressed in the host cell. Examples of host cells are provided that allow efficient and cost-effective expression of gene products, including components of multimeric products. Host cells can include cells that are part of a multicellular organism, or cells grown in different organisms or biological systems, in addition to isolated cells in culture. In certain embodiments of the present disclosure, the host cell is a microbial cell, such as a yeast (such as Saccharomyces, Schizosaccharomyces, etc.), or a bacterial cell, 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. Growth experiments with E. coli host cells with oxidizing cytoplasm, specifically the E. coli B strains SHuffle® Express (NEB Catalog No. C3028H) and SHuffle® T7 Express (NEB Catalog No. C3029H), and the E. coli K strain SHuffle® T7 (NEB Catalog No. C3026H), have shown that these E. coli B strains with oxidizing cytoplasm can grow to much higher cell densities than their closest corresponding E. coli K strain (WO / 2017 / 106583).

[0052] Modifications to host cell gene function. Certain modifications can be made to gene function 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%, and most preferably at least 95%) of the cells in culture expressing a gene product from each of the induced promoters, as measured by the method of Khlebnikov et al., described in Example 9 of WO / 2017 / 106583. For host cells other than E. coli, these modifications can involve genes whose functions are structurally similar to E. coli genes, or genes that perform a function in the host cell similar to that of E. coli genes. Modifications to host cell gene function include eliminating or reducing gene function by deleting the entire gene protein-encoding sequence, or deleting a sufficiently large portion of the gene, inserting a sequence within the gene, or otherwise modifying the gene sequence such that a reduced level of functional gene product is produced from that gene. Modifications to host cell gene function also include increasing gene function, for example by modifying a native promoter to create a stronger promoter that increases the transcription level of the gene, or by introducing a missense mutation in the protein coding sequence that results in a more active gene product. Modifications to host cell gene function also include modifying gene function in any way, including, for example, modifying a native inducible promoter to create a constitutively activated promoter. In addition to modifying gene function for inducer transport and metabolism, as described herein with respect to modifying the expression of inducible promoters and / or chaperone proteins, it is also possible to modify the reduced-oxidative environment of the host cell.

[0053] Host cell reducing-oxidizing environment. In bacterial cells such as E. coli, proteins that require disulfide bonds are typically transported to the periplasm where disulfide bond formation and isomerization are catalyzed by the Dsb system, including DsbABCD and DsbG. Increasing expression of cysteine ​​oxidase DsbA, disulfide isomerase DsbC, or combinations of Dsb proteins, all of which are normally transported into the periplasm, has been used to express heterologous proteins that require disulfide bonds (Makino et al., Microb Cell Fact 2011 May 14;10:32). It is also possible to express cytoplasmic forms of these Dsb proteins, such as cytoplasmic DsbA and / or DsbC ("cDsbA" or "cDsbC"), which lack signal peptides and therefore are not transported into the periplasm. Cytoplasmic Dsb proteins such as cDsbA and / or cDsbC are useful in making the host cell cytoplasm more oxidizing and thus more conducive to disulfide bond formation in heterologous proteins produced in the cytoplasm. The host cell cytoplasm can also be made less reducing and therefore more oxidizing by directly modifying the thioredoxin and glutaredoxin / glutathione enzyme systems. Mutants defective in glutathione reductase (gor) or glutathione synthetase (gshB), together with thioredoxin reductase (trxB), make the cytoplasm more oxidizing. These strains cannot reduce ribonucleotides and therefore cannot grow in the absence of exogenous reducing agents such as dithiothreitol (DTT). Suppressor mutations in the gene ahpC encoding the peroxiredoxin AhpC (such as ahpC* and ahpCA, Lobstein et al., Microb Cell Fact 2012 May 8;11:56;doi:10.1186 / 1475-2859-11-56) convert it into a disulfide reductase that generates reduced glutathione, allowing channeling of electrons onto the enzyme ribonucleotide reductase, allowing cells defective in gor and trxB, or defective in gshB and trxB, to grow in the absence of DTT.Different classes of mutant forms of AhpC can allow strains defective in gamma-glutamylcysteine ​​synthetase (gshA) activity and defective in trxB to grow in the absence of DTT. These include AhpC V164G, AhpC S71F, AhpC E173 / S71F, AhpC E171Ter, and AhpC dup162-169 (Faulkner et al., Proc Natl Acad Sci USA 2008 May 6;105(18):6735-6740, Epub 2008 May 2). In such strains with an oxidative cytoplasm, exposed protein cysteines are readily oxidized, leading to the formation of disulfide bonds, in a process catalyzed by thioredoxin, reversing their physiological function. Other proteins that may help reduce the effects of oxidative stress in host cells with an oxidative cytoplasm are HPI (hydroperoxidase I) catalase peroxidase, encoded by E. coli katG, and HPII (hydroperoxidase II) catalase peroxidase, encoded by E. coli katE, which disproportionate peroxides to water and 02 (Farr and Kogoma, Microbiol Rev. 1991 Dec;55(4):561-585; Review). Increasing the levels of KatG and / or KatE proteins in a host cell through induced co-expression or through increased levels of constitutive expression is an aspect of some embodiments of the present disclosure.

[0054] Another modification that can be made to the host cell is to express the sulfhydryl oxidase Ervlp from the inner membrane space of yeast mitochondria in the host cell cytoplasm, which has been shown to increase production of a variety of complex 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, Microb Cell Fact 2011 Jan 7;10:1).

[0055] Host cells containing the expression constructs preferably also express cDsbA and / or cDsbC and / or Ervlp, are defective in trxB gene function, are defective in any of gor, gshB or gshA gene function, optionally have increased levels of katG and / or katE gene function, and express an appropriate mutant form of AhpC such that the host cells can grow in the absence of DTT.

[0056] Chaperones. In some embodiments, the desired gene product is co-expressed with other gene products, e.g., chaperones, that are beneficial to the production of the desired gene product. Chaperones are proteins that assist in the folding or unfolding and / or assembly or disassembly of other gene products, but do not occur in the resulting monomeric or multimeric gene product structures when the structures are performing their normal biological function (completing the folding and / or assembly process). Chaperones can be expressed from an inducible or constitutive promoter in an expression construct, or expressed from a host cell chromosome, and preferably, expression of the chaperone protein 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 folding factors DnaK / DnaJ / GrpE, DsbC / DsbG, GroEL / GroES, IbpA / IbpB, Skp, Tig (trigger factor), and FkpA, which have been used to prevent protein aggregation of cytoplasmic or periplasmic proteins. Expression of these chaperones in combination has been shown to be beneficial for protein expression, as DnaK / DnaJ / GrpE, GroEL / GroES, and ClpB can function synergistically in supporting protein folding (Makino et al., Microb Cell Fact 2011 May 14;10:32). When expressing eukaryotic proteins in prokaryotic host cells, eukaryotic chaperone proteins such as protein disulfide isomerase (PDI) from the same or related eukaryotic species are expressed or inducibly co-expressed with the desired gene product in certain embodiments.

[0057] One chaperone that can be expressed in a host cell is a protein disulfide isomerase from the soil nematode (soft-rot fungus) Humicola insolens. The amino acid sequence of Humicola insolens PDI is set forth as SEQ ID NO:1 in WO / 2017 / 106583 and lacks the signal peptide of the native protein, thereby remaining in the host cytoplasm. The nucleotide sequence encoding PDI was optimized for expression in E. coli and an expression construct for PDI is set forth as SEQ ID NO:2 in WO / 2017 / 106583. SEQ ID NO:2 contains a GCTAGC Nhel restriction site at its 5' end, an AGGAGG ribosome binding site at nucleotides 7-12, the PDI coding sequence at nucleotides 21-1478, and a GTCGAC Sail restriction site at its 3' end. The nucleotide sequence of SEQ ID NO:2 was designed to be inserted immediately downstream of a promoter, such as an inducible promoter. The Nhel and Sail restriction sites of SEQ ID NO:2 can be used to insert this into a vector multiple cloning site, such as that of the pSOL expression vector (SEQ ID NO:3 of WO / 2017 / 106583) described in published U.S. Patent Application No. 2015 / 353940(A1), which is incorporated by reference in its entirety. Other PDI polypeptides can also be expressed in host cells, including PDI polypeptides from various species (Saccharomyces cerevisiae (UniProtKB PI 7967), Homo sapiens (UniProtKB P07237), Mus musculus (UniProtKB P09103), Caenorhabditis elegans (UniProtKB Q 17770 and Q 17967), Arabdopsis thaliana (UniProtKB 048773, Q9XI01, Q9S G3, Q9LJU2, Q9MAU6, Q94F09, and Q9T042), Aspergillus niger (UniProtKB Q12730) and modified forms of such PDI polypeptides.In certain embodiments of the present disclosure, the PDI polypeptide expressed in a host cell of the present disclosure shares at least 70%, or 80%, or 90%, or 95% 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 SEQ ID NO:1 of WO / 2017 / 106583, where the amino acid sequence identity is determined according to Example 10 of WO / 2017 / 106583.

[0058] Cellular transport of cofactors. When using the expression system of the present disclosure to produce enzymes that require cofactors for function, it is useful to use host cells that can synthesize the cofactor from available precursors or import it from the environment. Common cofactors include ATP, coenzyme A, flavin adenine dinucleotide (FAD), NAD+ / NADH, and heme. Polynucleotides encoding cofactor transporting and / or cofactor synthesising polypeptides can be introduced into the host cell, and such polypeptides can be constitutively expressed or inducibly co-expressed with the gene products produced by the methods of the present disclosure.

[0059] Glycosylation of polypeptide gene products. Host cells may have modifications in their ability to glycosylate polypeptides. For example, eukaryotic host cells may have eliminated or reduced gene functions in glycosyltransferase and / or oligo-saccharyltransferase genes, thereby impairing normal eukaryotic glycosylation of polypeptides to form glycoproteins. Prokaryotic host cells, such as E. coli, that do not normally glycosylate polypeptides, can be modified to express a set of eukaryotic and prokaryotic genes that provide glycosylation functions (DeLisa et al., WO2009089154A2, 2009 Jul 16).

[0060] Available host cell lines with altered genetic function. To generate preferred strains of host cells for use in the expression systems and methods of the present disclosure, it is useful to start with a strain that already contains the desired genetic alterations (Table A, WO / 2017 / 106583). [Table 2]

[0061] Expression constructs In some embodiments of the present disclosure, an inducible promoter is contemplated for use with the expression construct. Exemplary promoters are described herein and also in WO / 2016 / 205570, which is incorporated herein by reference in its entirety. As described herein, a cell comprising one or more expression constructs may optionally comprise one or more inducible promoters to express a gene product of interest. In one embodiment, the gene product is a fusion protein as described herein. In other embodiments, the gene product is a protein, e.g., a therapeutic protein.

[0062] Expression constructs. An expression construct is not a naturally occurring molecule, since it is a polynucleotide designed for the expression of one or more gene products of interest. An expression construct can be integrated into a host cell chromosome or maintained in a host cell as a polynucleotide molecule that replicates independently of the host cell chromosome, such as a plasmid or artificial chromosome. 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 modifies the expression of the 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 artificial chromosome. The following is a description of certain types of polynucleotide sequences that can be used in an expression construct for the expression or co-expression of gene products, including fusion proteins, as described herein.

[0063] Origins of replication. Expression constructs must contain origins of replication, also called replicons, to be maintained in a host cell as independently replicating polynucleotides. Different replicons that use the same replication mechanism cannot be maintained together in a single host cell through repeated cell divisions. As a result, plasmids can be classified into incompatibility groups depending on the origins of replication they contain, as shown in Table 2 of WO / 2016 / 205570. Origins of replication can be selected for use in expression constructs based on incompatibility group, copy number, and / or host range, among other criteria. As mentioned above, when two or more different expression constructs are used in the same host cell for co-expression of multiple gene products, it is best for the different expression constructs to contain origins of replication from different incompatibility groups, e.g., a pMB1 replicon in one expression construct and a pl5A replicon in another expression construct. The average copy number of an expression construct in a cell relative to the number of host chromosomal molecules is determined by the origin of replication contained in that expression construct. The copy number can vary from a few copies to several hundred per cell (Table 2 in WO / 2016 / 205570). In one embodiment of the present disclosure, different expression constructs are used that contain inducible promoters activated by the same inducer, but with different origins of replication. By selecting origins of replication that maintain each of the different expression constructs at a certain approximate copy number in the cell, it is possible to adjust the overall production level of a gene product expressed from one expression construct relative to another gene product expressed from a different expression construct. As an example, to co-express subunits A and B of a multimeric protein, an expression construct "colEl-Para-A" is created that contains the colEl replicon, the am promoter, and the coding sequence of subunit A expressed from the am promoter.

[0064] Another expression construct, "pl5A-Para-B," is created that contains the pl 5A replicon, the am promoter, and the coding sequence for subunit B. These two expression constructs can be maintained together in the same host cell, and expression of both subunit A and subunit B is induced by adding one inducer, arabinose, to the growth medium. If the expression level of subunit A needs to be significantly increased relative to that of subunit B, a new expression construct, pUC9ori-Para-A, for subunit A can be created, for example, with a modified pMB1 as found in the origin of replication of the pUC9 plasmid ("pUC9ori"), to bring the stoichiometric ratio of the expression amounts of the two subunits closer to the desired ratio. Expressing subunit A from a high copy number expression construct such as pUC9ori-Para-A should increase the amount of subunit A produced relative to the expression of subunit B from pl5A-Para-B. Similarly, using an origin of replication that maintains an expression construct at a lower copy number, such as pSOOl (WO / 2016 / 205570), may reduce the overall level of gene product expressed from the construct. The choice of origin of replication may determine which host cells are able to maintain an expression construct containing the replicon.For example, an expression construct containing the colEl origin of replication has a relatively narrow range of available hosts that are species within the family Enterobacteriaceae, whereas an expression construct containing the RK2 replicon can be maintained in E. coli, Pseudomonas aeruginosa, Pseudomonas putida, Azotobacter vinelandii, and Alcaligenes eutrophus, and if the expression construct contains the RK2 replicon and several regulator genes from the RK2 plasmid, it can be maintained in hosts as diverse as Sinorhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus, and Rhodobacter sphaeroides (Kiies and Stahl, Microbiol Rev 1989 Dec;53(4):491-516).

[0065] Similar considerations can be used to create expression constructs for inducible or co-expression in eukaryotic cells. For example, the 2 micron circular plasmid of Saccharomyces cerevisiae is compatible with plasmids from other yeast strains, such as pSRl (ATCC Accession Nos. 48233 and 66069, Araki et al., J Mol Biol 1985 Mar 20;182(2):191-203) and pKDl (ATCC Accession No. 37519, Chen et al., Nucleic Acids Res 1986 Jun 11;14(11):4471-4481).

[0066] Selectable markers. Expression constructs usually contain a selection gene, also referred to as a selectable marker, that encodes a protein necessary for the survival or growth of host cells in a selective culture medium. Host cells that do not contain an expression construct containing a selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins or complement auxotrophic defects of the host cells. One example of a selection scheme utilizes a drug, such as an antibiotic, to arrest the growth of the host cells. Cells that contain an expression construct containing a selectable marker produce a protein that confers drug resistance and survive the selection regimen. Some examples of antibiotics (and abbreviations that indicate genes that provide an antibiotic resistance phenotype) commonly used for selection of selectable markers are ampicillin (AmpR), chloramphenicol (CmlR or CmR), kanamycin (KanR), spectinomycin (SpcR), streptomycin (StrR), and tetracycline (TetR). Many of the plasmids in Table 2 of WO / 2016 / 205570 contain a selectable marker, such as pBR322 (AmpR, TetR), pMOB45 (CmR, TetR), pACYClW (AmpR, KanR), and pGBMl (SpcR, StrR). The native promoter region of the selection gene is usually included as part of the selectable marker portion of the expression construct, along with the coding sequence for its gene product. Alternatively, the coding sequence for the selection gene may be expressed from a constitutive promoter.

[0067] In various embodiments, suitable selectable markers include, but are not limited to, neomycin phosphotransferase (npt II), hygromycin phosphotransferase (hpt), dihydrofolate reductase (dhfr), zeocin, phleomycin bleomycin resistance gene (ble), gentamicin acetyltransferase, streptomycin phosphotransferase, mutant forms of acetolactate synthase (als), bromoxynil nitrilase, phosphinothricin acetyltransferase (bar), enolpyruvylshikimate-3-phosphate (EPSP) synthase (aro A), muscle specific tyrosine kinase receptor molecule (MuSK-R), copper zinc superoxide dismutase (sod1), metallothionein (cup1, MT1), beta-lactamase (BLA), puromycin N-acetyl-transferase (pac), blasticidin acetyltransferase (bls), blasticidin deaminase (bsr), histidinol dehydrogenase (HDH), N-succinyl-5-aminoimidazole-4-carboxamide ribotide (SAICAR) synthase (ade1), argininosuccinic acid degrading enzyme (arg4), beta-isopropylmalate dehydrogenase (leu2), invertase (suc2), orotidine-5'-phosphatase (OMP), decarboxylase (ura3), and orthologs of any of the above.

[0068] Inducible promoter. As described herein, there are several different inducible promoters that can be included in the expression construct as part of the inducible co-expression system of the present disclosure. A preferred inducible promoter shares at least 80% polynucleotide sequence identity (more preferably at least 90% identity, most preferably at least 95% identity) for 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 WO / 2016 / 205570 with reference to the E. coli K-12 substrain MG1655 genome sequence, where the percentage of polynucleotide sequence identity is determined using the method of Example 11 of WO / 2016 / 205570. Under "standard" induction conditions (see Example 5 of WO / 2016 / 205570), preferred inducible promoters have a strength that is at least 75% (more preferably at least 100%, most preferably at least 110%) 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. (Example 6 of WO / 2016 / 205570). Within an expression construct, an inducible promoter is positioned 5' (or "upstream of") to the coding sequence for a gene product that is to be expressed upon induction, such that the presence of the inducible promoter directs transcription of the gene product coding sequence in the 5' to 3' direction relative to the coding strand of a polynucleotide encoding the gene product.

[0069] Ribosome Binding Site. For a polypeptide gene product, the nucleotide sequence of the region between the transcription initiation site and the start codon of the coding sequence of the gene product to be expressed upon induction corresponds to the 5' untranslated region ("UTR") of the mRNA of the polypeptide gene product. Preferably, the region of the expression construct corresponding to the 5'UT contains a nucleotide sequence similar to a consensus ribosome binding site (RBS, also called Shine-Dalgarno sequence) found in the host cell species. In prokaryotes (archaea and bacteria), the RBS consensus sequence is GGAGG or GGAGGU, and in bacteria such as E. coli, the RBS consensus sequence is AGGAGG or AGGAGGU. The RBS is typically separated from the start codon by 5-10 intervening nucleotides. In the expression construct, the RBS sequence is preferably at least 55% identical, more preferably at least 70% identical, and most preferably at least 85% identical to the AGGAGGU consensus sequence and is separated from the start codon by 5-10 intervening nucleotides, more preferably 6-9 intervening nucleotides, and most preferably 6 or 7 intervening nucleotides. The ability of a given RBS to generate a desired translation initiation rate can be calculated using the RBS Calculator at the website salis.psu.edu / software / RBSLibraryCalculatorSearchMode, and the same tool can be used to optimize synthetic RBSs for translation rate over a range of 100,000 fold or more (Salis, Methods Enzymol 2011;498:19-42).

[0070] Multiple Cloning Site. A multiple cloning site (MCS), also called a polylinker, is a polynucleotide that contains multiple restriction sites adjacent to or overlapping each other. The restriction sites of an MCS typically occur once within the MCS sequence and preferably do not occur within the remainder of a plasmid or other polynucleotide construct, thereby allowing a restriction enzyme to cut the plasmid or other polynucleotide construct only within the MCS. Examples of MCS sequences are those in the pBAD series of expression vectors, including pBAD18, pBAD18-Cm, pBAD18-Kan, pBAD24, pBAD28, pBAD30, and pBAD33 (Guzman et al., J Bacteriol 1995 Jul;177(14):4121-4130), or those in the pPRO series of expression vectors derived from the pBAD vector, such as pPR018, pPR018-Cm, pPR018-Kan, pPR024, pPRO30, and pPR033 (U.S. Patent No. 8,178,338 B2; May 15, 2012; Keasling, Jay). Multiple cloning sites can be used to create expression constructs. By locating a multiple cloning site 3' (or downstream) of a promoter sequence, the MCS can be used to insert coding sequences for gene products to be expressed or co-expressed into the construct at the appropriate location relative to the promoter such that transcription of the coding sequences occurs. Depending on which restriction enzyme is used to cut within the MCS, a portion of the MCS sequence may remain in the expression construct after a coding sequence or other polynucleotide sequence is inserted into the expression construct. Any remaining MCS sequence may be upstream, downstream, or on either side of the inserted sequence. The ribosome binding site may be located upstream of the MCS, preferably immediately adjacent to the MCS or separated from it by only a few nucleotides, in which case the RBS is upstream of any coding sequence inserted into the MCS. Another alternative is to include the ribosome binding site within the MCS, in which case the choice of restriction enzyme used to cut within the MCS will determine whether the RBS is retained and in what relationship to the inserted sequence.A further alternative is to include a RBS within the polynucleotide sequence inserted into the expression construct at the MCS, preferably in appropriate relationship with any coding sequence to stimulate initiation of translation from the transcribed messenger RNA.

[0071] Expression from a constitutive promoter. The expression constructs of the present disclosure can also include a coding sequence expressed from a constitutive promoter. Unlike an inducible promoter, a constitutive promoter initiates continuous production of a gene product under most growth conditions. One example of a constitutive promoter is the constitutive promoter of the Tn3 bla gene, which encodes beta-lactamase and is responsible for the ampicillin resistance (AmpR) phenotype conferred to host cells by many plasmids, including pBR322 (ATCC 31344), pACYClW (ATCC 37031), and pBAD24 (ATCC 87399). Another constitutive promoter that can be used in the expression construct is the promoter of the E. coli lipoprotein gene Ipp, located at positions 1755731-1755406 (plus strand) of E. coli K-12 substrain MG1655 (Inouye and Inouye, Nucleic Acids Res 1985 May 10;13(9):3101-3110). A further example of a constitutive promoter that has been used for heterologous gene expression in E. coli is the trpLEDCBA promoter, located at positions 1321169-1321133 (minus strand) of E. coli K-12 substrain MG1655 (Windass et al., Nucleic Acids Res 1982 Nov 11;10(21):6639-6657). Constitutive promoters can be used in the expression construct for the expression of selectable markers as described herein, as well as for the constitutive expression of other gene products useful for co-expression of the desired product. For example, transcriptional regulators of inducible promoters such as AraC, PrpR, RhaR, and XylR, if not expressed from a bidirectional inducible promoter, can alternatively be expressed from a constitutive promoter, either in the same expression construct as the inducible promoter they regulate, or in a different expression construct. Similarly, gene products useful for the production or transport of inducers, such as PrpEC, AraE, or Rha, or proteins that modify the reduced-oxidative environment of the cell, to name a few, can be expressed from a constitutive promoter in an expression construct.Co-expressed gene products, and the resulting gene products useful for producing a desired product, also include chaperone proteins, cofactor transporters, and the like.

[0072] Signal peptides. Polypeptide gene products expressed or co-expressed by the disclosed methods can contain or lack a signal peptide, depending on whether such gene products are desired to be exported from the host cell cytoplasm into the periplasm or retained in the cytoplasm, respectively. Signal peptides (also referred to as signal sequences, leader sequences, or leader peptides) are structurally characterized by a stretch of hydrophobic amino acids, approximately 5-20 amino acids long, often about 10-15 amino acids long, that has a tendency 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 cleaved from mature polypeptides typically end with a stretch of amino acids that is recognized and cleaved by a signal peptidase. Signal peptides are functionally characterized by their ability to direct the transport of polypeptides, either co-translationally or post-translationally, through the cell membrane of prokaryotes (or the inner membrane of gram-negative bacteria such as E. coli) or 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, for example using methods such as those described in Example 12 of WO / 2016 / 205570.

[0073] Below are some descriptions of inducible promoters that can be used in expression constructs for expression or co-expression of gene products, as well as genetic modifications that can be made to host cells containing such expression constructs. These examples of inducible promoters and associated genes are derived from Escherichia coli (E. coli) strain MG1655 (American Type Culture Collection Accession No. ATCC 700926), which is a substrain of E. coli K-12 (American Type Culture Collection Accession No. ATCC 10798), unless otherwise specified. Table 1 of WO / 2016 / 205570 lists the genomic locations in E. coli MG1655 of the nucleotide sequences of these examples of inducible promoters and associated genes. The nucleotide and other gene sequences are referenced by their genomic locations in Table 1 of WO / 2016 / 205570 and are expressly incorporated herein by reference. Additional information regarding the E. coli promoters, genes, and strains described herein can be found in many public resources, including the online EcoliWiki resource at ecoliwiki.net.

[0074] 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), typically the terms "arabinose promoter" and "ara promoter" are used to designate the araBAD promoter. Several additional terms, such as Para, ParaB, ParaBAD, and PBAD, have been used to indicate the E. coli araBAD promoter. Use of "ara promoter" or any of the above alternative terms refers to the E. coli araBAD promoter. As can be seen from the use of the alternative term "araC-araBAD promoter," the araBAD promoter is considered to be part of a bidirectional promoter, where the araBAD promoter controls expression of the araBAD operon in one direction, and the araC promoter, which is adjacent to the araBAD promoter and on the opposite strand to the araBAD promoter, controls expression of the araC coding sequence in the other direction. The AraC protein is both a positive and negative transcriptional regulator of the araBAD promoter. In the absence of arabinose, the AraC protein represses transcription from the PBAD, whereas in the presence of arabinose, the AraC protein, which changes its conformation upon binding arabinose, becomes a positive regulatory element that allows transcription from the PBAD. The araBAD operon encodes proteins that metabolize L-arabinose by converting it to D-xylulose-5-phosphate via the intermediates L-ribulose and L-ribulose-phosphate. In order to maximize induction of expression from an arabinose-inducible promoter, it is useful to remove or reduce the function of AraA, which catalyzes the conversion of L-arabinose to L-ribulose, and optionally also remove or reduce the function of at least one of AraB and AraD.By eliminating or reducing the host cell's ability to reduce the effective concentration of arabinose in the cell, more arabinose can be made available for induction of arabinose-inducible promoters by eliminating or reducing the cell's ability to convert arabinose to other sugars. Genes encoding transporters that move arabinose into the host cell are araE, which encodes the low-affinity L-arabinose proton symporter, and the araFGH operon, which encodes subunits of the ABC superfamily high-affinity L-arabinose transporter. Other proteins that can transport L-arabinose into cells are certain variants of the LacY lactose permease: the LacY(AlWC) and LacY(AlWV) proteins, which have a cysteine ​​or valine amino acid instead of alanine at position 177, respectively (Morgan-Kiss et al., Proc Natl Acad Sci USA 2002 May 28;99(11):7373-7377). To achieve uniform induction of an arabinose-inducible promoter, it is useful to make the transport of arabinose into the cell independent of regulation by arabinose. This can be achieved by removing or reducing the activity of the AraFGH transporter protein and modifying the expression of araE so that it is only transcribed from a constitutive promoter. Constitutive expression of araE can be achieved by removing or reducing the function of the native araE gene and introducing into the cell an expression construct that contains the coding sequence of the AraE protein expressed from a constitutive promoter. Alternatively, in cells lacking AraFGH function, the promoter controlling the expression of the chromosomal araE gene of the host cell can be changed from an arabinose-inducible promoter to a constitutive promoter. In a similar manner, as a further alternative for uniform induction of an arabinose-inducible promoter, a host cell lacking AraE function can have any functional AraFGH coding sequence present in the cell expressed from a constitutive promoter.As another alternative, it is possible to express both the araE gene and the araFGH operon from a constitutive promoter by replacing the native araE and araFGH promoters with a constitutive promoter in the host chromosome. It is also possible to use a mutation in the LacY lactose permease that removes or reduces the activity of both the AraE and AraFGH arabinose transporters, allowing this protein to transport arabinose in that situation. Because expression of the lacY gene is not normally regulated by arabinose, the use of LacY mutants such as LacY(A177C) or LacY(A177V) does not result in an "all-or-none" 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 over 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.

[0075] Propionate promoter. The "propionate promoter" or "prp promoter" is the promoter of the E. coli prpBCDE operon, also called ΡΡΦΒara. Like 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, and activates transcription from the prp promoter when the PrpR protein binds 2-methylcitrate ("2-MC"). Propionate (also called propanoate) is the ion of propionic acid (or "propanoic acid"), CH3CH2COO-, with the general formula sharing certain properties of this class of molecules. [ka] It is the smallest of the "fatty" acids, having a carboxylate moiety, which produces an oily layer when salted out of water and has a soapy potassium salt. Commercially available propionate is generally sold as a monovalent cation salt of propionic acid, e.g., sodium propionate (CH3CH2COONa), or as a divalent cation salt, e.g., calcium propionate (Ca(CH3CH2COO)2). Propionate is membrane permeable and is metabolized to 2-MC by conversion of propionate to propionyl-CoA by PrpE (propionyl-CoA synthetase) and then conversion of propionyl-CoA to 2-MC 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 catabolism of 2-MC to smaller products such as pyruvate and succinate. Therefore, to maximize the induction of the propionate-inducible promoter by propionate added to the cell growth medium, it is desirable to have a host cell with PrpC and PrpE activities to convert propionate to 2-MC, but also with eliminated or reduced PrpD activity, and optionally eliminated or reduced PrpB activity, to prevent 2-MC from being metabolized. Another operon encoding proteins involved in 2-MC biosynthesis is the scpA-argK-scpBC operon, also referred to as the sbm-yg / DGH operon. These genes encode proteins required for the conversion of succinate to propionyl-CoA, which can then be converted to 2-MC by PrpC. Removal or reduction of the function of these proteins removes parallel pathways 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.It was found that the sbm-ygfD-ygfG-ygfH-ygfl deletion (Lee and Keasling, "A propionate-inducible expression system for enteric bacteria", Appl Environ Microbiol 2005 Nov;71(11):6856-6862), introduced into E. coli BL21(DE3) to create strain JSB, was useful in reducing background expression in the absence of exogenously supplied inducer, but this deletion also reduced overall expression from the prp promoter in strain JSB. Note, however, that the deletion sbm-ygfD-ygfG-ygfH-ygfl also appears to affect ygfl, which encodes a putative LysR family transcriptional regulator of unknown function. The genes sbm-yg / DGH are transcribed as one operon, with ygfl transcribed from the opposite strand. Because the 3' ends of the ygfti and ygfl coding sequences overlap by a few base pairs, a deletion that removes all of the sbm-yg / DGH operon is likely to remove ygfl coding function as well. Removing 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-yg / DGH (or scpA-argK-scpBC) operon, while leaving enough of the 3' end of the ygfli (or scpC) gene so as not to affect expression of ygfl, may be sufficient to sufficiently reduce background expression from a propionate-inducible promoter without reducing the maximum level of induced expression.

[0076] Rhamnose promoter. (As used herein, "rhamnose" means L-rhamnose.) "Rhamnose promoter" or "rha promoter" or PrhaSR is the promoter of 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, RhaR and RhaS, involved in regulating expression. The RhaR protein activates expression of the rhaSR operon in the presence of rhamnose, while the RhaS protein activates expression of the L-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively (Wickstrum et al, J Bacteriol 2010 Jan;192(1):225-232). The RhaS protein can also activate expression of the rhaSR operon, but in fact RhaS negatively autoregulates this expression by interfering with the ability of the cyclic AMP receptor protein (CRP) to coactivate expression with RhaR to a much higher level. 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 aldolase), which converts L-rhamnulose-1-P to L-lactaldehyde and DHAP (dihydroxyacetone phosphate). To maximize the amount of rhamnose in a 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 RhaA and / or 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 the rhamnose-inducible promoter and to enhance the sensitivity of the rhamnose-inducible promoter to induction by exogenously supplied rhamnose, RmlA, RmlB, RmlC, and RmlD are expressed in the rhamnose-inducible promoter.

[0077] It may be useful to eliminate or reduce the function of one or more of the RmlD proteins. L-rhamnose is transported into the cell by RhaT, rhamnose permease, or the L-rhamnose:proton symporter. As described above, expression of RhaT is activated by the transcription regulator RhaS. To make expression of RhaT independent of induction by rhamnose (which induces expression of RhaS), the host cell can be modified so that all functional RhaT coding sequences in the cell are expressed from a constitutive promoter. In addition, the coding sequence 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 increases due to the lack of negative autoregulation by RhaS, the level of expression of the rhamnose catalytic operon rhaBAD decreases, and the ability of rhamnose to induce expression from the rha promoter is further increased.

[0078] Xylose promoter. (As used herein, "xylose" means D-xylose.) Xylose promoter, or "xyl promoter", or PxylA, refers 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 operon and the xylFGHR operon are expressed in opposite directions on E. coli from adjacent xylose-inducible promoters (Song and Park, J Bacteriol. 1997 Nov;179(22):7025-7032). The transcriptional regulator for both the PxylA and PxylF 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 induced by xylose, 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 then 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 a xylose-inducible promoter, it is desirable to reduce the amount of xylose catalytically degraded by removing 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 a separate operon, the expression of which is also inducible by xylose. To make expression of the xylose transporters independent of induction by xylose, the host cells can be engineered so that all functional xylose transporters are expressed from a constitutive promoter.For example, the xylFGH coding sequence may be deleted, making XylR the only active protein expressed from the xylose-inducible PxylF promoter, and the xylE coding sequence may be modified to be expressed from a constitutive promoter rather than its native promoter. As another example, the xylR coding sequence may be expressed from PxylA or a promoter in the expression construct, while the xylFGHR operon may be deleted and xylE may be expressed constitutively, or alternatively, the xylFGH operon (lacking the xylR coding sequence because it is present in the expression construct) may be expressed from a constitutive promoter and the xylE coding sequence may be deleted or modified so that it does not produce an active protein.

[0079] Lactose promoter. The term "lactose promoter" refers to the lactose-inducible promoter for the lacZYA operon, also known as lacZp1. This lactose promoter is located at approximately 365603-365568 in the genomic sequence of E. coli K-12 substrain MG1655 (NCBI Reference Sequence NC 000913.2, 11-JAN-2012) (minus strand, NA polymerase binding ("-35") site is at approximately 365603-365598, Pribnow box ("-10") is at 365579-365573, and transcription start site is at 365567). In some embodiments, the inducible expression system of the present disclosure may include a lactose-inducible promoter, such as the lacZYA promoter. In other embodiments, the inducible expression system of the present disclosure includes one or more inducible promoters that are not lactose-inducible promoters.

[0080] Alkaline phosphatase promoter. The terms "alkaline phosphatase promoter" and "phoA promoter" refer to the promoter of the phoApsiF operon, which is a promoter that is induced under phosphatase starvation conditions. The phoA promoter region is located at approximately 401647-401746 (positive strand, Pribnow box ("-10") is at 401695-401701 (Kikuchi et al., Nucleic Acids Res 1981 Nov 11;9(21):5671-5678)) in the genome sequence of E. coli K-12 substrain MG1655 (NCBI Reference Sequence NC 000913.3, 16-DEC-2014). The transcriptional activator of the phoA promoter is PhoB, which, together with the sensor protein PhoR, is a transcriptional regulator that forms a two-component signal transduction system in E. coli. PhoB and PhoR are transcribed from the phoBR operon located at approximately 417050-419300 (positive strand, PhoB coding sequence is at 417,142-417,831 and PhoR coding sequence is 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 is generally used to direct the transcription of gene products produced at stages when the host cell is starved for phosphate, e.g., at later stages of fermentation. In some embodiments, the inducible co-expression system of the present disclosure can include a phoA promoter. In other embodiments, the inducible co-expression system of the present disclosure includes one or more inducible promoters that are not the phoA promoter.

[0081] As described herein, for example, when a cell line carrying the expression construct is or will be used for commercial purposes, it may be advantageous or desirable to remove the expression construct described herein (e.g., by an inducible or constitutive "hardening" mechanism). Thus, in some embodiments, the expression construct may include a "kill switch." For example, in embodiments, the expression construct includes a temperature-sensitive origin of replication. Additional hardening methods are known in the art and include the use of detergents and intercalating agents, drugs, and antibiotics (Buckner, MMC, et al., FEMS Microbiology Reviews, fuy031, 42, 2018, 781-804).

[0082] Before the present disclosure is further described, it is to be understood that this disclosure is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present disclosure will be limited only by the appended claims.

[0083] When a range of values ​​is provided, it is understood that each intervening value between the upper and lower limit of that range, to one-tenth of the unit of the lower limit unless otherwise clearly indicated by context, and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0084] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of this disclosure, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference to disclose and describe the relevant methods and / or materials for which the publications are cited.

[0085] Please note that, as used herein, the singular forms "a," "and," and "the" include plural referents unless the context clearly indicates otherwise. Thus, for example, a reference to a "conformation-switching probe" includes a plurality of such conformation-switching probes, a reference to a "microfluidic device" includes a reference to one or more microfluidic devices and equivalents thereof known to those skilled in the art, and so forth. Furthermore, please note that the claims may be drafted to exclude any element, e.g., any optional element. Thus, this statement is intended to serve as a predicate for the use of exclusive language, such as "solely," "only," and the like, or the use of "negative" limitations in connection with the recitation of claim elements.

[0086] As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has distinct components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the disclosure. Any recited method may be carried out in the order of events recited or in any other order which is logically possible. This is intended to provide support for all such combinations. EXAMPLES

[0087] Example 1 Growth / no growth selection assay of high-expressing E. coli strains This example describes and implements a simple growth / no growth selection strategy to identify soluble protein high-expressing E. coli strains in a mixed population (library).

[0088] Here, using an E. coli strain harboring a plasmid encoding arabinose promoter-driven expression of an mCherry-FolA fusion protein, we show that (1) trimethoprim by itself has a very limited selectable dynamic range and (2) the addition of sulfamethoxazole sensitizes the mCherry-FolA strain to trimethoprim.

[0089] Strains: Strain #1. E. coli harboring a plasmid encoding an antibiotic target gene (folA) fusion under the control of the pBAD promoter. Strain #2. Control E. coli harboring a plasmid encoding a different antibiotic target gene (murA).

[0090] Experimental: Prepare trimethoprim antibiotic gradient plates with arabinose. The antibiotic gradient is made identical across the plates. However, the concentration of the second antibiotic (sulfamethoxazole) is uniform across each plate but varies between plates. Inoculate strains across the antibiotic gradient. Length of growth is a measure of relative antibiotic resistance. For strains harboring the pBAD plasmid with the folA gene, length of growth may be inversely proportional to sulfamethoxazole concentration. Plates containing the highest sulfamethoxazole concentration should have the shortest length of growth.

[0091] As shown in Figure 1, cells carrying the mCherry-FolA fusion are highly resistant to trimethoprim. Figure 1 shows that resistance to 100ug / ml trimethoprim (mCherry-FolA) is dependent on arabinose and the mCherry-FolA fusion. Sulfamethoxazole up to 16ug / ml did not inhibit growth.

[0092] Synergy with sulfamethoxazole improves the trimethoprim dynamic range as shown in Figure 2. Figure 2 shows that trimethoprim has strong synergy with sulfamethoxazole, with susceptibility to trimethoprim being restored at greater than 2ug / ml of sulfamethoxazole.

[0093] Example 2 Soluble protein selection assay-insulin-FolA fusions This example describes and performs the assay from Example 1 using two insulin-FolA fusion constructs (insulin variants) and using two E. coli chaperone libraries.

[0094] Agar plates were prepared covering a matrix of the two drugs trimethoprim and sulfamethoxazole in the combinations described herein and in Example 1 and Figures 1-2. The combination matrix is ​​necessary because the actual concentration of antibiotic tolerated by the best performing strain in the library is unknown. Two control strains carrying empty (chaperone-free) plasmids and an experimental strain carrying a chaperone plasmid were each individually plated across a set of plates covered with a combination matrix (e.g., trimethoprim-sulfamethoxazole matrix) as shown in Figure 3. Approximately 300 million cells per plate from each of the six strains were plated individually on all 16 plates. The plates were incubated for 5 days before final analysis.

[0095] After 5 days of incubation, differences were observed between the control and experimental strains in some of the agar plates. These differences could be slight, where the experimental plates showed larger colonies than the control, or significant, where growth was observed only in the experimental plates and not in the control. For each of the two insulin-FolA fusion / chaperone plasmid pairs, seven positive hits were randomly picked for further characterization using the soluble insulin HiPrBind assay (see, e.g., WO / 2021 / 163349). Of the seven strains picked for each of the two insulin-FolA fusions, two strains showed increased amounts of soluble insulin compared to the control strain.

[0096] In conclusion, the FolA selection assay allows for growth / no growth selection of strains that produced higher amounts of the soluble protein-FolA fusion of interest. In addition, a ranking of low to very high expressing strains can be obtained by modifying the selection conditions to increase the concentration of the synergistic antibiotic. Finally, large libraries containing billions of cells can be tested in parallel to identify the best performing subset of strains.

[0097] Example 3 Soluble Protein Selection Assay-Alpha Fetoprotein This example describes and performs the above-mentioned assays, which incorporate solid-phase assays for further analysis of populations of cells. Solid-phase assays are described herein and in, for example, PCT / US22 / 53107.

[0098] Applying the assay described in the above examples to two different target proteins (insulin and alpha-fetoprotein), the assay showed that the assay gave rise to hundreds of growing colonies (candidates) after selection on selective plates. The ideal goal would be to test all candidates to determine whether they were true winners or mutants that had acquired an escape mechanism. Here, a solid-phase assay was used to test the entire growing population and identify winners that actually produced more fusion protein than the background control.

[0099] Determination of relative expression of soluble alpha-fetoprotein by capillary immunoblot using the Protein Simple Jess automated instrument. Wet cell paste from 100 μL of culture was resuspended in lysis buffer (50 mM Tris-HCl, 200 mM sodium chloride, 10.0 g / L octylglucoside, 2400 U / mL lysozyme, 2 U / mL benzonase, pH 7.2) and lysed on ice for 1 h. After lysis, samples were diluted to an OD600 of 0.008 with 0.1x sample buffer (Protein Simple) and centrifuged at 3300×g for 30 min at 4° C. After centrifugation, samples were mixed with fluorescent master mix (Protein Simple) containing 100 mM iodoacetamide to an OD600 of 0.0064, vortexed, and heated to 95° C. for 5 min. The prepared samples were loaded onto a 12-230 kDa Separation Module plate (Protein Simple) along with rabbit monoclonal anti-alpha 1 fetoprotein primary antibody (abcam item number ab169552) and the necessary reagents to perform a chemiluminescence immunoassay. The Separation Module plate was loaded onto the Jess along with a 25 capillary cartridge and run using the default chemiluminescence detection method. The Simple Western assay results were analyzed using Protein Simple Compass for Simple Western software.

[0100] Case Study: Alpha-Fetoprotein A plasmid library was constructed with the following features: (Figure 4) An arabinose promoter controls a target protein (alpha-fetoprotein) genetically fused in-frame to folA at its C-terminus. In addition, plasmids were designed to carry two chaperones driven by propionate promoters, randomly selected from a pool of approximately 1200 unique chaperone genes. The theoretical diversity is 1.44 million unique combinations (1200 x 1200).

[0101] A set of agar plates was prepared containing 10 μg / ml kanamycin, 125 mM arabinose, 1 mM propionate, and one of 64 different combinations of trimethoprim / sulfamethoxazole according to Table B below. [Table 3]

[0102] Approximately 300 million cells of this library were plated onto each of 64 plates. A control strain carrying the library and the exact plasmid except that the plasmid does not contain the chaperone gene was also plated onto each of the 64 plates. The entire collection of plates was incubated at 30°C for 1 week. When the plates were examined, only a subset of the plates (inside the bold box in Table C) contained visible colonies; the others either had no change in growth compared to day 0 or were spread of bacteria with no robust colonies present. [Table 4]

[0103] An example of a plate with colonies after one week of incubation is shown in FIG.

[0104] Eight of the nine plates were analyzed by solid phase assay.

[0105] Round 1 Solid-phase assay

[0106] A nitrocellulose membrane (Cytiva Protran BA 83 Cat. No. 10401316) is layered on top of the agar. A sewing needle was used to apply latex paint to the agar for use in positional alignment of the agar plate with the image obtained from the nitrocellulose membrane after processing. This was done by dipping the needle into light-colored latex paint and then using the paint-covered needle tip to pierce the membrane through the agar, leaving some of the paint in the agar and a puncture mark on the membrane. The membrane was then gently lifted off the agar surface and placed on Whatman paper (Cytiva Cat. No. 1001-090) with the side containing the bound cells facing up. In all subsequent steps, the membrane is always placed with the cell side facing up. Excess paint was removed from the nitrocellulose membrane using Whatman paper. The nitrocellulose was then transferred to a clean dish. 2 ml of fixative solution [2.6% (w / v) paraformaldehyde, 0.04% (w / v) glutaraldehyde, 32.25 mM Na 3 PO 4, pH 7.4] was applied to the membrane and allowed to incubate for 3 minutes at room temperature. The membrane was then washed three times with 1x PBS [135 mM NaCl, 2.7 mM KCl, 11 mM phosphate buffer, pH 7.4]. The membrane was then incubated overnight in blocking solution [0.1 M NaHCO3 pH 8.6, 1 mM EDTA, 5 mg / ml bovine serum albumin (fraction V), 1 μM biotin]. The next day, the membrane was transferred to a fresh dish and treated with 5 ml of lysozyme solution [Millipore Sigma catalog number 71110-4, diluted 10,000 times in 1x Immunoassay buffer (PerkinElmer catalog number AL000F)] for 10 minutes. Subsequently, the membrane was transferred to a fresh dish and incubated overnight with detection reagents for alpha-fetoprotein [0.25 nM anti-alpha-fetoprotein (abcam catalog no. ab130748), 0.25 nM anti-rabbit antibody-alkaline phosphatase (Millipore Sigma catalog no. A2556)]. The next day, the membrane was washed 6-8 times, draining excess liquid each time, and the membrane was transferred to a fresh dish containing 20-50 ml of 1x wash buffer (Azure Biosystems catalog no. AC2113). After the last wash, the membrane was treated with 2 ml of alkaline phosphatase substrate (SeraCare KPL PhosphaGLO, catalog no. 5430-0055) for about 3 min. The membrane was drained and placed on Whatman paper (Cytiva catalog no. 1001-090). The puncture marks on the membrane are highlighted by hand using a black pen. The membrane was then placed in the imaging chamber of an Azure 600 imager (Azure Biosystems) and imaged for (1) fluorescence (excitation 732 / emission 832) and (2) chemiluminescence. The fluorescent image captured the pen markings and was merged with the chemiluminescent image using Azure Biosystems image capture software. The fluorescent image contains markings to align the chemiluminescent image with the agar plate. The merged image was printed onto a clear transparency film using a laser printer.

[0107] An example of the image obtained is shown in FIG.

[0108] Examination of the eight analyzed membranes (bold letters in the matrix in Table D below) showed that only three of the eight membranes contained positive hits. The three membranes that contained positive hits were from agar plates that contained the trimethoprim / sulfamethoxazole combination from the blue box in the matrix below. This result suggests that only colonies appearing at the highest tolerated concentration of trimethoprim / sulfamethoxazole contain the true winners of this selection strategy. [Table 5]

[0109] Recovery and characterization of hits The resulting images were used to pick up 50 hits from the corresponding plates to guide hit recovery. The merged image (fluorescence + chemiluminescence image) contains both the location of the hits and alignment information to recover live hits from the source agar plate. The agar is aligned with the printed transparency film using the paint marks in the agar and the dots captured by fluorescence imaging on the transparency film. Putative hits are recovered by gently touching the loop to the surface of the agar using the blunt end of a sterile loop and then touching the surface of a fresh induction plate [LB / Kan (10 μg / ml) / arabinose (125 μM) / propionate (1 mM)] gridded into 8 sectors. Each putative hit is given one sector. In each sector, a sterile loop is then used to streak out the initial inoculum and isolate for single colonies by spreading the recovered E. coli. The induction plate is incubated at 30° C. for 2 days. The plate is then processed using the same procedure as described for round 1 solid-phase hit identification.

[0110] Round 2 Results Illustrative images of test plates compared to control plates Images from round 2 in FIG. 7, where the alpha-fetoprotein signal from the eight picked hits is greater than the alpha-fetoprotein signal obtained from either the naive library, the no-chaperone control strain, or the positive control strain.

[0111] To ensure strain purity and stable genotype, singles were picked from each dark sector from the round 2 image and passaged on LB kanamycin (50 μg / ml) agar plates by streaking out single colonies. The aim is to identify stable strains that improve alpha-fetoprotein expression without any selective pressure (i.e., the presence of trimethoprim / sulfamethoxazole in the medium). After overnight growth, a repeat streak on LB kanamycin (50 μg / ml) agar was performed on a fresh plate. Well-isolated single colonies after the second round of streak purification were patched onto fresh sectored induction plates, with each sector populated by a single colony. Plates were incubated for 2 days and processed by solid phase as described above. Images of processed membranes shown in Figure 8 show that 22 of the sectors are still positive for alpha-fetoprotein expression, with the signal from many sectors exceeding that of the positive control strain.

[0112] Plasmids from 22 positive strains were recovered and analyzed. Twelve unique plasmids were identified. The folA gene was excised from these 12 plasmids and recircularized to create new expression plasmids lacking folA but still harboring the original chaperone (Figure 9).

[0113] The new plasmid was transformed back into a fresh host E. coli strain, and the newly created strain was streaked onto fresh sectored induction plates and incubated at 30°C for 24 h for analysis of alpha-fetoprotein expression by solid-phase assay.

[0114] Solid-phase assays showed that two of the newly created strains (#4 and #5) clearly expressed more alpha-fetoprotein on agar plates than the negative control (N, no chaperone). (Figure 10) To determine whether these strains would perform better in liquid culture, the same 12 strains were cultured in liquid induction medium containing 50 μg / ml kanamycin, 125 μM arabinose, and 1 mM propionate. After 24 hours of growth at 30°C, the cultures were harvested by pelleting the cells, and soluble monomeric alpha-fetoprotein was quantified by automated capillary Western analysis. The relative abundance of alpha-fetoprotein was normalized to that present in the negative control strain (no chaperone).

[0115] In liquid culture, six strains (#1, 2, 8, 9, 11) clearly expressed more alphafetoprotein than the negative control strain (no chaperone). The winners in liquid culture were not the same as the two strains (#5 and 6) identified on agar plates. This finding is not uncommon. (Figure 11)

[0116] These data demonstrated that the folA fusion assay identifies higher performing strains within pooled libraries, especially when coupled in tandem with the solid-phase assay.

[0117] The various embodiments described above can be combined to provide further embodiments. All U.S. patents, U.S. patent application publications, U.S. patent applications, foreign patents, foreign patent applications, and non-patent publications mentioned herein and / or listed in the Application Data Sheet are incorporated herein by reference in their entirety. Aspects of the embodiments can be modified as necessary to use concepts from the various patents, applications, and publications to provide still further embodiments.

[0118] These and other changes can be made to the embodiments in light of the above detailed description. In general, in the following claims, the terms used should not be construed to limit the claims to the specific embodiments disclosed in the specification and claims, but rather to include all possible embodiments, along with the full scope of equivalents to which such claims are entitled. Accordingly, the claims are not limited by this disclosure.

Claims

1. 1. A method for identifying a host cell capable of producing a soluble protein of interest, comprising the steps of: (a) preparing a population of host cells, each host cell of said population comprising an expression construct capable of expressing (i) a protein of interest and (ii) a fusion protein comprising a selection protein; (b) incubating the host cells of (a) under conditions that allow expression of the fusion protein, said conditions including a growth substance that includes at least two synergistic selection agents; (c) visualizing the host cells capable of growing; This method identifies the ability to produce a soluble protein of interest.

2. 1. A method for identifying a host cell which produces the greatest amount of a soluble protein of interest among a population of host cells, comprising: (a) preparing a population of host cells, each host cell of said population comprising an expression construct capable of expressing (i) a protein of interest and (ii) a fusion protein comprising a selection protein; (b) incubating the host cells of (a) under conditions that allow expression of the fusion protein, said conditions including a growth substance that includes at least two synergistic selection agents; (c) visualizing the host cells capable of growing; This method identifies the host cell that produces the greatest amount of the soluble protein of interest among a population of host cells.

3. 3. The method of claim 1 or 2, further comprising the step of binning the host cells based on the amount of soluble protein produced.

4. The method according to any one of claims 1 to 3, wherein the selection protein is a target of an antibiotic or an antibiotic resistance protein.

5. The method of claim 4, wherein the selection protein is FolA.

6. The method of claim 5, wherein the FolA is from E. coli.

7. The method of claim 6, wherein the FolA is set forth in SEQ ID NO:

1.

8. 8. The method of any one of claims 1 to 7, comprising using two synergistic selection agents, said synergistic selection agents being selected from the group consisting of antibiotics, sugars, chemical agents, enzyme substrate analogues, enzyme inhibitors, agents that sequester biomolecules, chelating agents, and agents that damage cell walls or cell membranes.

9. 9. The method of claim 8, wherein the two synergistic selective agents are trimethoprim and sulfamethoxazole.

10. The method according to any one of claims 1 to 9, wherein the protein of interest is a heterologous protein.

11. 11. The method of claim 10, wherein the heterologous protein is selected from the group consisting of an antibody, a Fab, a scFv, a nanobody, a T-cell receptor, and a chimeric antigen receptor, a growth factor, a cytokine, a hormone, an enzyme, or a functional fragment thereof.

12. 12. The method of any one of claims 1 to 11, wherein the population of host cells comprises a library of host cells, the library being composed of cells with unique genotypes and / or unique genetically engineered cells.

13. The method of claim 10, wherein the library comprises from about 1,000 to about 1 billion host cells.

14. The method according to any one of claims 1 to 13, wherein the host cell is selected from the group consisting of a eukaryotic cell, a prokaryotic cell, a bacterial cell, a mammalian cell, and an insect cell.

15. 15. The method of claim 14, wherein the bacterial cell is an E. coli cell.

16. 16. The method of claim 15, wherein the E. coli cell comprises: (a) an altered gene function of at least one gene encoding a transporter protein for an inducer of the at least one inducible promoter; (b) a reduced level of gene function of at least one gene encoding a protein that metabolizes the inducer of the at least one inducible promoter; (c) a reduced level of gene function of at least one gene encoding a protein involved in the biosynthesis of the inducer of the at least one inducible promoter; (d) an altered gene function of a gene that affects the reducing / oxidizing environment of the cytoplasm of the host cell; (e) a reduced level of gene function of a gene encoding a reductase; (f) at least one expression construct encoding at least one disulfide bond isomerase protein; (g) at least one polynucleotide encoding a form of DsbC lacking a signal peptide; and / or (h) at least one polynucleotide encoding an Ervlp.

17. The method of any one of claims 1 to 16, wherein the expression construct is an extrachromosomal construct selected from the group consisting of a polynucleotide, a plasmid, and an artificial chromosome.

18. The method of claim 17 , wherein the expression construct comprises an inducible promoter.

19. 20. The method of claim 17, wherein the expression construct comprises two or more inducible promoters.

20. 20. The method of claim 19, wherein at least one inducible promoter is a propionate-inducible promoter and at least one other inducible promoter is an L-arabinose-inducible promoter.

21. The method of any one of claims 1 to 20, wherein the growth substrate is selected from the group consisting of selective media.

22. The method of any one of claims 1 to 21, wherein the growth substrate comprises a matrix of two or more synergistic selection agents.

23. 23. The method of claim 22, wherein the two synergistic selective agents are trimethoprim and sulfamethoxazole, and said agents are present in the growth medium at concentration ranges of 1 ug / ml to 1000 ug / ml and 1 ug / ml to 1000 ug / ml, respectively.

24. The method according to any one of claims 1 to 23, wherein the conditions allowing expression of the fusion protein comprise the presence of one or more inducers of expression of the fusion protein.

25. 25. The method of any one of claims 1 to 24, wherein the host cells are identified using a technique selected from the group consisting of visual inspection, chemiluminescence, radiography, fluorescence, and colorimetry.

26. 26. The method of any one of claims 1 to 25, wherein visualising the host cells in step (c) comprises detecting growth on an agar plate.

27. (a) plating the host cells onto a growth substrate and incubating the host cells under conditions that permit host cell growth on the growth substrate; (b) optionally preparing at least one replicate plate and incubating said replicate plate under conditions that allow host cell growth and production of said protein of interest; (c) transferring host cells from said growth substrate or at least one replicate plate of (b) to a membrane; (d) preparing the host cells transferred to the membrane in (c) for probing, comprising: (i) optionally fixing the host cells under conditions allowing immobilization of cellular components; (ii) blocking the host cells; and (iii) optionally lysing the host cells under conditions allowing permeabilization of the host cells; (e) contacting the permeabilized host cells with a probe solution comprising the at least one probe under conditions that allow binding of the at least one probe to the protein of interest, thereby forming a probe-protein of interest complex; 27. The method of any one of claims 1 to 26, further comprising the step of: (f) imaging the host cells under conditions that allow identifying the host cells that produce the protein of interest.

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