WO2017106583A1 - Système d'expression cytoplasmique - Google Patents

Système d'expression cytoplasmique Download PDF

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Publication number
WO2017106583A1
WO2017106583A1 PCT/US2016/067064 US2016067064W WO2017106583A1 WO 2017106583 A1 WO2017106583 A1 WO 2017106583A1 US 2016067064 W US2016067064 W US 2016067064W WO 2017106583 A1 WO2017106583 A1 WO 2017106583A1
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host cells
promoter
gene
inducible promoter
cells
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PCT/US2016/067064
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English (en)
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Sean Mcclain
Philip BARISH
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Absci, Llc
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Publication of WO2017106583A1 publication Critical patent/WO2017106583A1/fr
Priority to US16/009,147 priority Critical patent/US20180282405A1/en
Priority to US16/871,736 priority patent/US20200270338A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/241—Tumor Necrosis Factors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/10—Immunoglobulins specific features characterized by their source of isolation or production
    • C07K2317/14—Specific host cells or culture conditions, e.g. components, pH or temperature
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value

Definitions

  • This application includes a sequence listing submitted electronically, in a file entitled “AbSci-002PCT_ST25.txt”, created on December 15, 2016 and having a size of 24 kilobytes (KB), which is incorporated by reference herein.
  • the present invention is in the general technical fields of molecular biology and biotechnological manufacturing. More particularly, the present invention is in the technical field of recombinant protein expression.
  • Disulfide bonds form more readily in an oxidizing environment, in which the sulfur atoms participating in a disulfide bond are less likely to be reduced to form sulfhydryl groups, which would eliminate the disulfide bond.
  • An oxidizing environment can be found within certain subcellular compartments of cells, such as the secretory pathway and certain topologically 'extracellular' compartments of eukaryotic cells (Go and Jones, "Redox compartmentalization in eukaryotic cells", Biochim Biophys Acta 2008 Nov; 1780(11): 1273-1290; doi: 10.1016/j.bbagen.2008.01.011 ; Epub 2008 Jan 26; Review), and the periplasm of bacteria such as E. coli.
  • the cell cytoplasm is normally maintained in a relatively reduced state by the thioredoxin and the glutaredoxin/glutathione enzyme systems. This generally inhibits the formation of disulfide bonds in the cytoplasm, and most proteins that need disulfide bonds to function are exported into the eukaryotic secretory pathway or the bacterial periplasm where disulfide bond formation can readily occur.
  • the present invention provides host cells having an altered gene function of at least one gene that makes the reduction/oxidation environment of the host cell cytoplasm more oxidizing, which are capable of growing to high cell densities and producing recombinant proteins in soluble form. Also provided are methods for growing such host cells to high densities, for optionally modulating the growth rates of the host cells, and for inducing the host cells to produce the desired gene product in soluble form. Further aspects of the invention relate to host cell preparation and storage methods, which utilize the advantages of producing gene products in host cell cytoplasm to provide methods for long-term storage and/or transport of expressed gene products retained in a stable and soluble form within the host cells.
  • Method I is a method of producing at least one gene product; Method I comprises a set of combinations of steps A - F, as described below. Method I can be symbolized by the following schema:
  • Method I ⁇ A[l-8], B[l-3], C[l] 0-1 , D[l-3] 0-1 , E[l-2] 0-1 , F[l] 0-1 ⁇ wherein A - F represent steps of Method I.
  • a subscript of 0-1 indicates that a step can be present or absent in Method I, and if present, with no limitation on the number of times the step can be performed.
  • a letter followed by bracketed numbers, for example A[l-3], indicates that step A has attributes Al, A2, and A3, and step A can optionally include any combination of attributes Al, A2, and/or A3.
  • Each attribute, such as Al for example may have a number of alternative instances, such as Al . l, A1.2, and A1.3 for example.
  • Alternative instances such as Al .l can be presented as a list wherein every member of the list is an alternative instance of Al and can therefore be combined with any instance of any other attributes of step A, and/or with any other instances and/or attribute(s) of any other step of Method I.
  • Step A of Method I providing one or more host cells.
  • Al providing host cells comprising at least one expression construct.
  • Al . l providing host cells comprising at least one expression construct comprising a polynucleotide sequence selected from the group consisting of: pSOL (SEQ ID NO:3) or fragments thereof.
  • A.1.2 providing host cells comprising at least two expression constructs.
  • A2 providing host cells comprising at least one expression construct comprising at least one inducible promoter.
  • A2.1 providing host cells comprising at least one expression construct comprising at least one inducible promoter, wherein the inducible promoter is not a lactose-inducible promoter.
  • A2.2 providing host cells comprising at least one expression construct comprising at least one inducible promoter, wherein the inducible promoter is selected from the group consisting of an L-arabinose-inducible promoter, a propionate-inducible promoter, a rhamnose-inducible promoter, a xylose-inducible promoter, a lactose-inducible promoter, and a promoter inducible by phosphate depletion.
  • the inducible promoter is selected from the group consisting of an L-arabinose-inducible promoter, a propionate-inducible promoter, a rhamnose-inducible promoter, a xylose-inducible promoter, a lactose-inducible promoter, and a promoter inducible by phosphate depletion.
  • A2.3 providing host cells comprising at least one expression construct comprising at least one inducible promoter, wherein the inducible promoter is selected from the group consisting of the araBAD promoter, the prpBCDE promoter, the rhaSR promoter, the xlyA promoter, the lacZYA promoter, and the phoA promoter.
  • A3 providing host cells comprising at least one expression construct comprising at least one inducible promoter and at least one polynucleotide sequence encoding a gene product to be transcribed from at least one said inducible promoter.
  • A3.1 providing host cells comprising at least one expression construct comprising at least one inducible promoter and at least one polynucleotide sequence encoding a gene product to be transcribed from at least one said inducible promoter, wherein the gene product lacks a signal sequence.
  • A3.2 providing host cells comprising at least one expression construct comprising at least one inducible promoter and at least one polynucleotide sequence encoding a gene product to be transcribed from at least one said inducible promoter, wherein the gene product further comprises a tag selected from the group consisting of: polyhistidine, pestivirus N pro , CSFV N pro , CSFV N pro (strain Alfort), BDV N pro , BVDV N pro , SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, and fragments thereof.
  • A3.3 providing host cells comprising at least one expression construct comprising at least one inducible promoter and at least one polynucleotide sequence encoding a gene product to be transcribed from at least one said inducible promoter, wherein the gene product forms a number of disulfide bonds selected from the group consisting of: at least one and fewer than twenty disulfide bonds; at least two and fewer than seventeen disulfide bonds; at least eighteen and fewer than one hundred disulfide bonds; at least three and fewer than ten disulfide bonds; at least three and fewer than eight disulfide bonds; one disulfide bond; two disulfide bonds; three disulfide bonds; four disulfide bonds; five disulfide bonds; six disulfide bonds; seven disulfide bonds; eight disulfide bonds; and nine disulfide bonds.
  • A3.4 providing host cells comprising at least one expression construct comprising at least one inducible promoter and at least one polynucleotide sequence encoding a gene product to be transcribed from at least one said inducible promoter, wherein the gene product is an insulin polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NO: 11, SEQ ID NO: 12, and fragments thereof.
  • A3.5 providing host cells comprising at least one expression construct comprising at least one inducible promoter and at least one polynucleotide sequence encoding a gene product to be transcribed from at least one said inducible promoter, wherein the gene product is selected from the group consisting of: an immunoglobulin heavy chain, an immunoglobulin light chain, and fragments thereof.
  • A3.6 providing host cells comprising at least one expression construct comprising at least one inducible promoter and at least one polynucleotide sequence encoding a gene product to be transcribed from at least one said inducible promoter, wherein the gene product is selected from the group consisting of: 1 -antitrypsin; 2C4; activin; addressins; alkaline phosphatase; anti- CDl la; anti-CD18; anti-CD20; anti-clotting factors such as Protein C; anti-HER- 2 antibody; anti-IgE; anti-IgG; anti-VEGF; antibodies and antibody fragments; antibodies to ErbB2 domain(s) such as 2C4 (WO 01/00245 hybridoma ATCC HB-12697), which binds to a region in the extracellular domain of ErbB2 (e.g., any one or more residues in the region from about residue 22 to about residue 584 of ErbB2, inclusive); Apo2 ligand (Apo2L); atrial
  • A4 providing host cells having an altered gene function of at least one gene that affects the reduction/oxidation environment of the host cell cytoplasm.
  • A4.1 providing host cells having an altered gene function of at least one gene that increases the oxidizing environment of the host cell cytoplasm.
  • A4.2 providing host cells having an altered gene function of at least one gene that increases the oxidizing environment of the host cell cytoplasm selected from the group consisting of gor, gshA, gshB, and trxB.
  • A4.3 providing host cells having an altered gene function of at least one gene selected from the group consisting of ahpC, katG, and katE.
  • A4.4 providing host cells having an ahpC A gene.
  • A5 providing host cells having a reduced level of gene function of at least one gene encoding a protein that metabolizes an inducer of at least one inducible promoter.
  • A5.1 providing host cells having a reduced level of gene function of at least one gene encoding a protein that metabolizes an inducer of at least one inducible promoter, wherein the gene is selected from the group consisting of araA, arciB, arciD, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB.
  • A6 providing host cells having an altered level of gene function of at least one gene encoding a transporter protein for an inducer of at least one inducible promoter.
  • A6.1 providing host cells having an altered level of gene function of at least one gene encoding a transporter protein for an inducer of at least one inducible promoter, wherein the gene is selected from the group consisting of araE, araF, araG, araH, rhaT, xylF, xylG, and xylH.
  • A6.2 providing host cells having an altered level of gene function of at least one gene encoding a transporter protein for an inducer of at least one inducible promoter, wherein the altered gene function is expressing the transporter protein from a constitutive promoter.
  • A7 providing host cells further comprising a polynucleotide encoding a polypeptide selected from the group consisting of: cDsbA, cDsbC, a protein disulfide isomerase, Ervlp, a chaperone, and a transporter protein for a cofactor of the gene product to be transcribed from at least one inducible promoter.
  • A8 providing host cells wherein the host cells are prokaryotic cells.
  • A8.1 providing host cells wherein the host cells are E. coli cells.
  • A8.2 providing host cells wherein the host cells are E. coli B strain cells.
  • A8.3 providing host cells wherein the host cells are E. coli EB0001 cells.
  • A8.3 providing host cells wherein the host cells are E. coli EB0002 cells.
  • Step B of Method I growing the host cells.
  • B 1 growing the host cells until the host cells reach a density greater than 50 (OD 600 ).
  • Bl . l growing the host cells until the host cells reach a density greater than a cell density selected from the group consisting of: 60 (OD 60 o); 70 (OD 600 ); 80 (OD 600 ); 90 (OD 600 ); 95 (OD 600 ); 100 (OD 600 ); 105 (OD 600 ); 110 (OD 600 ); 115 (OD 600 ); 120 (OD 600 ); 125 (OD 600 ); 130 (OD 600 ); 135 (OD 600 ); 140 (OD 600 ); 145 (OD 600 ); 150 (OD 600 ); 155 (OD 600 ); 160 (OD 600 ); 165 (OD 600 ); 170 (OD 600 ); and 175 (OD 600 ).
  • B2 growing the host cells at a specific cell growth rate between 0.01 and
  • B2.1 growing the host cells at a specific cell growth rate selected from the group consisting of: 0.01 to 0.7; 0.05 to 0.3; 0.1 to 0.2; approximately 0.15 (0.15 plus-or-minus 10%); and 0.15.
  • B3 growing the host cells in a fermentation volume between 0.1 L and 1,000,000L.
  • B 3.1 growing the host cells in a fermentation volume selected from the group consisting of: 0.1L, 0.25L, 0.5 L; 0.6L; 0.75 L; 0.8L; 1L; 2L; 3L; 4L; 5L; 7.5L; 10L; 15L; 20L; 25L; 30L; 40L; 50L; 60L; 70L; 80L; 90L; 100L; 200L; 250L; 300L; 500L; 750L; 1000L; 1500L; 2000L; 2500L; 3000L; 5000L; 7500L; 10,000L; 15,000L; 20,000L; 25,000L; 50,000L; 75,000L; 100,000L; greater than 100,000L and less than 1,000,000L, and 1,000,000L.
  • a fermentation volume selected from the group consisting of: 0.1L, 0.25L, 0.5 L; 0.6L; 0.75 L; 0.8L; 1L; 2L; 3L; 4L; 5L; 7.5L; 10L; 15L; 20L; 25L; 30L;
  • Step C of Method I adding at least one inducer to the host cells.
  • CI adding at least one inducer to the host cells per host cell density (OD 600 ) unit.
  • Step D of Method I collecting the host cells and storing them.
  • D2 collecting the host cells and storing them at a temperature of less than 0 degrees C.
  • D3 collecting the host cells and storing them for a period longer than 24 hours.
  • Step E of Method I lysing the host cells.
  • El .1 lysing the host cells using lysozyme.
  • E2 lysing the host cells by mechanical disruption.
  • Step F of Method I purifying the gene product.
  • Fl .l purifying active gene product from the soluble cell lysate fraction.
  • F1.2 purifying gene product from the soluble cell lysate fraction, wherein the gene product contains properly formed disulfide bonds.
  • Fig. 1 shows the growth of E. coli EB0001(pBAD24-Infliximab_HC/pPRO33- Infliximab LC) cells over time, measured as OD 60 o, in five fermentation runs performed under different conditions.
  • the cells in Run D reached an OD 60 o of 167.2 at 18.25 hours.
  • Fig. 2 shows the growth of E. coli EB0001(pBAD24-Infliximab_HC/pPRO33- Infliximab LC) cells over time, measured as OD 60 o, for the exponential growth phase portion of fermentation Run D shown in Fig. 1.
  • the growth curve was fit to an exponential curve to determine a specific growth rate indicator, 0.0625/hour, for the cells in this fermentation run.
  • Fig. 3 shows the growth of E. coli EB0001(pBAD24-Infliximab_HC/pPRO33- Infliximab LC) cells over time, measured as OD 60 o, for the exponential growth phase portion of fermentation Run F.
  • the growth curve was fit to an exponential curve to determine a specific growth rate indicator, 0.112/hour, for the cells in this fermentation run.
  • Fig. 4 is a schematic representation of a proinsulin glargine polypeptide.
  • the amino acids of the A and B chains are shown as light gray and dark gray circles, respectively.
  • the N-terminal propeptide and the C-peptide (or 'connecting peptide') that connects the A and B chains are shown as dashed lines.
  • the solid dark gray lines between cysteine residues in the A and B chains, and connecting two cysteines within the A chain, represent the disulfide bonds present in correctly folded mature insulin glargine.
  • Fig. 5 shows the growth of EB0001(pSOL-proglargine/Ervlp) cells, also referred to as strain AbS0092, measured as optical density at 600 nm (OD 60 o).
  • Cell growth was measured for the period of time (EIT or Elapsed Induction Time) in which the EB0001(pSOL-proglargine/Ervlp) cells were induced to express proinsulin glargine.
  • the time point at 0 hours indicates the time at which induction was started.
  • the points plotted are the average of optical density measurements taken from two separate bioreactors; the error bars indicate the range of values at each time point.
  • FIG. 6 is a schematic diagram showing the digestion of purified proinsulin glargine with glutamyl endopeptidase ('Glu-C') and with trypsin to generate cross- linked peptide fragments for characterization by reverse-phase chromatography and by mass spectometry. Disulfide bonds are represented by solid dark gray lines connecting cysteine residues.
  • the problem of producing gene products such as therapeutic proteins at commercial scale and in soluble form is addressed by providing suitable host cells capable of growth at high cell density in fermentation culture, and which can produce soluble gene products in the oxidizing host cell cytoplasm through highly controlled inducible gene expression.
  • Host cells of the invention with these qualities are produced by combining some or all of the following characteristics.
  • the host cells are genetically modified to have an oxidizing cytoplasm, through increasing the expression or function of oxidizing polypeptides in the cytoplasm, and/or by decreasing the expression or function of reducing polypeptides in the cytoplasm. Specific examples of such genetic alterations are provided herein.
  • host cells can also be genetically modified to express chaperones and/or cofactors that assist in the production of the desired gene product(s), and/or to glycosylate polypeptide gene products.
  • the host cells comprise one or more expression constructs designed for the expression of one or more gene products of interest; in certain embodiments, at least one expression construct comprises an inducible promoter and a polynucleotide encoding a gene product to be expressed from the inducible promoter.
  • the host cells contain additional genetic modifications designed to improve certain aspects of gene product expression from the expression construct(s).
  • the host cells (A) have an alteration of gene function of at least one gene encoding a transporter protein for an inducer of at least one inducible promoter, and as another example, wherein the gene encoding the transporter protein is selected from the group consisting of araE, araE, araG, araH, rhaT, xylF, xylG, and xylH, or particularly is araE, or wherein the alteration of gene function more particularly is expression of araE from a constitutive promoter; and/or (B) have a reduced level of gene function of at least one gene encoding a protein that metabolizes an inducer of at least one inducible promoter, and as further examples, wherein the gene encoding a protein that metabolizes an inducer of at least one said inducible promoter is selected from the group consisting of araA, araB, araD, prpB, prpD, rhaA, rhaB,
  • Host Cells with Oxidizing Cytoplasm are designed to express gene products; in certain embodiments of the invention, the gene products are expressed in a host cell.
  • host cells are provided that allow for the efficient and cost-effective expression of gene products, including components of multimeric products.
  • Host cells can include, in addition to isolated cells in culture, cells that are part of a multicellular organism, or cells grown within a different organism or system of organisms.
  • the host cells are microbial cells such as yeasts (Saccharomyces, Schizosaccharomyces, etc.) or bacterial cells, or are gram- positive bacteria or gram-negative bacteria, or are E. coli, or are an E.
  • E. coli B strain or are E. coli (B strain) EB0001 cells (also called E. coli ASE(DGH) cells), or are E. coli (B strain) EB0002 cells.
  • E. coli host cells having 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)
  • Prokaryotic host cells expression constructs designed for expression of gene products are provided in host cells, such as prokaryotic host cells.
  • Prokaryotic host cells can include archaea (such as Haloferax volcanii, Sulfolobus solfataricus), Gram-positive bacteria (such as Bacillus subtilis, Bacillus licheniformis, Brevibacillus choshinensis, Lactobacillus brevis, Lactobacillus buchneri, Lactococcus lactis, and Streptomyces lividans), or Gram-negative bacteria, including Alphaproteobacteria (Agrobacterium tumefaciens, Caulobacter crescentus, Rhodobacter sphaeroides, and Sinorhizobium meliloti), Betaproteobacteria (Alcaligenes eutrophus), and Gammaproteobacteria (Acinetobacter calcoaceticus, Azotobacter vine), or the like.
  • Preferred host cells include Gammaproteobacteria of the family Enterobacteriaceae, such as Enterobacter, Erwinia, Escherichia (including E. coli), Klebsiella, Proteus, Salmonella (including Salmonella typhimurium), Serratia (including Serratia marcescans), and Shigella. Eukaryotic host cells.
  • host cells can be used for the expression systems of the invention, 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, Dekkera/Brettanomyces species, and Yarrowia lipolyticd); other fungi (Aspergillus nidulans, Aspergillus niger, Neurospora crassa, Penicillium, Tolypocladium, Trichoderma reesia); insect cell lines (Drosophila melanogaster Schneider 2 cells and Spodoptera frugiperda Sf9 cells); and mammalian cell lines including immortalized cell lines (Chinese hamster ovary (CHO) cells, HeLa
  • alterations can be made to the gene functions of host cells comprising inducible expression constructs, to promote efficient and homogeneous induction of the host cell population by an inducer.
  • the combination of expression constructs, 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 the culture expressing gene product from each induced promoter, as measured by the method of Khlebnikov et al. described in Example 9.
  • these alterations can involve the function of genes that are structurally similar to an E. coli gene, or genes that carry out a function within the host cell similar to that of the E. coli gene.
  • Alterations to host cell gene functions include eliminating or reducing gene function by deleting the gene protein-coding sequence in its entirety, or deleting a large enough portion of the gene, inserting sequence into the gene, or otherwise altering the gene sequence so that a reduced level of functional gene product is made from that gene. Alterations to host cell gene functions also include increasing gene function by, for example, altering the native promoter to create a stronger promoter that directs a higher level of transcription of the gene, or introducing a missense mutation into the protein-coding sequence that results in a more highly active gene product. Alterations to host cell gene functions include altering gene function in any way, including for example, altering a native inducible promoter to create a promoter that is constitutively activated. In addition to alterations in gene functions for the transport and metabolism of inducers, as described herein with relation to inducible promoters, and/or an altered expression of chaperone proteins, it is also possible to alter the reduction- oxidation environment of the host cell.
  • proteins that need disulfide bonds are typically exported into the periplasm where disulfide bond formation and isomerization is catalyzed by the Dsb system, comprising DsbABCD and DsbG.
  • Dsb system comprising DsbABCD and DsbG.
  • Increased expression of the cysteine oxidase DsbA, the disulfide isomerase DsbC, or combinations of the Dsb proteins, which are all normally transported into the periplasm has been utilized in the expression of heterologous proteins that require disulfide bonds (Makino et al., "Strain engineering for improved expression of recombinant proteins in bacteria", Microb Cell Fact 2011 May 14; 10: 32).
  • cytoplasmic forms of these Dsb proteins such as a cytoplasmic version of DsbA and/or of DsbC ('cDsbA or 'cDsbC'), that lacks a signal peptide and therefore is not transported into the periplasm.
  • Cytoplasmic Dsb proteins such as cDsbA and/or cDsbC are useful for making the cytoplasm of the host cell more oxidizing and thus more conducive to the formation of disulfide bonds in heterologous proteins produced in the cytoplasm.
  • the host cell cytoplasm can also be made less reducing and thus more oxidizing by altering the thioredoxin and the glutaredoxin/glutathione enzyme systems directly: mutant strains defective in glutathione reductase (gor) or glutathione synthetase (gshB), together with thioredoxin reductase (trxB), render the cytoplasm oxidizing. These strains are unable to reduce ribonucleotides and therefore cannot grow in the absence of exogenous reductant, such as dithiothreitol (DTT).
  • DTT dithiothreitol
  • ahpC* and ahpC A Suppressor mutations (such as ahpC* and ahpC A , Lobstein et al., "SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm", Microb Cell Fact 2012 May 8; 11 : 56; doi: 10.1186/1475-2859-11-56) in the gene ahpC, which encodes the peroxiredoxin AhpC, convert it to a disulfide reductase that generates reduced glutathione, allowing the channeling of electrons onto the enzyme ribonucleotide reductase and enabling the cells defective in gor and trxB, or defective in gshB and trxB, to grow in the absence of DTT.
  • AhpC can allow strains, defective in the activity of gamma- glutamylcysteine synthetase (gshA) and defective in trxB, to grow in the absence of DTT; these include AhpC V164G, AhpC S71F, AhpC E173/S71F, AhpC E171Ter, and AhpC dupl62-169 (Faulkner et al., "Functional plasticity of a peroxidase allows evolution of diverse disulfide-reducing pathways", Proc Natl Acad Sci U S A 2008 May 6; 105(18): 6735-6740, Epub 2008 May 2).
  • Another alteration that can be made to host cells 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 the 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, "Pre- expression of a sulfhydryl oxidase significantly increases the yields of eukaryotic disulfide bond containing proteins expressed in the cytoplasm of E. coli" Microb Cell Fact 2011 Jan 7; 10: 1).
  • Host cells comprising expression constructs preferably also express cDsbA and/or cDsbC and/or Ervlp; are deficient in trxB gene function; are also deficient in the gene function of either gor, gshB, or gshA; optionally have increased levels of katG and/or katE gene function; and express an appropriate mutant form of AhpC so that the host cells can be grown in the absence of DTT.
  • Chaperones In some embodiments, desired gene products are coexpressed with other gene products, such as chaperones, that are beneficial to the production of the desired gene product. Chaperones are proteins that assist the non-covalent folding or unfolding, and/or the 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 functions (having completed the processes of folding and/or assembly).
  • Chaperones can be expressed from an inducible promoter or a constitutive promoter within an expression construct, or can be expressed from the host cell chromosome; preferably, expression of chaperone protein(s) in the host cell is at a sufficiently high level to produce coexpressed gene products that are properly folded and/or assembled into the desired product.
  • Examples of chaperones present in E. coli host cells are the folding factors DnaK/DnaJ/GrpE, DsbC/DsbG, GroEL/GroES, IbpA/IbpB, Skp, Tig (trigger factor), and FkpA, which have been used to prevent protein aggregation of cytoplasmic or periplasmic proteins.
  • a eukaryotic chaperone protein such as protein disulfide isomerase (PDI) from the same or a related eukaryotic species, is in certain embodiments of the invention coexpressed or inducibly coexpressed with the desired gene product.
  • PDI protein disulfide isomerase
  • One chaperone that can be expressed in host cells is a protein disulfide isomerase from Humicola insolens, a soil hyphomycete (soft-rot fungus).
  • An amino acid sequence of Humicola insolens PDI is shown as SEQ ID NO: l ; it lacks the signal peptide of the native protein so that it remains in the host cell cytoplasm.
  • the nucleotide sequence encoding PDI was optimized for expression in E. coli; the expression construct for PDI is shown as SEQ ID NO: 2.
  • SEQ ID NO: 2 contains a GCTAGC Nhel restriction site at its 5' end, an AGGAGG ribosome binding site at nucleotides 7 through 12, the PDI coding sequence at nucleotides 21 through 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.
  • SEQ ID NO: 2 The Nhel and Sail restriction sites in SEQ ID NO: 2 can be used to insert it into a vector multiple cloning site, such as that of the pSOL expression vector (SEQ ID NO: 3), described in published US patent application US2015353940A1 , which is incorporated by reference in its entirety herein.
  • PDI polypeptides can also be expressed in host cells, including PDI polypeptides from a variety of 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 also modified forms of such PDI polypeptides.
  • species Sacharomyces cerevisiae (UniProtKB PI 7967)
  • Homo sapiens UniProtKB P07237)
  • Mus musculus UniProtKB P09103
  • a PDI polypeptide expressed in host cells of the invention shares at least 70%, or 80%, or 90%, or 95% amino acid sequence identity across 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: l , where amino acid sequence identity is determined according to Example 10.
  • Cellular transport of cofactors When using the expression systems of the invention to produce enzymes that require cofactors for function, it is helpful to use a host cell capable of synthesizing the cofactor from available precursors, or taking it up from the environment.
  • cofactors include ATP, coenzyme A, flavin adenine dinucleotide (FAD), NAD + /NADH, and heme.
  • Polynucleotides encoding cofactor transport polypeptides and/or cofactor synthesizing polypeptides can be introduced into host cells, and such polypeptides can be constitutively expressed, or inducibly coexpressed with the gene products to be produced by methods of the invention.
  • Host cells can have alterations in their ability to glycosylate polypeptides.
  • eukaryotic host cells can have eliminated or reduced gene function in glycosyltransf erase and/or oligo- saccharyltransferase genes, impairing the normal eukaryotic glycosylation of polypeptides to form glycoproteins.
  • Prokaryotic host cells such as E. coli, which do not normally glycosylate polypeptides, can be altered to express a set of eukaryotic and prokaryotic genes that provide a glycosylation function (DeLisa et al., "Glycosylated protein expression in prokaryotes", WO2009089154A2, 2009 Jul 16).
  • the function of one or more genes of host cells is eliminated or reduced by identifying a nucleotide sequence within the coding sequence of the gene to be disrupted, such as one of the E. coli K-12 substrain MG1655 coding sequences incorporated herein by reference to the genomic location of the sequence, and more specifically by selecting two adjacent stretches of 50 nucleotides each within that coding sequence.
  • the Quick & Easy E. coli Gene Deletion Kit is then used according to the manufacturer's instructions to insert a polynucleotide construct containing a selectable marker between the selected adjacent stretches of coding sequence, eliminating or reducing the normal function of the gene.
  • Red/ET recombination methods can also be used to replace a promoter sequence with that of a different promoter, such as a constitutive promoter, or an artificial promoter that is predicted to promote a certain level of transcription (De Mey et al., "Promoter knock-in: a novel rational method for the fine tuning of genes", BMC Biotechnol 2010 Mar 24; 10: 26).
  • the function of host cell genes can also be eliminated or reduced by RNA silencing methods (Man et al , "Artificial trans-encoded small non-coding RNAs specifically silence the selected gene expression in bacteria", Nucleic Acids Res 2011 Apr; 39(8): e50, Epub 2011 Feb 3). Further, known mutations that alter host cell gene function can be introduced into host cells through traditional genetic methods.
  • Expression constructs are polynucleotides designed for the expression of one or more gene products of interest, and thus are not naturally occurring molecules. Expression constructs can be integrated into a host cell chromosome, or maintained within the host cell as polynucleotide molecules replicating independently of the host cell chromosome, such as plasmids or artificial chromosomes.
  • An example of an expression construct is a polynucleotide resulting from the insertion of one or more polynucleotide sequences into a host cell chromosome, where the inserted polynucleotide sequences alter the expression of chromosomal coding sequences.
  • An expression vector is a plasmid expression construct 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 be maintained on an extrachromosomal polynucleotide such as a plasmid or artificial chromosome.
  • an extrachromosomal polynucleotide such as a plasmid or artificial chromosome.
  • the expression construct is the pSOL expression vector (SEQ ID NO: 3), described in published US patent application US2015353940A1 , which is incorporated by reference in its entirety herein.
  • inducible Promoters The following is a description of inducible promoters that can be used in expression constructs for expression of gene products, along with some of the genetic modifications that can be made to host cells that contain such expression constructs. Examples of these inducible promoters and related genes are, unless otherwise specified, those derived from Escherichia coli (E. coli) strain MG1655 (American Type Culture Collection deposit ATCC 700926), which is a substrain of E. coli K-12 (American Type Culture Collection deposit ATCC 10798). Table 1 of International Application PCT/US 13/53562 (published as WO2014025663A1) lists the genomic locations, in E.
  • E. coli MG1655 of the nucleotide sequences for these examples of inducible promoters and related genes; the WO2014025663A1 publication is incorporated by reference in its entirety herein. Nucleotide and other genetic sequences, referenced by genomic location as in Table 1 of WO2014025663A1, are expressly incorporated by reference herein. Additional information about E. coli promoters, genes, and strains described herein can be found in many public sources, including the online EcoliWiki resource, located at ecoliwiki.net.
  • Arabinose promoter (As used herein, 'arabinose' means L-arabinose.)
  • Several E. coli operons involved in arabinose utilization are inducible by arabinose— araBAD, araC, araE, and araFGH— but the terms 'arabinose promoter' and 'am promoter' are typically used to designate the araBAD promoter.
  • Several additional terms have been used to indicate the E. coli araBAD promoter, such as ara , V araB , P ARABAD , and P BAD -
  • the use herein of 'am promoter' or any of the alternative terms given above, means the E. coli araBAD promoter.
  • the araBAD promoter is considered to be part of a bidirectional promoter, with the araBAD promoter controlling expression of the araBAD operon in one direction, and the araC promoter, in close proximity to and on the opposite strand from the araBAD promoter, controlling expression of the araC coding sequence in the other direction.
  • the AraC protein is both a positive and a negative transcriptional regulator of the araBAD promoter.
  • the AraC protein In the absence of arabinose, the AraC protein represses transcription from P BAD , but in the presence of arabinose, the AraC protein, which alters its conformation upon binding arabinose, becomes a positive regulatory element that allows transcription from P BAD -
  • the araBAD operon encodes proteins that metabolize L-arabinose by converting it, through the intermediates L-ribulose and L-ribulose-phosphate, to D-xylulose-5-phosphate.
  • AraA which catalyzes the conversion of L-arabinose to L-ribulose
  • AraB and AraD optionally to eliminate or reduce the function of at least one of AraB and AraD, as well. Eliminating or reducing the ability of host cells to decrease the effective concentration of arabinose in the cell, by eliminating or reducing the cell's ability to convert arabinose to other sugars, allows more arabinose to be available for induction of the arabinose-inducible promoter.
  • the genes encoding the transporters which move arabinose into the host cell are araE, which encodes the low-affinity L- arabinose proton symporter, and the araFGH operon, which encodes the subunits of an ABC superfamily high-affinity L-arabinose transporter.
  • LacY lactose permease the LacY(AlWC) and the LacY(AlWV) proteins, having a cysteine or a valine amino acid instead of alanine at position 177, respectively
  • LacY(AlWC) and the LacY(AlWV) proteins having a cysteine or a valine amino acid instead of alanine at position 177, respectively
  • Morgan-Kiss et al. "Long-term and homogeneous regulation of the Escherichia coli araBAD promoter by use of a lactose transporter of relaxed specificity", Proc Natl Acad Sci U S A 2002 May 28; 99(11): 7373-7377).
  • arabinose-inducible promoter In order to achieve homogenous induction of an arabinose-inducible promoter, it is useful to make transport of arabinose into the cell independent of regulation by arabinose. This can be accomplished by eliminating or reducing the activity of the AraFGH transporter proteins and altering the expression of araE so that it is only transcribed from a constitutive promoter. Constitutive expression of araE can be accomplished by eliminating or reducing the function of the native araE gene, and introducing into the cell an expression construct which includes a coding sequence for the AraE protein expressed from a constitutive promoter.
  • the promoter controlling expression of the host cell's chromosomal araE gene can be changed from an arabinose-inducible promoter to a constitutive promoter.
  • a host cell that lacks AraE function can have any functional AraFGH coding sequence present in the cell expressed from a constitutive promoter.
  • LacY(AlWC) protein appears to be more effective in transporting arabinose into the cell, use of polynucleotides encoding the LacY(AlWC) protein is preferred to the use of polynucleotides encoding the LacY(AlWV) protein.
  • the 'propionate promoter' or 'prp promoter' is the promoter for the E. coli prpBCDE operon, and is also called ⁇ ⁇ ⁇ Like the ara promoter, the prp promoter is part of a bidirectional promoter, controlling expression of the prpBCDE operon in one direction, and with the prpR promoter controlling expression of the prpR coding sequence in the other direction.
  • the PrpR protein is the 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, CH 3 CH 2 COO — , of propionic acid (or 'propanoic acid'), and is the smallest of the 'fatty' acids having the general formula H(CH 2 ) w COOH that shares certain properties of this class of molecules: producing an oily layer when salted out of water and having a soapy potassium salt.
  • Commercially available propionate is generally sold as a monovalent cation salt of propionic acid, such as sodium propionate (CH 3 CH 2 COONa), or as a divalent cation salt, such as calcium propionate (Ca(CH 3 CH 2 COO) 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).
  • PrpE propionyl-CoA synthetase
  • PrpC 2-methylcitrate synthase
  • a host cell with PrpC and PrpE activity, to convert propionate into 2-MC, but also having eliminated or reduced PrpD activity, and optionally eliminated or reduced PrpB activity as well, to prevent 2-MC from being metabolized.
  • Another operon encoding proteins involved in 2-MC biosynthesis is the scpA-argK-scpBC operon, also called 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.
  • Elimination or reduction of the function of these proteins would remove a parallel pathway for the production of the 2-MC inducer, and thus might reduce background levels of expression of a propionate-inducible promoter, and increase sensitivity of the propionate-inducible promoter to exogenously supplied propionate. It has been found that a deletion of sbm-yg/D-ygfG-ygfH-ygfl, introduced into E.
  • genes sbm-yg/DGH are transcribed as one operon, and ygfl is transcribed from the opposite strand.
  • the 3' ends of the ygfti and ygfl coding sequences overlap by a few base pairs, so a deletion that takes out all of the sbm- yg/DGH operon apparently takes out ygfl coding function as well.
  • Eliminating or reducing the function of a subset of the sbm-yg/DGH gene products such as YgfG (also called ScpB, methylmalonyl-CoA decarboxylase), or deleting the majority of the sbm-yg/DGH (or scpA-argK-scpBC) operon while leaving enough of the 3' end of the ygfti (or scpC) gene so that the expression of ygfl is not affected, could be sufficient to reduce background expression from a propionate-inducible promoter without reducing the maximal level of induced expression.
  • YgfG also called ScpB, methylmalonyl-CoA decarboxylase
  • deleting the majority of the sbm-yg/DGH or scpA-argK-scpBC
  • ygfti or scpC
  • Rhamnose promoter (As used herein, 'rhamnose' means L-rhamnose.)
  • the 'rhamnose promoter' or 'rha promoter', or P r haSR is the promoter for the E. coli rhaSR operon.
  • the rha promoter is part of a bidirectional promoter, controlling expression of the rhaSR operon in one direction, and with the rhaBAD promoter controlling expression of the rhaBAD operon in the other direction.
  • the rha promoter however, has two transcriptional regulators involved in modulating expression: RhaR and RhaS.
  • RhaR protein activates expression of the rhaSR operon in the presence of rhamnose
  • RhaS protein activates expression of the L-rhamnose catabolic and transport operons, rhaBAD and rhaT, respectively
  • RhaC/XylS family activator RhaS negatively autoregulates rhaSR expression by preventing cyclic AMP receptor protein activation
  • RhaS protein can also activate expression of the rhaSR operon, in effect 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 greater level.
  • CRP cyclic AMP receptor protein
  • the rhaBAD operon encodes the rhamnose catabolic proteins RhaA (L-rhamnose isomerase), which converts L-rhamnose 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).
  • RhaA L-rhamnose isomerase
  • RhaB rhamnulokinase
  • RhaD rhamnulose-1 -phosphate aldolase
  • E. coli cells can also synthesize L-rhamnose from alpha-D-glucose-l-P through the activities of the proteins RmlA, RmlB, RmlC, and RmlD (also called RfbA, RfbB, RfbC, and RfbD, respectively) encoded by the rmlBDACX (or rfbBDACX) operon.
  • L- rhamnose is transported into the cell by RhaT, the rhamnose permease or L-rhamnose:proton symporter. As noted above, the expression of RhaT is activated by the transcriptional regulator RhaS.
  • RhaT independent of induction by rhamnose (which induces expression of RhaS)
  • the host cell can be altered so that all functional RhaT coding sequences in the cell are expressed from constitutive promoters. Additionally, the coding sequences for RhaS can be deleted or inactivated, so that no functional RhaS is produced.
  • the level of expression from the rhaSR promoter is increased due to the absence of negative autoregulation by RhaS, and the level of expression of the rhamnose catalytic operon rhaBAD is decreased, further increasing the ability of rhamnose to induce expression from the rha promoter.
  • Xylose promoter (As used herein, 'xylose' means D-xylose.)
  • the xylose promoter region is similar in organization to other inducible promoters in that the xylAB operon and the xylFGHR operon are both expressed from adjacent xylose-inducible promoters in opposite directions on the E. coli chromosome (Song and Park, "Organization and regulation of the D-xylose operons in Escherichia coli K-12: XylR acts as a transcriptional activator", J Bacteriol.
  • the transcriptional regulator of both the PxyiA and PxyiF promoters is XylR, which activates expression of these promoters in the presence of xylose.
  • the xylR gene is expressed either as part of the xylFGHR operon or from its own weak promoter, which is not inducible 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.
  • XylA D-xylose isomerase
  • XylB xylulokinase
  • the xylFGHR operon encodes XylF, XylG, and XylH, the subunits of an ABC super- family 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.
  • the host cell can be altered so that all functional xylose transporters are expressed from constitutive promoters.
  • the xylFGHR operon could be altered so that the xylFGH coding sequences are deleted, leaving XylR as the only active protein expressed from the xylose-inducible P ⁇ IF promoter, and with the xylE coding sequence expressed from a constitutive promoter rather than its native promoter.
  • the xylR coding sequence is expressed from the P ⁇ IA or the P ⁇ IF promoter in an expression construct, while either the xylFGHR operon is deleted and xylE is constitutively expressed, or alternatively an xylFGH operon (lacking the xylR coding sequence since that is present in an expression construct) is expressed from a constitutive promoter and the xylE coding sequence is deleted or altered so that it does not produce an active protein.
  • Lactose promoter refers to the lactose-inducible promoter for the lacZYA operon, a promoter which is also called lacZpl ; this lactose promoter is located at ca. 365603 - 365568 (minus strand, with the RNA polymerase binding ('-35') site at ca. 365603-365598, the Pribnow box ('-10') at 365579-365573, and a transcription initiation site at 365567) in the genomic sequence of the E. coli K-12 substrain MG1655 (NCBI Reference Sequence NC 000913.2, l l-JAN-2012).
  • expression systems of the invention can comprise a lactose-inducible promoter such as the lacZYA promoter. In other embodiments, the expression systems of the invention comprise one or more inducible promoters that are not lactose-inducible promoters.
  • Alkaline phosphatase promoter Alkaline phosphatase promoter.
  • the terms 'alkaline phosphatase promoter' and 'phoA promoter' refer to the promoter for the phoApsiF operon, a promoter which is induced under conditions of phosphate starvation.
  • the phoA promoter region is located at ca.
  • the transcriptional activator for the phoA promoter is PhoB, a transcriptional regulator that, along with the sensor protein PhoR, forms a two-component signal transduction system in E. coli.
  • PhoB and PhoR are transcribed from the phoBR operon, located at ca. 417050 - 419300 (plus strand, with the PhoB coding sequence at 417,142 - 417,831 and the PhoR coding sequence at 417,889 - 419,184) in the genomic sequence of the 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 lack of a substance - intracellular phosphate - rather than by the addition of an inducer. For this reason the phoA promoter is generally used to direct transcription of gene products that are to be produced at a stage when the host cells are depleted for phosphate, such as the later stages of fermentation.
  • expression systems of the invention can comprise a phoA promoter.
  • the expression systems of the invention comprise one or more inducible promoters that are not phoA promoters.
  • inducible promoter As described above, there are several different inducible promoters that can be included in expression constructs as part of the expression systems of the invention. Preferred inducible promoters share at least 80% polynucleotide sequence identity (more preferably, at least 90% identity, and most preferably, at least 95% identity) to at least 30 (more preferably, at least 40, and most preferably, at least 50) contiguous bases of a promoter polynucleotide sequence as defined in Table 1 of WO2014025663A1, where percent polynucleotide sequence identity is determined using the methods of Example 10.
  • preferred inducible promoters have at least 75% (more preferably, at least 100%, and most preferably, at least 110%) of the strength of the corresponding 'wild-type' inducible promoter of E. coli K-12 substrain MG1655, as determined using the quantitative PCR method of De Mey et al. (Example 9).
  • an inducible promoter is placed 5' to (or 'upstream of) the coding sequence for the gene product that is to be inducibly expressed, so that the presence of the inducible promoter will direct transcription of the gene product coding sequence in a 5' to 3' direction relative to the coding strand of the polynucleotide encoding the gene product.
  • Carbon catabolite repression CC .
  • the presence of an active CCR regulatory system within a host can affect the ability of an inducer to activate transcription from an inducible promoter. For example, when a host cell such as E. coli is grown in a medium containing glucose, an active CCR regulatory system causes genes needed for the utilization of other carbon sources, such as the araBAD and prpBCDE operons, are expressed at a low level if at all, even if the arabinose or propionate inducer is also present in the growth medium.
  • host cells of the invention that have been genetically modified, so that they lack the ability to metabolize an inducer into another compound, do not necessarily exhibit CCR by that inducer on promoters regulated by less-preferred carbon sources.
  • This absence of a significant CCR effect is observed when very low concentrations of the non-metabolized inducer are used; surprisingly, those very low concentrations are also the most effective for producing optimal yields of gene product.
  • coexpression of gene products can be carried out by expressing one gene product component from the L-arabinose-inducible araBAD promoter and another gene product component from the propionate- inducible prpBCDE promoter.
  • host cells such as E.
  • coexpression typically produces the best yields of multimeric gene product at L-arabinose inducer concentrations of less than 100 micromolar (0.0015%) per OD unit of cells, and at these L-arabinose concentrations very little or no L-arabinose-mediated CC of the prpBCDE promoter is observed.
  • Gene products expressed by the methods of the invention are recombinant gene products, in that they are produced by recombinant engineering methods in which a polynucleotide encoding the gene product to be expressed is placed downstream of a promoter that is used to direct expression of the gene product in a host cell.
  • Gene products expressed by the methods of the invention can also be heterologous gene products, in that the gene product is native to a species other than that of the host cell.
  • the methods of the invention can be used to express mammalian polypeptides in microbial cells.
  • gene products expressed by the methods of the invention can also be artificial (non-naturally occurring) gene products, in that the polynucleotide and/or amino acid sequences of the gene products are the product of human invention and do not occur in nature.
  • Gene products expressed by the methods of the invention are in some instances polypeptides, such as any, or more than one, of the following: 1 -antitrypsin; 2C4; activin; addressins; alkaline phosphatase; anti-CDl la; anti-CD18; anti-CD20; anti-clotting factors such as Protein C; anti-HER-2 antibody; anti-IgE; anti-IgG; anti-VEGF; antibodies and antibody fragments; antibodies to ErbB2 domain(s) such as 2C4 (WO 01/00245 hybridoma ATCC HB-12697), which binds to a region in the extracellular domain of ErbB2 (e.g., any one or more residues in the region from about residue 22 to about residue 584 of ErbB2, inclusive); Apo2 ligand (Apo2L); atrial naturietic factor; BDNF; beta-lactamase; bombesin; bone morphogenetic protein (BMP); brain IGF-I; calc
  • Gene products expressed by the methods of the invention can include any, or more than one, of the following insulin polypeptides.
  • An insulin polypeptide produced by the methods of the invention comprises in some embodiments the amino acid sequence of a mature A chain or of a mature B chain of insulin, and in other embodiments comprises both a mature A chain and a mature B chain.
  • a proinsulin polypeptide comprises a mature A chain of insulin and a mature B chain of insulin.
  • Insulin polypeptide chains in certain embodiments comprise one or more of any of the naturally occurring amino acid sequences of insulins, or fragments thereof, and in other embodiments comprise one or more insulin analogue amino acid sequences, or fragments thereof, and in further embodiments comprise combinations of naturally occurring insulin amino acid sequences and/or insulin analogue amino acid sequences.
  • insulin amino acid sequences examples include insulin amino acid sequences and insulin analogue amino acid sequences.
  • Insulin degludec and insulin detemir have modified B29 lysine residues as described in the "Sequences Presented in The Sequence Listing" table below.
  • A mature A chain
  • B mature B chain
  • Disulfide Bonds Gene products expressed by the methods of the invention are in some instances polypeptides that form disulfide bonds.
  • the numbers and locations of disulfide bonds formed by a polypeptide can be determined by methods such as that of Example 7).
  • the number of disulfide bonds for a gene product such as a polypeptide is the total number of intramolecular and intermolecular bonds formed by that gene product when it is present in a functional product.
  • a light chain of a human IgG antibody typically has three disufide bonds (two intramolecular bonds and one intermolecular bond)
  • a heavy chain of a human IgG antibody typically has seven disufide bonds (four intramolecular bonds and three intermolecular bonds).
  • a gene product expressed by methods of the invention is a polypeptide that forms at least one and fewer than twenty disulfide bonds, or at least two and fewer than seventeen disulfide bonds, or at least eighteen and fewer than one hundred disulfide bonds, or at least three and fewer than ten disulfide bonds, or at least three and fewer than eight disulfide bonds, or is a polypeptide that forms a number of disulfide bonds selected from the group consisting of one, two, three, four, five, six, seven, eight, and nine disulfide bonds.
  • Signal Peptides Polypeptide gene products expressed by the methods of the invention typically lack signal peptides, as it is desirable for such gene products to be retained in the oxidizing cytoplasm of the host cell.
  • Signal peptides also termed signal sequences, leader sequences, or leader peptides
  • Signal peptides are characterized structurally by a stretch of hydrophobic amino acids, approximately five to twenty amino acids long and often around ten to fifteen amino acids in length, that has a tendency to form a single alpha-helix. This hydrophobic stretch is often immediately preceded by a shorter stretch enriched in positively charged amino acids (particularly lysine).
  • Signal peptides that are to be cleaved from the mature polypeptide typically end in a stretch of amino acids that is recognized and cleaved by signal peptidase.
  • Signal peptides can be characterized functionally by the ability to direct transport of a polypeptide, either co-translationally or post- translationally, through the plasma membrane of prokaryotes (or the inner membrane of gram negative bacteria like E. coli), or into the endoplasmic reticulum of eukaryotic cells.
  • the degree to which a signal peptide enables a polypeptide to be transported into the periplasmic space of a host cell like E. coli, for example, can be determined by separating periplasmic proteins from proteins retained in the cytoplasm, using a method such as that provided in Example 8.
  • gene products to be expressed by the methods of the invention can be designed to include molecular moieties that aid in the purification and/or detection of the gene products.
  • molecular moieties are known in the art; as one example, a polypeptide gene product can be designed to include a polyhistidine 'tag' sequence - a run of six or more histidines, preferably six to ten histidine residues, and most preferably six histidines - at its N- or C-terminus. The presence of a polyhistidine sequence on the end of a polypeptide allows it to be bound by cobalt- or nickel-based affinity media, and separated from other polypeptides.
  • the polyhistidine tag sequence can be removed by exopeptidases.
  • Additional tags, expressed at the N-terminal end of the amino acid sequence of a polypeptide gene product produced by the methods of the invention comprise in certain embodiments: (1) the self-cleaving N-terminal portions (N pro ) of polyproteins from pestiviruses such as Hog cholera virus (strain Alfort) (SEQ ID NO: 13), also called classical swine fever virus (CSFV), and from border disease virus (BDV) and bovine viral diarrhea virus (BVDV), and fragments thereof; (2) the N-terminal portion of carboxypeptidase B ('CPB') precursor, which is for example SEQ ID NO: 14 (amino acids 21-110 of Sus scrofa CPB, SwissProt P09955.5), and fragments thereof; and/or (3) small ubiquitin-related modifier (SUMO) (SEQ ID NO: 15, SwissProt P55853.1) Any N-terminal tag may itself be further tagged at its N-
  • the SUMO protease polypeptides are also fusion proteins comprising 6xHis tags, allowing for a two-step purification: in the first step, the expressed 6xHis-SUMO-tagged polypeptide is purified by binding to a nickel column, followed by elution from the column. In the second step, the SUMO tags on the purified polypeptides are cleaved by the 6xHis-tagged SUMO protease, and the SUMO protease - polypeptide reaction mixture is run through a second nickel column, which retains the SUMO protease but allows the now untagged polypeptide to flow through.
  • fluorescent protein sequences can be expressed as part of a polypeptide gene product, with the amino acid sequence for the fluorescent protein preferably added at the N- or C-terminal end of the amino acid sequence of the polypeptide gene product.
  • the resulting fusion protein fluoresces when exposed to light of certain wavelengths, allowing the presence of the fusion protein to be detected visually.
  • a well-known fluorescent protein is the green fluorescent protein of Aequorea victoria, and many other fluorescent proteins are commercially available, along with nucleotide sequences encoding them.
  • Glycosylation Gene products expressed by the methods of the invention may be glycosylated or unglycosylated.
  • the expressed gene products are polypeptides.
  • Glycosylated polypeptides are polypeptides that comprise a covalently attached glycosyl group, and include polypeptides comprising all the glycosyl groups normally attached to particular residues of that polypeptide (fully glycosylated polypeptides), partially glycosylated polypeptides, polypeptides with glycosylation at one or more residues where glycosylation does not normally occur (altered glycosylation), and polypeptides glycosylated with at least one glycosyl group that differs in structure from the glycosyl group normally attached to one or more specified residues (modified glycosylation).
  • modified glycosylation is the production of "defucosylated” or "fucose-deficient" polypeptides, polypeptides lacking fucosyl moieties in the glycosyl groups attached to them, by expression of polypeptides in host cells lacking the ability to fucosylate polypeptides.
  • Unglycosylated polypeptides are polypeptides that do not comprise a covalently bound glycosyl group.
  • An unglycosylated polypeptide can be the result of deglycosylation of a polypeptide, or of production of an aglycosylated polypeptide.
  • Deglycosylated polypeptides can be obtained by enzymatically deglycosylating glycosylated polypeptides, whereas aglycosylated polypeptides can be produced by expressing polypeptides in host cells that do not have the capability to glycosylate polypeptides, such as prokaryotic cells or cells in which the function of at least one glycosylation enzyme has been eliminated or reduced.
  • the expressed polypeptides are aglycosylated, and in a more specific embodiment, the aglycosylated polypeptides are expressed in prokaryotic cells such as E. coli.
  • Gene products expressed by the methods of the invention may be covalently linked to other types of molecules.
  • molecules that may be covalently linked to expressed gene products include polypeptides (such as receptors, ligands, cytokines, growth factors, polypeptide hormones, DNA- binding domains, protein interaction domains such as PDZ domains, kinase domains, antibodies, and fragments of any such polypeptides); water-soluble polymers (such as polyethylene glycol (PEG), carboxymethylcellulose, dextran, polyvinyl alcohol, polyoxyethylated polyols (such as glycerol), polyethylene glycol propionaldehyde, and similar compounds, derivatives, or mixtures thereof); and cytotoxic agents (such as chemotherapeutic agents, growth-inhibitory agents, toxins (such as enzymatically active toxins of bacterial, fungal, plant, or animal origin, or fragments thereof), and radioactive isotopes
  • polypeptides such as receptors, ligands
  • the expressed gene products are antibodies.
  • the term 'antibody' is used in the broadest sense and specifically includes 'native' antibodies, fully-human antibodies, humanized antibodies, chimeric antibodies, multispecific antibodies (such as bispecific antibodies), monoclonal antibodies, polyclonal antibodies, antibody fragments, and other polypeptides derived from antibodies that are capable of binding antigen.
  • the numbering of the residues in an immunoglobulin heavy chain is that of the EU index (the residue numbering of the human IgGl EU antibody) as in Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, 1991, National Institute of Health, Bethesda, Maryland.
  • 'Native' antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of inter-chain disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at its N- terminal end a variable domain (V H ) followed by a number of constant domains.
  • V H variable domain
  • Each light chain has a variable domain at it N-terminal end (V L ) and a constant domain at its C-terminal end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light-chain variable domain is aligned with the variable domain of the heavy chain.
  • V L N-terminal end
  • HV s hypervariable regions
  • FR framework regions
  • the term 'Fc region' refers to a C-terminal region of an immunoglobulin heavy chain, and includes native Fc regions and variant Fc regions.
  • the human IgG heavy-chain Fc region can be defined to stretch from an amino acid residue at position Cys226, or from Pro230, to the carboxyl -terminus thereof.
  • the Fc region can be defined to extend from the N-terminal residue (Ala231) of the conserved C H 2 immunoglobulin domain to the C-terminus, and may include multiple conserved domains such as C H 2, C H 3, and C H 4.
  • the C- terminal lysine (residue 447 according to the EU numbering system) of the native Fc region may be removed, for example, during production or purification of the antibody, or by recombinantly engineering the nucleic acid encoding a heavy chain of the antibody. Accordingly, a composition of intact antibodies may comprise antibody populations with all K447 residues removed, antibody populations with no K447 residues removed, and antibody populations having a mixture of antibodies with and without the K447 residue.
  • the Fc region of an antibody is crucial for recruitment of immunological cells and antibody dependent cytotoxicity (ADCC). In particular, the nature of the ADCC response elicited by antibodies depends on the interaction of the Fc region with receptors (Fc s) located on the surface of many cell types.
  • Humans contain at least five different classes of Fc receptors.
  • the binding of an antibody to FcRs determines its ability to recruit other immunological cells and the type of cell recruited.
  • the ability to engineer antibodies with altered Fc regions that can recruit only certain kinds of cells can be critically important for therapy (US Patent Application 20090136936 Al , 05-28-2009, Georgiou, George).
  • Native antibodies produced by mammalian cells typically comprise a branched, biantennary oligosaccharide that is generally attached by an N-linkage to Asn297 of the CH2 domain of the Fc region.
  • antibodies produced by the methods of the invention are not glycosylated or are aglycosylated, for example, due to a substitution at residue 297 of the Fc region, or to expression in a host cell that does not have the capability to glycosylate polypeptides. Due to altered ADCC responses, unglycosylated antibodies may stimulate a lower level of inflammatory responses such as neuroinflammation. Also, since an antibody having an aglycosylated Fc region has very low binding affinity for Fc receptors, such antibodies would not bind to the large number of immune cells that bear these receptors. This is a significant advantage since it reduces non-specific binding, and also increases the half-life of the antibody in vivo, making this attribute very beneficial in therapeutics.
  • antibody fragments' comprise a portion of an intact antibody, preferably comprising the antigen-binding region thereof.
  • antibody fragments include Fab, Fab', F(ab') 2 , Fc, Fd, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules such as scFv; and multispecific antibodies formed from antibody fragments.
  • a 'human antibody' is one that possesses an amino-acid sequence corresponding to that of an antibody produced by a human.
  • a 'chimeric' antibody is one in which a portion of the heavy and/or light chain is identical to, or shares a certain degree of amino acid sequence identity with, corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical to, or shares a certain degree of amino acid sequence identity with, corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies.
  • a 'humanized' antibody is a chimeric antibody that contains minimal amino acid residues derived from non- human immunoglobulin molecules.
  • a humanized antibody is a human immunoglobulin (recipient antibody) in which HVR residues of the recipient antibody are replaced by residues from an immunoglobulin HVR of a non-human species (donor antibody) such as mouse, rat, rabbit, or nonhuman primate.
  • donor antibody such as mouse, rat, rabbit, or nonhuman primate.
  • FR residues of the human recipient antibody are replaced by corresponding non-human residues.
  • humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody.
  • the term 'monoclonal antibody' refers to an antibody obtained from a population of substantially homogeneous antibodies, in that the individual antibodies comprising the population are identical except for possible mutations, such as naturally occurring mutations, that may be present in minor amounts.
  • the modifier 'monoclonal' indicates the character of the antibody as not being a mixture of discrete antibodies.
  • polyclonal antibody preparations which typically include different antibodies directed against different determinants (epitopes)
  • each monoclonal antibody of a monoclonal antibody preparation is directed against the same single determinant on an antigen.
  • monoclonal antibody preparations are advantageous in that they are typically uncontaminated by other immunoglobulins .
  • the 'binding affinity' of a molecule such as an antibody generally refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule and its binding partner (such as an antibody and the antigen it binds). Unless indicated otherwise, 'binding affinity' refers to intrinsic binding affinity that reflects a 1 : 1 interaction between members of a binding pair (such as antibody and antigen).
  • the affinity of a molecule X for its partner Y can generally be represented by the dissociation constant (Kd).
  • Kd dissociation constant
  • binding affinity A variety of ways to measure binding affinity are known in the art, any of which can be used for purposes of the present invention. Specific illustrative methods for measuring binding affinity are described in Example 6. Antibodies and antibody fragments produced by and/or used in methods of the invention preferably have binding affinities of less than 100 nM, more preferably have binding affinities of less than 10 nM, and most preferably have binding affinities of less than 2 nM, as measured by a surface- plasmon resonance assay as described in Example 6.
  • Enzymes Used in Industrial Applications Many industrial processes utilize enzymes that can be produced by the methods of the invention. These processes include treatment of wastewater and other bioremediation and/or detoxification processes; bleaching of materials in the paper and textile industries; and degradation of biomass into material that can be fermented efficiently into biofuels. In many instances it would be desirable to produce enzymes for these applications in microbial host cells or preferably in bacterial host cells, but the active enzyme is difficult to express in large quantities due to problems with enzyme folding and/or a requirement for a cofactor. In certain embodiments of the invention, the expression methods of the invention are used to produce enzymes with industrial applications, such as arabinose- and xylose-utilization enzymes (e.g.
  • xylose isomerase (EC 5.3.1.5)) or lignin-degrading peroxidases (e.g. lignin peroxidase (EC 1.11.1.14), manganese peroxidase (EC 1.11.1.13), versatile peroxidase (EC 1.11.1.16), or laccase (EC 1.10.3.2)).
  • lignin peroxidase EC 1.11.1.14
  • manganese peroxidase EC 1.11.1.13
  • versatile peroxidase EC 1.11.1.16
  • laccase EC 1.10.3.2
  • the methods of the invention can be used to produce a wide variety of gene products in soluble and active form, as opposed to in inclusion bodies, which are insoluble and in which the gene product is inactive.
  • the production of gene products in soluble form can be confirmed using the methods described in Example 4.
  • the selection of methods for characterizing gene product activity will depend on the nature of the gene product. For example, if the gene product is an antibody, then the methods of Example 6 can be used to measure the degree to which the antibody can bind antigen, an important aspect of antibody activity. If the gene product is an enzyme, the ability of the enzyme to catalyze an appropriate biochemical reaction can be measured.
  • disulfide bonds one indication that a polypeptide has been produced in active form is the presence of correctly positioned disulfide bonds in the polypeptide, as can be determined using the methods of Example 7.
  • a batch process is characterized by inoculation of the sterile culture medium (batch medium) with microorganisms at the start of the process, cultivated for a specific reaction period. During cultivation, cell concentrations, substrate concentrations (carbon source, nutrient salts, vitamins, etc.) and product concentrations change. Good mixing ensures that there are no significant local differences in composition or temperature of the reaction mixture.
  • the reaction is non- stationary and cells are grown until the growth-limiting substrate (generally the carbon source) has been consumed.
  • Continuous operation is characterized in that fresh culture medium (feed medium) is added continuously to the fermentor and spent media and cells are drawn continuously from the fermentor at the same rate.
  • growth rate is determined by the rate of medium addition, and the growth yield is determined by the concentration of the growth limiting substrate (i.e. carbon source). All reaction variables and control parameters remain constant in time and therefore a time-constant state is established in the fermentor followed by constant productivity and output.
  • Semi-continuous operation can be regarded as a combination of batch and continuous operation.
  • the fermentation is started off as a batch process and when the growth-limiting substrate has been consumed, a continuous feed medium containing glucose and minerals is added in a specified manner (fed-batch).
  • this operation employs both a batch medium and a feed medium to achieve cell growth and efficient production of the desired protein. No cells are added or taken away during the cultivation period and therefore the fermentor operates batchwise as far as the microorganisms are concerned.
  • the present invention can be utilized in a variety of processes, including those mentioned above, a particular utilization is in conjunction with a fed-batch process.
  • cell growth and product accumulation can be monitored indirectly by taking advantage of a correlation between metabolite formation and some other variable, such as medium H, optical density, color, and titrable acidity.
  • optical density provides an indication of the accumulation of insoluble cell particles and can be monitored on-stream using a micro-OD unit coupled to a display device or a recorder, or off-line by sampling.
  • Optical density readings at 600 nanometers (OD600) are used as a means of determining dry cell weight.
  • High-cell-density fermentations are generally described as those processes which result in a yield of >30 g cell dry weight/liter (OD 60 o >60) at a minimum, and in certain embodiments result in a yield of >40 g cell dry weight/liter (OD 60 o >80).
  • All high-cell-density fermentation processes employ a concentrated nutrient media that is gradually metered into the fermentor in a "fed-batch" process.
  • a concentrated nutrient feed media is required for high-cell-density processes in order to minimize the dilution of the fermentor contents during feeding.
  • a fed- batch process is required because it allows the operator to control the carbon source feeding, which is important because if the cells are exposed to concentrations of the carbon source high enough to generate high cell densities, the cells will produce so much of the inhibitory byproduct, acetate, that growth will stop (Majewski and Domach, "Simple constrained-optimization view of acetate overflow in E. coli Biotechnol Bioeng 1990 Mar 25; 35(7): 732-738).
  • Acetic acid and its deprotonated ion, acetate together represent one of the main inhibitory byproducts of bacterial growth and recombinant protein production in bioreactors.
  • acetate is the most prevalent form of acetic acid. Any excess carbon energy source may be converted to acetic acid when the amount of the carbon energy source greatly exceeds the processing ability of the bacterium.
  • the choice of growth medium may affect the level of acetic acid inhibition; cells grown in defined media may be affected by acetic acid more than those grown in complex media. Replacement of glucose with glycerol may also greatly decrease the amount of acetic acid produced.
  • glycerol produces less acetic acid than glucose because its rate of transport into a cell is much slower than that of glucose.
  • glycerol is more expensive than glucose, and may cause the bacteria to grow more slowly.
  • the use of reduced growth temperatures can also decrease the speed of carbon source uptake and growth rate thus decreasing the production of acetic acid.
  • Bacteria produce acetic acid not only in the presence of an excess carbon energy source or during fast growth, but also under anaerobic conditions. When bacteria such as E. coli are allowed to grow too fast, they may exceed the oxygen delivery ability of the bioreactor system which may lead to anaerobic growth conditions. To prevent this from happening, a slower constant growth rate may be maintained through nutrient limitation.
  • E. coli BL21(DE3) is one of the strains that has been shown to produce lower levels of acetic acid because of its ability to use acetic acid in its glyoxylate shunt pathway.
  • Small-scale fed-batch fermentors are available for production of recombinant proteins.
  • Larger fermentors have at least 1000 liters of capacity, preferably about 1000 to 100,000 liters of capacity (i.e. working volume), leaving adequate room for headspace.
  • These fermentors use agitator impellers or other suitable means to distribute oxygen and nutrients, especially glucose (the preferred carbon/energy source).
  • Small-scale fermentation refers generally to fermentation in a fermentor that is no more than approximately 100 liters in volumetric capacity, and in some specific embodiments no more than approximately 10 liters.
  • Standard reaction conditions for the fermentation processes used to produce recombinant proteins generally involve maintenance of pH at about 5.0 to 8.0 and cultivation temperatures ranging from 20 to 50 degrees C for microbial host cells such as E. coli.
  • fermentation is performed at an optimal pH of about 7.0 and an optimal cultivation temperature of about 30 degrees C.
  • the standard nutrient media components in these fermentation processes generally include a source of energy, carbon, nitrogen, phosphorus, magnesium, and trace amounts of iron and calcium.
  • the media may contain growth factors (such as vitamins and amino acids), inorganic salts, and any other precursors essential to product formation.
  • the media may contain a transportable organophosphate such as a glycerophosphate, for example an alpha-glycerophos- phate and/or a beta-glycerophosphate, and as a more specific example, glycerol-2- phosphate and/or glycerol-3 -phosphate.
  • the elemental composition of the host cell being cultivated can be used to calculate the proportion of each component required to support cell growth.
  • the component concentrations will vary depending upon whether the process is a low-cell-density or a high-cell-density process.
  • the glucose concentrations in low-cell-density batch fermentation processes range from 1 to 5 g/L
  • high-cell-density batch processes use glucose concentrations ranging from 45 g/L to 75 g/L.
  • growth media may contain modest concentrations (for example, in the range of 0.1 - 5 mM, or 0.25 mM, 0.5 mM, 1 mM, 1.5 mM, or 2 mM) of protective osmolytes such as betaine, dimethylsulfoniopropionate, and/or choline.
  • the metabolic rate is directly proportional to availability of oxygen and a carbon/energy source; thus, reducing the levels of available oxygen or carbon/energy sources, or both, will reduce metabolic rate.
  • Manipulation of fermentor operating parameters such as agitation rate or back pressure, or reducing 0 2 pressure, modulates available oxygen levels and can reduce host cell metabolic rate. Reducing concentration or delivery rate, or both, of the carbon/energy source(s) has a similar effect.
  • induction of expression can lead to a decrease in host cell metabolic rate.
  • the growth rate stops or decreases dramatically.
  • Reduction in host cell metabolic rate can result in more controlled expression of the gene product(s) of interest, including the processes of protein folding and assembly.
  • Host cell metabolic rate can be assessed by measuring cell growth rates, either specific growth rates or instantaneous growth rates (by measuring optical density (OD) such as OD600 and or optionally by converting OD to biomass), as described in Example 1 below.
  • Desirable growth rates are, in certain embodiments of the invention, in the range of 0.01 to 0.7, or are in the range of 0.05 to 0.3, or are in the range of 0.1 to 0.2, or are approximately 0.15 (0.15 plus-or-minus 10%), or are 0.15.
  • the E. coli EB0001 strain which is also called ASE(DGH), was prepared as described in Example 3 of International Application PCT/US13/53562 (published as WO2014025663A1) and in Example l .B. of International Application PCT/US 14/14968 (published as WO2015020690A1); the WO2014025663A1 and WO2015020690A1 publications are incorporated by reference in their entirety herein.
  • coli EB0001 can be expressed as: AaraBAD fliuA2 [Ion] ompT ahpC gal Xatt::pNEB3-rl-cDsbC (Spec, lacI) AtrxB sulAl l i?( ⁇ cr-73: :miniTnlO-Tet s )2 [dcm] R(zgb-210: :TnlO- -Tet s ) Aara£p: :J23104 AscpA-argK-scpBC endAl rpsL- Arg43 Agor A(mcrC-mrr) ⁇ ⁇ 4: :IS ⁇ 0
  • Strain EB0002 has a genotype which can be expressed as EBOOOl prpD, or as:
  • Infliximab is a chimeric monoclonal antibody that binds to TNF-alpha. Codon- optimized polynucleotide sequences encoding the infliximab heavy chain and the infliximab light chain were cloned into the pBAD24 and pPR033 vectors, respectively, as described in Example 5 of WO2014025663 Al .
  • the resulting pBAD24-Infliximab_HC and pPR033-Infliximab_LC expression constructs were transformed into E. coli EBOOOl by heat shock at 42 degrees C followed by incubation at 37 degrees C overnight to create E. coli EB0001(pBAD24- Infliximab_HC/pPR033-Infliximab_LC) host cells.
  • E. coli EB0001(pBAD24-Infliximab_HC/pPRO33-Infliximab_LC) host cells were grown to high cell densities using the following methods.
  • the equipment and media common to six fermentation runs, referred to as Run A - Run F, are described below, followed by the unique aspects of each fermentation run for the growth periods shown in Figs. 1 - 3.
  • the host cells were grown in a New Brunswick BioFlo/CelliGen 115 water jacketed fermentor (Eppendorf North America, Hauppauge, New York), 1L vessel size with a 2X Rushton impeller and a BioFlo/CelliGen 115 Fermentor/Bioreactor controller; temperature, pH, and dissolved oxygen (DO) were monitored.
  • New Brunswick BioFlo/CelliGen 115 water jacketed fermentor Eppendorf North America, Hauppauge, New York
  • 1L vessel size with a 2X Rushton impeller and a BioFlo/CelliGen 115 Fermentor/Bioreactor controller temperature, pH, and dissolved oxygen (DO) were monitored.
  • the bottle was filled to 1L with MilliQ double-distilled H 2 0, and the pH adjusted to 7.0 with ammonium hydroxide.
  • 900 mL of the fermentation media solution was transferred to the fermentor vessels and autoclaved for 45 minutes. After autoclaving, the fermentor vessels were allowed to cool, the sensors were calibrated, and a further six hours were allowed for equilibration after calibration.
  • antibiotics 100 micrograms/mL ampicillin (AMP) for pBAD24 and 34 micrograms/mL chloramphenicol (CAM) for pP 033)
  • the initial set points for the fermenter conditions were:
  • Inoculum and sampling a feeder culture of E. coli EB0001(pBAD24- Infliximab_HC/pPR033-Infliximab_LC) host cells was prepared and used to inoculate the fermentation medium at an initial OD 60 o of 0.1. After an initial fermentation period of 9 to 10 hours, samples of the fermentation culture were collected at 15-minute intervals and the OD 60 o of the samples was determined. Growth rates for each fermentation run were determined by plotting the change in OD 6 oo over time for the exponential phase of the growth curve, and then determining the best-fit exponential curve, as shown in Figs. 2 and 3.
  • the value of the exponent for the best-fit exponential curve is one indicator of the specific growth rate per hour, and is referred to herein as a 'specific growth rate indicator'. Changes in biomass over time can also be used to determine specific growth rates.
  • instantaneous growth rates for each successive point assayed during the fermentation run can be determined as follows. For a pair of time points at which the fermentation culture has been assayed, with 'time t .
  • the instantaneous growth rate at time t is calculated as: ( ln(biomass at time t ) - ln(biomass at time ⁇ ) ) / (time t - time t .i).
  • Instantaneous growth rates are often used to determine whether a fermentation culture is achieving growth rates comparable to a desired specific growth rate in response to an automated feeding schedule.
  • Glucose was added to 2% at time points (hours) Glucose was added to 2.25% at 15.8 hours.
  • the cells demonstrated a specific growth rate indicator of 0.24/hour.
  • Glucose was added to 2% at time points (hours): 12, 14.6, 16.3, and 17.75.
  • Nitrogen feed solution (see below) was added at 12.6 hours.
  • MgS0 4 (6 ml of 1M MgS0 4 solution) was added at 15 hours.
  • the cells demonstrated a specific growth rate indicator of 0.21/hour.
  • Nitrogen feed when the culture reached approximately 20 OD 60 o, 125 mL salt solution containing (amounts per 125 mL) was added:
  • Glucose was added to 2% at time points (hours): 10.7, 13.1, 14.4, 15.35, 16.3, 16.8, 17.3, and 18.1.
  • MgS0 4 (6 ml of 1M MgS0 4 solution) was added at 12.9 hours.
  • Nitrogen feed solution (see above) was added automatically between 12.5 and 13.6 hours, at a pump output of 33% to deliver approximately 67 ml of nitrogen feed solution in 60.1 minutes.
  • a maximum OD of 124.68 was obtained in 18.5 hours with a specific growth rate indicator of 0.21/hour.
  • Glucose was added to 2% at time points (hours): 10.14, 11.9, 13, 14.26, 16.1 , 17.25, 17.8, 18.3, and 18.8.
  • Glucose was added to 1% at time points (hours): 15.1, 15.45, and 15.83.
  • MgS0 4 (6 ml of 1M MgS0 4 solution) was added at 12.8 hours.
  • Nitrogen feed solution (see above) was added automatically between 12.5 and 13.6 hours, as in Run C. A maximum OD of 167.2 was obtained in 18.25 hours, with a specific growth rate indicator of 0.0625/hour (see Figs. 1 and 2).
  • Oygen tank pressure brought down to 10.0 - 8 kPa.
  • 0 2 high limit initially set at 20 L/min, then to 40 L/min mid-run; low limit at 0.2 L/min.
  • MgS0 4 (6 ml of 1M MgS0 4 solution) was added at 13.3 hours.
  • Nitrogen feed solution (see above) was added automatically between 12.5 and 13.6 hours, as in Runs C and D.
  • a maximum OD of 132.1 was obtained in 19.0 hours, with a specific growth rate indicator of 0.2393/hour.
  • 0 2 high limit initially set at 20 L/min, then to 40 L/min mid-run; low limit at 0.2 L/min.
  • the nitrogen feed solution was changed from that shown in Run B, in that the ferrous sulfate and trace elements were removed from the solution; these compounds were added in the glucose feed instead.
  • Two periods of automatic nitrogen feeding were performed, at 11.75 - 12.75 hours and at 19.25 - 20.25 hours, with a 27% pump output.
  • E. coli EB0001(pBAD24-Infliximab_HC/pP O33-Infliximab_LC) host cells for example in 200- to 500-ml shake flasks and generally as described in Example 3 below, are used to determine optimal growth times and inducer concentrations for expression of infliximab by the host cells.
  • E. coli EB0001(pBAD24-Infliximab_HC/pPRO33-Infliximab_LC) host cells are grown to high cell density as described above, until the cells reach a suitable growth phase for induction of protein expression, which is generally performed when cells have reached or are nearing the end of the exponential growth phase.
  • a suitable growth phase for induction of protein expression which is generally performed when cells have reached or are nearing the end of the exponential growth phase.
  • an inoculum of these cells in fermentation media, producing an initial OD 60 o of 0.1 is grown in a ferm enter until the cell culture reaches an OD 60 o between 80 and 90; at a specific growth rate of approximately 0.15/hour, with growth taking roughly 24 hours.
  • Host cells such as the E.
  • coli EB0001(pBAD24- Infliximab_HC/pPR033-Infliximab_LC) host cells can also be induced at higher cell densities, for example up to an OD 60 o of 120 or more, by adjusting the feeding conditions described above, producing higher cell densities at the end of the exponential growth phase.
  • the inducers L-arabinose and propionate are introduced into the fermentation media, at concentrations determined by smaller-volume inducer titration experiments, with application of an appropriate scaling factor, which is less than 1. On this basis, the optimal concentrations of L-arabinose and propionate for induction of E.
  • coli EB0001(pBAD24- Infliximab_HC/pPR033-Infliximab_LC) host cell fermentation cultures are in the range of 1 to 266 micromolar (approximately 0.000015 to 0.004%) per OD unit of cells, and more specifically 6 to 67 micromolar (approximately 0.00009 to 0.001%) per OD unit of cells for L-arabinose, and 1 to 100 mM per OD unit of cells, and more specifically 5 to 50 mM per OD unit of cells for propionate.
  • the host cells are allowed to grow under inducing conditions for a length of time sufficient to produce the desired protein, with longer growth times used to produce proteins having longer polypeptide chains or a more complex structure, such as a multimeric structure; for the production of infliximab, the host cells are allowed to grow for 14-24 hours post-induction.
  • the fermentation culture is then centrifuged at 4300 RPM for seven minutes to harvest the cells.
  • the host cells containing infliximab in the cytoplasm can be frozen at that point for storage and/or transport, or can be lysed to release the expressed protein.
  • Host cells can be lysed using known chemical and enzymatic methods, for example by resuspending the cells in a solution containing lysozyme.
  • coli EB0001(pBAD24-Infliximab_HC/pP O33-Infliximab_LC) host cells are preferrably lysed by mechanical methods, such as cell disruption using a Microfluidics model LV1 microfluidizer for volumes up to 60 ml, or a Microfluidics model M-110Y microfluidizer for volumes greater than 60 mL (Microfluidics International Corp., Westwood, Massachusetts).
  • protein can be separated from cell membranes and other insoluble components in the mixture of lysis products using an extraction solution comprising Triton X-100 (0.5% to 15%; for most applications 1% to 8%) and urea (0.1M to 8M; for most applications 1M to 5M).
  • Triton X-100 0.5% to 15%; for most applications 1% to 8%
  • urea 0.1M to 8M; for most applications 1M to 5M.
  • the most effective concentrations of Triton X-100 and urea in the extraction solution will vary depending on the type of protein to be extracted and other factors, so testing different concentrations would be advantageous.
  • This optional extraction procedure is not sufficent to solubilize inclusion bodies. Centrifugation at 20,000 x g for 15 minutes at room temperature is then used to separate out the insoluble fraction, and the supernatant containing soluble protein including the expressed infliximab is collected.
  • the infliximab antibodies are detected and quantified using a capillary electrophoresis Western blot, run on a WES system (ProteinSimple, San Jose, California), according to the manufacturer's instructions. Soluble protein extracts are loaded into the capillary set, proteins are electrophoretically separated by size, and then the infliximab antibodies in the samples are detected with a blocking step (instead of the use of a primary antibody), and incubation with an HRP- conjugated goat anti-human secondary antibody that recognizes human antibody heavy and light chains. Antibody detection is accomplished by addition of the chemiluminescent substrate to the capillary and the direct capture of the light emitted during the enzyme-catalyzed reaction.
  • Molecular weight estimates are calculated using a standard curve generated using six biotinylated proteins ranging from 12 k to 230 kDa for each run. Fluorescent standards are included in the sample loading buffer, giving each sample an internal standard that is used to align the sample with the molecular weight standard.
  • serial dilutions are prepared of commerically available infliximab having a known protein concentration, starting for example at 10 micrograms/mL and diluted down to 1.0 nanograms/mL. Approximately five WES system capillaries are used to run the serial dilution.
  • a curve is generated by the WES system software representing the protein band's chemiluminescence intensity, and the area under each curve is evaluated, with a standard curve of these areas plotted for the infliximab protein bands in the infliximab serial dilution capillaries.
  • the area under each curve representing the chemiluminescence intensity of an experimental infliximab sample can the compared to the standard curve generated for the samples of known infliximab concentration.
  • the infliximab antibodies can be further purified as described in Example 5, and additional characterization of the infliximab antibodies is described in Example 6 (measurement of antibody binding affinity) and Example 7 (characterizing the disulfide bonds present in coexpression products).
  • the form of insulin glargine that was expressed lacks the signal sequence that is present in preproglargine, and will be referred to herein as proglargine.
  • the proglargine polypeptide that was expressed has the following structure, and is shown schematically in Fig. 4: an N-terminal propeptide, the insulin glargine B chain (SEQ ID NO: 10), a connecting C-peptide, and the insulin glargine A chain (SEQ ID NO: 9) at its C-terminus.
  • the N-terminal propeptide portion of the insulin glargine polypeptide comprises an N-terminal methionine residue and an arginine residue as the C-terminal residue of the N-terminal propeptide, located immediately upstream of the insulin glargine B chain (SEQ ID NO: 10), so that tryptic cleavage releases the N-terminal propeptide from the B chain.
  • the insulin glargine B chain (SEQ ID NO: 10) and the C-peptide each also have an arginine residue as the C-terminal residue of those portions of the proglargine polypeptide; as a result, digestion with trypsin also releases the C-peptide from the insulin glargine B and A chains.
  • the pSOL expression vector (SEQ ID NO:3), also described in published US patent application US2015353940A1 , has two multiple cloning sites: one downstream of the L-arabinose-inducible araBAD promoter, and another downstream of the propionate-inducible prpBCDE promoter.
  • a polynucleotide encoding the above proglargine polypeptide was cloned into the pSOL expression vector (SEQ ID NO:3) downstream of the araBAD promoter.
  • EB0001 cells also called E. coli ASE(DGH) cells, described in Example 1 above
  • EB0001(pSOL-proglargine/Ervlp) cells were transformed into EB0001 cells (also called E. coli ASE(DGH) cells, described in Example 1 above) by heat shock at 42 degrees C, followed by incubation at 37 degrees C overnight, to create EB0001(pSOL-proglargine/Ervlp) cells.
  • the combination of EB0001 host cells with the expression vector pSOL- proglargine/Ervlp is also referred to as AbS0092.
  • the E. coli EB0001(pSOL- proglargine/Ervlp) host cells were grown to high cell densities, greater than 170 OD 6 oo, and produced soluble proglargine with properly formed disulfide bonds.
  • the EB0001(pSOL-proglargine/Ervlp) host cells were grown in a DASGIP fermentation system (Eppendorf North America, Hauppauge, New York) in two separate 250-ml DASbox fermentation vessels, which are further described in Example 1.
  • the bioreactors used for these host cells were bioreactor 3 ('BR3') and bioreactor 4 ('BR4'); the cells in each bioreactor were grown under conditions designed to minimize any differences in the growth of the cells when compared to those in the other bioreactor.
  • the bioreactors were calibrated as follows: B 3: BR4:
  • Fermentation included cascaded dissolved oxygen (DO) control, with the following varying conditions: agitation (400-1600 rpm), concentration of added oxygen (' ⁇ 02', 21-100%), with total input gas flow at 6-12 sLph.
  • DO dissolved oxygen
  • the fermentation conditions for the initial growth stage were 30.0 degrees C, DO 30%, pH 7.0, growth feed with 70% glucose at an initial feed rate of 0.6 mL/hr, for a set growth rate of 0.15/hr.
  • the fermentation conditions for the induction stage were 27.0 degrees C, DO 30%, pH 7.0, induction feed with 70% glycerol at an induction feed coefficient of 0.15 mL/g DC w*hr ('DCW is dry cell weight).
  • Fermentation medium pre- sterilization components, concentration in g/L per 90 mL volume added to each bioreactor:
  • Fermentation medium post-sterilization components (sterile stock concentration), amount in mL added to reach total volume of ca. 100 mL in each bioreactor:
  • Induction feed components (sterile stock concentration), amount
  • the cells were grown under the growth stage conditions (30.0 degrees C, DO 30%, pH 7.0, growth feed containing 70% glucose at an initial feed rate of 0.6 mL/hr, for a set growth rate of 0.15/hr) for 26 hours, and were sampled (OD 60 o 136.4 and 131.2, respectively).
  • 5 mL of lOx Tremendous Broth was added to each bioreactor.
  • Induction was initiated; the fermentation conditions were set to the induction stage conditions: 27.0 degrees C, DO 30%, pH 7.0, and induction feed containing 70% glycerol at an induction feed coefficient of 0.15 mL/g DC w/hr.
  • the induction feed also contained the inducer L-arabinose, at a concentration calculated as follows from the total volume of components added to create the induction feed:
  • the host cells in each bioreactor were sampled again at 27, 37, 39, 42, and 45 hours total fermentation time; these time points when expressed in terms of elapsed induction time (EIT(hrs)) are 0, 10, 12, 15, and 18.
  • the mean optical densities (the average of the two OD 60 o readings, one from each bioreactor) observed at each time point are shown graphically in Fig. 5, with the time points labeled in terms of elapsed induction time (EIT(hrs)).
  • EIT(hrs) elapsed induction time
  • the flow rate (F) in mL/hr is the flow rate of induction feed with inducer set manually based on an induction feed coefficient of 0.15 mL/g DC w/hr and OD measurement.
  • the volume (V) in mL is the volume of fermentation medium in the bioreactor, calculated from data generated by the DASbox fermenter system and taking into account the starting volume; minus the volume of media lost during autoclaving; plus any additions that the bioreactor made, such as growth feed, induction feed, and base and/or acid; plus the volume of nutrient solution (TB) added; minus the total volume of cumulative samples removed.
  • the cumulative grams of L-arabinose added (g ara ) in grams is calculated using the concentration of L-arabinose in the induction feed as calculated above, 5.4 g/L, multiplied by the flow rate (F) in mL/hr during the time period preceding the time of sampling, multiplied by the total duration of the induction feed (EIT) in hr, then divided by 1000 mL/L.
  • F flow rate
  • EIT total duration of the induction feed
  • the L-arabinose concentration ([ara]) in g/L is the grams of L-arabinose added (gara) divided by the volume (V) in mL and multiplied by the conversion factor 1000 ml/L.
  • the L-arabinose concentration / OD 60 o ([ara]/OD 60 o) is calculated by dividing the L-arabinose concentration ([ara]) in g/L or mg/L by the OD 60 o, then converting to % per OD 6 oo or mM per OD 60 o or micromolar ( ⁇ ) per OD 60 o using the following conversion factors:
  • each bioreactor produced at least 0.87 g/L of proglargine, which when adjusted for cell density was at least 5.2 mg/L/OD 600 .
  • the host cells were harvested by centrifugation at 4300 RPM for seven minutes, and stored as frozen pellets.
  • the stored host cell pellet from Bioreactor 3 is thawed, and lOg wet cell pellet is suspended in 100 mL lysis buffer: 50 mM Tris, 8M urea, pH 7.5 with 4 microliters benzonase.
  • the suspension is passed twice through a Microfluidics model LV1 microfluidizer (Microfluidics International Corp., Westwood, Massachusetts), with a 3 x 6 mL lysis buffer chase.
  • the LV1 has a void volume and ca. 70-80% of the material makes it through in a single pass; passing through twice results in an average 1.5x lytic treatment of the cell suspension.
  • the cell lysate is centrifuged at 20,000 x g for 30 minutes at 4 degrees C to precipitate the insoluble material, and the supernatant - also termed the soluble fraction - is collected and passed through a 2-micron filter.
  • the filtered soluble fraction is then passed over a 25-mL DEAE Sepharose Fast Flow (GE Healthcare Life Sciences, Pittsburgh, Pennsylvania) column, with an equilibration/wash buffer: 50 mM Tris, 8M urea, pH 7.5, and an elution buffer: 50 mM Tris, 8M urea, 1M NaCl, pH 7.5. Proteins are eluted using a 0-40% NaCl gradient over 15 CV (column volumes), followed by 100% NaCl over 5 CV. Fractions eluted from the DEAE column that contain the proglargine product, as detected on a non-reducing SDS-PAGE gel, are pooled and diluted with 2-3 volumes of 50 mM Tris pH 7.5 to reduce conductivity.
  • DEAE Sepharose Fast Flow GE Healthcare Life Sciences, Pittsburgh, Pennsylvania
  • the pooled fractions containing proglargine are then passed over a 1.7-mL Mono Q column (GE Healthcare Life Sciences, Pittsburgh, Pennsylvania), with an equilibration/wash buffer: 50 mM Tris, 4M urea, pH 7.5 for 10 CV, and an elution buffer: 50 mM Tris, 4M urea, 1M NaCl, pH 7.5. Proteins are eluted using a 0- 40% NaCl gradient (step 1) over 40 CV, followed by 100% NaCl over 10 CV (step 2).
  • the concentrated proglargine fractions are passed over a 120-mL 16-by-600 Superdex 200-pg column (HiLoad, GE Healthcare Life Sciences, Pittsburgh, Pennsylvania), with an equilibration/wash buffer: 50 mM Tris, 4M urea, 200 mM NaCl, pH 7.5.
  • the method of preparation of the purified proglargine samples for analysis by mass spectrometry is summarized in Fig. 6.
  • the proglargine is first digested with glutamyl endopeptidase ('Glu-C'), which cleaves on the C-terminal side of glutamic acid residues.
  • Proglargine fragments produced by this digestion are shown in Fig. 6, along with the locations of the disulfide bonds in properly assembled insulin glargine that would be expected to be present in these fragments.
  • the fragment containing two disulfide bonds and the amino acid sequences of SEQ ID NO: 18 and SEQ ID NO:20 also has additional amino acid residues from the N-terminal propeptide attached to the N-terminal residue of SEQ ID NO: 18.
  • a trypsin digest is performed to cleave the propeptide from the amino acid sequence of SEQ ID NO: 18 prior to MS analysis, which is carried out as described in Example 7.
  • a sample containing an insulin glargine standard known to have disulfide bonds in the proper positions, such as commercially available insulin glargine, is prepared for MS analysis in the same way as the purified proglargine samples produced by the methods of the invention.
  • the results of the MS analysis are shown in base-peak chromatograms.
  • the MS peaks created by non-reduced proglargine peptide fragments are compared to those created by reduced and alkylated proglargine peptide fragments. Peaks corresponding to the disulfide-bonded proglargine fragments are expected to be seen in the non-reduced sample, but are absent in the reduced and alkylated sample.
  • the presence and retention time of the peptide fragments containing two crosslinks and a single crosslink, respectively, is a determinant of the disulfide bond confirmation.
  • the +4 charge state of the doubly crosslinked peptide is predominant and it elutes in the extracted ion chromatogram at a retention time similar to the properly folded insulin glargine standard.
  • the +2 charge state of the singly crosslinked species is also observed as the predominant species and elutes as a single peak at a retention time comparable to the properly folded insulin glargine standard.
  • Purification of the proglargine and mass spectrometry results as described above are to provide confirmation that the proglargine produced by fermentation of the EB0001(pSOL-proglargine/Ervlp) host cells is present in the soluble cell lysate fraction and has correctly formed disulfide bonds.
  • Host cells containing expression constructs comprising inducible promoters - such as L-arabinose-inducible, propionate-inducible, L-rhamnose- inducible, or D-xylose-inducible promoters - are grown to the desired density for small-volume titrations (such as an OD 60 o of approximately 0.5) in M9 minimal medium containing the appropriate antibiotics, then cells are aliquoted into small volumes of M9 minimal medium, optionally prepared with no carbon source such as glycerol, and with the appropriate antibiotics and varying concentrations of each inducer.
  • inducible promoters - such as L-arabinose-inducible, propionate-inducible, L-rhamnose- inducible, or D-xylose-inducible promoters - are grown to the desired density for small-volume titrations (such as an OD 60 o of approximately 0.5) in M9 minimal medium containing the appropriate antibiotics, then cells are aliquo
  • Small-volume titrations can be performed in 200- to 500-ml shake flasks.
  • concentration of L-arabinose, L-rhamnose, or D-xylose necessary to induce expression is typically less (and is often substantially less) than 0.02% per OD unit of cells.
  • the tested concentrations of L- arabinose can range from 2% to 1.5%, 1%, 0.5%, 0.2%, 0.1%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.005%, 0.002%, 0.001%, 0.0005%, 0.0002%, 0.0001%, 0.00005%, 0.00002%, 0.00001%, 0.000005%, 0.000002%, 0.000001%, 0.0000005%, 0.0000002%, 0.0000001%, 0.00000005%, 0.00000002%, and 0.00000001%, all per OD unit of cells.
  • a concentration of 66.61 micromolar L- arabinose corresponds to 0.001% L-arabinose.
  • concentrations to be tested can range from 1 M to 750 mM, 500 mM, 250 mM, 100 mM, 75 mM, 50 mM, 25 mM, 10 mM, 5 mM, 1 mM, 750 micromolar, 500 micromolar, 250 micromolar, 100 micromolar, 50 micromolar, 25 micromolar, 10 micromolar, 5.0 micromolar, 2.5 micromolar, 1.0 micromolar, 500 nM, 250 nM, 100 nM, 50 nM, 25 nM, 10 nM, 5.0 nM, 2.5 nM, and 1.0 nM all per OD unit of cells.
  • the concentration of a different inducer such as propionate, added to each of the tubes containing concentration Y of the first inducer is varied in each series of samples.
  • titration experiments can start at a 'standard' combination of inducer concentrations, which for host cells having a reduced level of gene function of at least one gene encoding a protein that metabolizes the inducer is 0.0015% (100 micromolar) of any of L-arabinose, L-rhamnose, or D- xylose per OD unit of cells, and/or 100 micromolar propionate per OD unit of cells.
  • the 'standard' combination of inducer concentrations is 0.0033% (220 micromolar) of any of L-arabinose, L-rhamnose, or D-xylose per OD unit of cells, and/or 83 mM propionate per OD unit of cells. Additional combinations of inducer concentrations that vary from that of the 'standard' combination are tested; in a series of titration experiments, the results from initial experiments can be used to 'fine-tune' the inducer concentrations used in later experiments.
  • Similar titration experiments can be performed with any combination of inducers used in an expression system of the invention, including but not limited to L-arabinose, propionate, L-rhamnose, and D-xylose.
  • inducers used in an expression system of the invention, including but not limited to L-arabinose, propionate, L-rhamnose, and D-xylose.
  • the cells are pelleted, the desired product is extracted from the cells, and the yield of product per mass value of cells is determined by a quantitative immunological assay such as ELISA, or by purification of the product and quantification by UV absorbance at 280 nm.
  • the proteins to be expressed are engineered to include a fluorescent protein moiety, such as that provided by the mKate2 red fluorescent protein (Evrogen, Moscow, Russia), or the enhanced green fluorescent proteins from Aequorea victoria and Bacillus cereus.
  • a fluorescent protein moiety such as that provided by the mKate2 red fluorescent protein (Evrogen, Moscow, Russia)
  • the enhanced green fluorescent proteins from Aequorea victoria and Bacillus cereus.
  • Another approach to determining the amount and activity of gene products produced by different concentrations of inducers in a high-throughput titration experiment is to use a sensor capable of measuring biomolecular binding interactions, such as a sensor that detects surface plasmon resonance, or a sensor that employs bio-layer interferometry (BLI) (for example, an Octet® QK system from forteBIO, Menlo Park, CA).
  • a sensor capable of measuring biomolecular binding interactions such as a sensor that detects surface plasmon resonance, or a sensor that employs bio-layer interferometry (BLI) (for example, an Octet® QK system from forteBIO, Menlo Park, CA).
  • BBI bio-layer interferometry
  • the gene product can be detected and quantified using a capillary electrophoresis Western blot, such as that run on a WES system as described in Example 1 above.
  • the most straightforward approach is to lyse cells using any effective method, such as enzymatic lysis with lysozyme, as described in more detail in Example 8 below, or by cell disruption with a microfluidizer, as described in Example 1 above.
  • the lysed cells are then centrifuged at 20,000 x g for 15 minutes at room temperature to separate out the insoluble fraction as a pellet; the soluble fraction (the supernatant) is collected.
  • Any method such as ELISA or capillary electrophoresis Western blots, that can be used to detect the gene product, and preferably to specifically and quantifiably detect the gene product in each fraction, is employed and the amounts present in the soluble and insoluble fractions are compared.
  • Inclusion bodies can be harvested by centrifugation of lysed host cells, stained with dyes such as Congo Red, and visualized using bright-field or cross-polarized light microscopy at modest (10X) magnification (Wang et al., "Bacterial inclusion bodies contain amyloid-like structure", PLoS Biol 2008 Aug 5; 6(8): el95; doi: 10.1371 /journal. pbio.0060195).
  • dyes such as Congo Red
  • inclusion bodies can also be resolubilized (Singh and Panda, "Solubilization and refolding of bacterial inclusion body proteins", J Biosci Bioeng 2005 Apr; 99(4): 303-310; Review) and tested, using specific binding assays or other methods of protein identification, for example, to determine if they include the gene products that are being produced.
  • Antibodies produced by the expression systems of the invention are purified by centrifuging samples of lysed host cells at 10,000 x g for 10 minutes to remove any cells and debris. The supernatant is filtered through a 0.45 micrometer filter.
  • a 1-ml HiTrap MabSelect Protein A column (GE Healthcare Life Sciences, Pittsburgh, PA) is set up to achieve flow rates of 1 ml/min, and is used with the following buffers: binding buffer: 0.02 M sodium phosphate, pH 7.0; elution buffer: 0.1 M glycine-HCl, pH 2.7; and neutralization buffer: 1 M Tris-HCl, pH 9.0. The column is equilibrated with 5 column volumes (5 ml) of binding buffer, and then the sample is applied to the column.
  • the column is washed with 5-10 column volumes of the binding buffer to remove impurities and unbound material, continuing until no protein is detected in the eluent (determined by UV absorbance at 280 nm).
  • the column is then eluted with 5 column volumes of elution buffer, and the column is immediately re-equilibrated with 5-10 column volumes of binding buffer.
  • the antibody binding affinity is measured by a radiolabeled antigen-binding assay (RIA) performed with the Fab version of an antibody of interest and its antigen as described by the following assay. Production of the Fab version of a full-length antibody is well known in the art.
  • RIA radiolabeled antigen-binding assay
  • Solution-binding affinity of Fabs for antigen is measured by equilibrating Fab with a minimal concentration of ( 125 I)-labeled antigen in the presence of a titration series of unlabeled antigen, then capturing bound antigen with an anti-Fab antibody-coated plate (see, for example, Chen et al., "Selection and analysis of an optimized anti-VEGF antibody: crystal structure of an affinity-matured Fab in complex with antigen", J Mol Biol 1999 Nov 5; 293(4): 865-881).
  • microtiter plates (DYNEX Technologies, Inc., Chantilly, Virginia) are coated overnight with 5 micrograms/ml of a capturing anti-Fab antibody (Cappel Labs, West Chester, Pennsylvania) in 50 mM sodium carbonate (pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin in PBS for two to five hours at room temperature (approximately 23 °C).
  • a non- adsorbent plate (Nunc #269620; Thermo Scientific, Rochester, New York), 100 pM or 26 pM [ 125 I]-antigen are mixed with serial dilutions of a Fab of interest (e.g.
  • Fab-12 in Presta et al. , "Humanization of an anti -vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders", Cancer Res 1997 Oct 15; 57(20): 4593-4599).
  • the Fab of interest is then incubated overnight; however, the incubation may continue for a longer period (e.g., about 65 hours) to ensure that equilibrium is reached. Thereafter, the mixtures are transferred to the capture plate for incubation at room temperature (e.g. , for one hour). The solution is then removed and the plate washed eight times with 0.1% TWEEN-20TM surfactant in PBS.
  • the Kd or Kd value is measured by using surface-plasmon resonance assays using a BIACORE®-2000 or a BIACORE®-3000 instrument (BIAcore, Inc., Piscataway, New Jersey) at 25°C with immobilized antigen CM5 chips at ⁇ 10 response units (RU).
  • CM5 carboxymethylated dextran biosensor chips
  • EDC N-ethyl-N'-(3-dimethylamino- propyl)-carbodiimide hydrochloride
  • NHS N-hydroxysuccinimide
  • Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 micrograms/ml ( ⁇ 0.2 micromolar) before injection at a flow rate of 5 microliters/minute to achieve approximately 10 RU of coupled protein. Following the injection of antigen, 1 M ethanolamine is injected to block unreacted groups. For kinetics measurements, two-fold serial dilutions of Fab (0.78 nM to 500 nM) are injected in PBS with 0.05% TWEEN 20TM surfactant (PBST) at 25°C at a flow rate of approximately 25 microliters /min.
  • PBST TWEEN 20TM surfactant
  • association rates (k on ) and dissociation rates (k off ) are calculated using a simple one-to-one Langmuir binding model (BIAcore® Evaluation Software version 3.2) by simultaneously fitting the association and dissociation sensorgrams.
  • the equilibrium dissociation constant (Kd) is calculated as the ratio k off /k on .
  • Kd equilibrium dissociation constant
  • the initial measurement step determines the baseline, followed by loading the His-tagged antigen at a concentration of 25nM onto Ni-NTA biosensors for 10 minutes in IX KB+ buffer (0.01% BSA, 0.002% Tween-20 in PBS, pH7.4), followed by another baseline measurement step (IX KB+ buffer only for 2 minutes).
  • the sensor is then dipped into a well containing antibody (the association step) for 10 minutes, followed by a 20-minute wash in IX KB+ buffer to measure dissociation.
  • the equilibrium dissociation constant (Kd) is calculated as the ratio of k off /k on , with the Octet software determining the k off and k on rates.
  • the number and location of disulfide bonds in protein expression products can be determined by digestion of the protein with a protease, such as trypsin, under non- reducing conditions, and subjecting the resulting peptide fragments to mass spectrometry (MS) combining sequential electron transfer dissociation (ETD) and collision-induced dissociation (CID) MS steps (MS2, MS3) (Nili et al., "Defining the disulfide bonds of insulin-like growth factor-binding protein-5 by tandem mass spectrometry with electron transfer dissociation and collision-induced dissociation", J Biol Chem 2012 Jan 6; 287(2): 1510-1519; Epub 2011 Nov 22).
  • a protease such as trypsin
  • any free cysteine residues are first blocked by alkylation: the expressed protein is incubated protected from light with the alkylating agent iodoacetamide (5 mM) with shaking for 30 minutes at 20°C in buffer with 4 M urea.
  • the alkylating agent iodoacetamide iodoacetamide
  • NEM is used as the alkylating reagent, with trypsin proteolysis in combination with reduction/alkylation conducted under denaturing conditions (6M GuaHCl).
  • the expressed protein is separated by non- reducing SDS-PAGE using precast gels.
  • the expressed protein is incubated in the gel after electrophoresis with iodoacetamide or NEM, or without as a control. Protein bands are stained, de-stained with double-deionized water, excised, and incubated twice in 500 microliters of 50 mM ammonium bicarbonate, 50% (v/v) acetonitrile while shaking for 30 minutes at 20°C. Protein samples are dehydrated in 100% acetonitrile for 2 minutes, dried by vacuum centrifugation, and rehydrated with 10 mg/ml of trypsin or chymotrypsin in buffer containing 50 mM ammonium bicarbonate and 5 mM calcium chloride for 15 minutes on ice.
  • Excess buffer is removed and replaced with 50 microliters of the same buffer without enzyme, followed by incubation for 16 hours at 37°C or 20°C, for trypsin and chymotrypsin, respectively, with shaking. Digestions are stopped by addition of 3 microliters of 88% formic acid, and after brief vortexing, the supernatant is removed and stored at -20°C until analysis.
  • Alternative protein fragmentation methods such as the use of endoproteinase Lys-C, glutamyl endopeptidase ('Glu-C'), or cyanogen bromide (CNBr) are used if trypsinolysis provides insufficient sequence coverage ( ⁇ 75%).
  • TCEP tris(2-carboxyethyl)phosphine
  • Peptides are injected onto a 1 mm x 8 mm trap column (Michrom Bio esources, Inc., Auburn, CA) at 20 microliters/minute in a mobile phase containing 0.1% formic acid.
  • the trap cartridge is then placed in-line with a 0.5 mm x 250 mm column containing 5 mm Zorbax SB-C18 stationary phase (Agilent Technologies, Santa Clara, CA), and peptides separated by a 2-30% acetonitrile gradient over 90 minutes at 10 microliters/minute with a 1100 series capillary HPLC (Agilent Technologies); alternatively, a CI 8 column suitable for UPLC is used.
  • Peptides are analyzed using a LTQ Velos linear ion trap with an ETD source (Thermo Fisher Scientific Inc., Waltham, Massachusetts). Electrospray ionization is performed using a Captive Spray source (Michrom Bioresources, Inc.), or preferably, an uncoated, pulled fused silica emitter (New Objective Inc., Woburn, Massachuetts) at 3.0 kV. Alternatively, analysis of medium-sized proteolytic fragments is performed using a Thermo LTQ-FT MS (7 Tesla) instrument, or a Synapt G2-Si quadrupole traveling wave ion mobility time-of-flight (ToF) mass spectrometer (Waters Corp., Milford, Massachusetts).
  • ETD Electrospray ionization is performed using a Captive Spray source (Michrom Bioresources, Inc.), or preferably, an uncoated, pulled fused silica emitter (New Objective Inc., Woburn, Massachuetts)
  • peptides are analyzed using an Orbitrap FusionTM TribridTM mass spectrometer (Thermo Fisher Scientific).
  • Disulfide-linked peptides have charge states of +4 or greater following trypsinization due to the presence of two N-termini and two basic residues (arginine or lysine) at the carboxy termini.
  • These disulfide-linked peptides are preferentially isolated by the Orbitrap FusionTM instrument so that the disulfide bonds can be broken using ETD fragmentation.
  • Survey MS scans are followed by seven data-dependant scans consisting of CID and ETD MS2 scans on the most intense ion in the survey scan, followed by five MS3 CID scans on the first- to fifth-most intense ions in the ETD MS2 scan.
  • CID scans use normalized collision energy of 35
  • ETD scans use a 100 ms activation time with supplemental activation enabled.
  • Minimum signals to initiate MS2 CID and ETD scans are 10,000, minimum signals for initiation of MS3 CID scans are 1000, and isolation widths for all MS2 and MS3 scans are 3.0 m/z.
  • the dynamic exclusion feature of the software is enabled with a repeat count of 1 , exclusion list size of 100, and exclusion duration of 30 seconds.
  • Inclusion lists to target specific cross-linked species for collection of ETD MS2 scans are used. Separate data files for MS2 and MS3 scans are created by Bioworks 3.3 (Thermo Fisher Scientific) using ZSA charge state analysis. Matching of MS2 and MS3 scans to peptide sequences is performed by Sequest (V27, Rev 12, Thermo Fisher Scientific). The analysis is performed without enzyme specificity, a parent ion mass tolerance of 2.5, fragment mass tolerance of 1.0, and a variable mass of +16 for oxidized methionine residues. Results are then analyzed using the program Scaffold (V3 00 08, Proteome Software, Portland, OR) with minimum peptide and protein probabilities of 95 and 99% being used.
  • Scaffold V3 00 08, Proteome Software, Portland, OR
  • Software tools for data interpretation also include Proteome DiscovererTM 2.0 with the Disulfinator node (Thermo Fisher Scientific). Peptides from MS3 results are sorted by scan number, and cysteine containing peptides are identified from groups of MS3 scans produced from the five most intense ions observed in ETD MS2 scans. The identities of cysteine peptides participating in disulfide-linked species are further confirmed by manual examination of the parent ion masses observed in the survey scan and the ETD MS2 scan.
  • the expression system of the invention can be used to express gene products that accumulate in different compartments of the cell, such as the cytoplasm or periplasm.
  • Host cells such as E. coli or S. cerevisiae have an outer cell membrane or cell wall, and can form spheroplasts when the outer membrane or wall is removed.
  • Expressed proteins made in such hosts can be purified specifically from the periplasm, or from spheroplasts, or from whole cells, using the following method (Schoenfeld, "Convenient, rapid enrichment of periplasmic and spheroplasmic protein fractions using the new PeriPrepsTM Periplasting Kit", Epicentre Forum 1998 5(1): 5; see www.epibio.com/newsletter/f5_l/f5_lpp.asp).
  • This method using the PeriPrepsTM Periplasting Kit (Epicentre® Biotechnologies, Madison WI; protocol available at www.epibio.com/pdftechlit/107pl0612.pdf), is designed for E. coli and other gram negative bacteria, but the general approach can be modified for other host cells such as S. cerevisiae.
  • the bacterial host cell culture is grown to late log phase only, as older cell cultures in stationary phase commonly demonstrate some resistance to lysozyme treatment. If the expression of recombinant protein is excessive, cells may prematurely lyse; therefore, cell cultures are not grown in rich medium or at higher growth temperatures that might induce excessive protein synthesis. Protein expression is then induced; the cells should be in log phase or early stationary phase.
  • the cell culture is pelleted by centrifugation at a minimum of 1,000 x g for 10 minutes at room temperature. Note: the cells must be fresh, not frozen. The wet weight of the cell pellet is determined in order to calculate the amount of reagents required for this protocol.
  • the cells are thoroughly resuspended in a minimum of 2 ml of PeriPreps Periplasting Buffer (200 mM Tris-HCl pH 7.5, 20% sucrose, 1 mM EDTA, and 30 U/microliter eady-Lyse Lysozyme) for each gram of cells, either by vortex mixing or by pipeting until the cell suspension is homogeneous. Note: excessive agitation may cause premature lysing of the spheroplasts resulting in contamination of the periplasmic fraction with cytoplasmic proteins.
  • PeriPreps Periplasting Buffer 200 mM Tris-HCl pH 7.5, 20% sucrose, 1 mM EDTA, and 30 U/microliter eady-Lyse Lysozyme
  • Step 2 Add 3 ml of purified water at 4°C for each gram of original cell pellet weight (Step 2) and mix by inversion.
  • the lysed cells are pelleted by centrifugation at a minimum of 4,000 x g for 15 minutes at room temperature.
  • OmniCleave Endonuclease is optionally added to PeriPreps Lysis Buffer. Inclusion of a nuclease will generally improve the yield of protein and the ease of handling of the lysates, but addition of a nuclease is undesirable in some cases: for example, the use of a nuclease should be avoided if residual nuclease activity or transient exposure to the magnesium cofactor will interfere with subsequent assays or uses of the purified protein.
  • EDTA to the lysate to inactivate OmniCleave Endonuclease, likewise, may interfere with subsequent assay or use of the purified protein.
  • 2 microliters of OmniCleave Endonuclease and 10 microliters of 1.0 M MgCl 2 are diluted up to 1 ml with PeriPreps Lysis Buffer (10 mM Tris-HCl pH 7.5, 50 mM KC1, 1 mM EDTA, and 0.1% deoxycholate) for each milliliter of Lysis Buffer needed in Step 10.
  • the pellet is resuspended in 5 ml of PeriPreps Lysis Buffer for each gram of original cell pellet weight.
  • the pellet is incubated at room temperature for 10 minutes (if included, OmniCleave Endonuclease activity will cause a significant decrease in viscosity; the incubation is continued until the cellular suspension has the consistency of water).
  • the cellular debris is pelleted by centrifugation at a minimum of 4,000 x g for 15 minutes at 4°C.
  • the supernatant containing the spheroplast fraction is transferred to a clean tube.
  • lysates may contain substantial amounts of mono- or oligonucleotides. The presence of these degradation products may affect further processing of the lysate: for example, nucleotides may decrease the binding capacity of anion exchange resins by interacting with the resin.
  • the above protocol can be used to prepare total cellular protein with the following modifications.
  • the cells pelleted in Step 2 can be fresh or frozen; at Step 4, the cells are incubated for 15 minutes; Steps 5 through 8 are omitted; at Step 10, 3 ml of PeriPreps Lysis Buffer is added for each gram of original cell pellet weight.
  • the samples can be analyzed by any of a number of protein characterization and/or quantification methods.
  • the successful fractionation of periplasmic and spheroplastic proteins is confirmed by analyzing an aliquot of both the periplasmic and spheroplastic fractions by SDS-PAGE (two microliters of each fraction is generally sufficient for visualization by staining with Coomassie Brilliant Blue). The presence of unique proteins or the enrichment of specific proteins in a given fraction indicates successful fractionation.
  • the host cell contains a high-copy number plasmid with the ampicillin resistance marker, then the presence of ⁇ -lactamase (31.5 kDa) mainly in the periplasmic fraction indicates successful fractionation.
  • ⁇ -lactamase 31.5 kDa
  • Other E. coli proteins found in the periplasmic space include alkaline phosphatase (50 kDa) and elongation factor Tu (43 kDa).
  • the amount of protein found in a given fraction can be quantified using any of a number of methods (such as SDS-PAGE and densitometry analysis of stained or labeled protein bands, scintillation counting of radiolabeled proteins, enzyme-linked immunosorbent assay (ELISA), or scintillation proximity assay, among other methods.) Comparing the amounts of a protein found in the periplasmic fraction as compared to the spheroplastic fraction indicates the degree to which the protein has been exported from the cytoplasm into the periplasm.
  • methods such as SDS-PAGE and densitometry analysis of stained or labeled protein bands, scintillation counting of radiolabeled proteins, enzyme-linked immunosorbent assay (ELISA), or scintillation proximity assay, among other methods.
  • the strength of a promoter is measured as the amount of transcription of a gene product initiated at that promoter, relative to a suitable control.
  • a suitable control could use the same expression construct, except that the 'wild- type' version of the promoter, or a promoter from a 'housekeeping' gene, is used in place of the promoter to be tested.
  • expression of the gene product from the promoter can be compared under inducing and non- inducing conditions.
  • RNA Two micrograms of RNA is used to synthesize cDNA using a random primer and RevertAid H Minus M-MulV reverse transcriptase (Fermentas, Glen Burnie, Maryland).
  • the strength of the promoter is determined by RT-qPCR carried out in an iCycler IQ ® (Bio-Rad, Eke, Belgium) using forward and reverse primers designed to amplify the cDNA corresponding to the transcript produced from the promoter. (For this purpose, the De Mey et al.
  • coli strains containing expression constructs comprising at least one inducible promoter controlling expression of a fluorescent reporter gene are grown at 37°C under antibiotic selection to an optical density at 600 nm (OD600) of 0.6 to 0.8.
  • OD600 optical density at 600 nm
  • Cells are collected by centrifugation (15,000 x g), washed in C medium without a carbon source, resuspended in fresh C medium containing antibiotics, glycerol, and/or inducer (for the induction of gene expression) to an OD600 of 0.1 to 0.2, and incubated for 6 h. Samples are taken routinely during the growth period for analysis.
  • Culture fluorescence is measured on a Versafluor Fluorometer (Bio- ad Inc., Hercules, California) with 360/40-nm-wavelength excitation and 520/10-nm- wavelength emission filters.
  • the strength of expression from an inducible promoter upon induction can be expressed as the ratio of the maximum population-averaged fluorescence (fluorescence/OD ratio) of the induced cells relative to that of control (such as uninduced) cells.
  • flow cytometry is performed on a Beckman-Coulter EPICS XL flow cytometer (Beckman Instruments Inc., Palo Alto, California) equipped with an argon laser (emission at a wavelength of 488 nm and 15 mW) and a 525-nm-wavelength band pass filter.
  • sampled cells Prior to the analysis, sampled cells are washed with phosphate- buffered saline that had been filtered (filter pore size, 0.22 micrometers), diluted to an OD600 of 0.05, and placed on ice. For each sample, 30,000 events are collected at a rate between 500 and 1,000 events/s. The percentage of induced (fluorescent) cells in each sample can be calculated from the flow cytometry data.
  • Percent polynucleotide sequence or amino acid sequence identity is defined as the number of aligned symbols, i.e. nucleotides or amino acids, that are identical in both aligned sequences, divided by the total number of symbols in the alignment of the two sequences, including gaps.
  • the degree of similarity (percent identity) between two sequences may be determined by aligning the sequences using the global alignment method of Needleman and Wunsch (J. Mol. Biol. 48:443, 1970), as implemented by the National Center for Biotechnology Information (NCBI) in the Needleman- Wunsch Global Sequence Alignment Tool, available through the website blast.ncbi.nlm.nih.gov/Blast.cgi.
  • the Needleman and Wunsch alignment parameters are set to the default values (Match/Mismatch Scores of 2 and -3, respectively, and Gap Costs for Existence and Extension of 5 and 2, respectively).
  • Other programs used by those skilled in the art of sequence comparison may also be used to align sequences, such as, for example, the basic local alignment search tool or BLAST® program (Altschul et al., "Basic local alignment search tool", J Mol Biol 1990 Oct 5; 215(3): 403-410), as implemented by NCBI at the blast.ncbi.nlm.nih.gov/Blast.cgi website, using the default parameter settings described.
  • the BLAST algorithm has multiple optional parameters including two that may be used as follows: (A) inclusion of a filter to mask segments of the query sequence that have low compositional complexity or segments consisting of short-periodicity internal repeats, which is preferably not utilized or set to Off, and (B) a statistical significance threshold for reporting matches against database sequences, called the 'Expect' or E-score (the expected probability of matches being found merely by chance; if the statistical significance ascribed to a match is greater than this E-score threshold, the match will not be reported).
  • E-score the statistical significance threshold for reporting matches against database sequences
  • preferred threshold values are 0.5, or in order of increasing preference, 0.25, 0.1, 0.05, 0.01, 0.001 , 0.0001, 0.00001 , and 0.000001.
  • nucleic acids for example, by in vitro amplification, purification from cells, or chemical synthesis
  • methods for manipulating nucleic acids for example, by site- directed mutagenesis, restriction enzyme digestion, ligation, etc.
  • various vectors, cell lines, and the like useful in manipulating and making nucleic acids are described in the above references.
  • polynucleotide including labeled or biotinylated polynucleotides

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Abstract

La présente invention concerne des cellules hôtes ayant un cytoplasme oxydant, qui sont capables de croître jusqu'à des densités cellulaires élevées et de produire des protéines recombinées sous forme soluble. L'invention concerne également des procédés de culture desdites cellules hôtes jusqu'à des densités élevées, des procédés pour éventuellement moduler les vitesses de croissance desdites cellules hôtes, et des procédés induisant les cellules hôtes à produire le produit génique recherché sous forme soluble. D'autres aspects de l'invention concernent des procédés de préparation et de stockage de cellules hôtes, qui utilisent les avantages de la production de produits géniques dans le cytoplasme d'une cellule hôte pour fournir des méthodes de stockage et/ou de transport à long terme de produits géniques exprimés qui sont retenus sous une forme soluble et stable dans les cellules hôtes.
PCT/US2016/067064 2012-08-05 2016-12-15 Système d'expression cytoplasmique WO2017106583A1 (fr)

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US16/871,736 US20200270338A1 (en) 2012-08-05 2020-05-11 Expression constructs, host cells, and methods for producing insulin

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020069011A1 (fr) * 2018-09-25 2020-04-02 Absci, Llc Procédés de purification de protéines
CN113260626A (zh) * 2018-11-05 2021-08-13 豪夫迈·罗氏有限公司 在原核宿主细胞中产生双链蛋白质的方法
WO2023114905A1 (fr) 2021-12-16 2023-06-22 Absci Corporation Protéines de fusion associées à une membrane pour améliorer la compétence de cellules
WO2023114452A1 (fr) 2021-12-17 2023-06-22 Absci Corporation Criblage en phase solide pour des souches bactériennes haute performance
WO2023122567A1 (fr) 2021-12-21 2023-06-29 Absci Corporation Analyse de sélection de fola pour identifier des souches ayant une expression de protéine cible soluble accrue
WO2023122448A1 (fr) 2021-12-23 2023-06-29 Absci Corporation Produits et procédés d'expression hétérologue de protéines dans une cellule hôte
WO2023129881A1 (fr) 2021-12-30 2023-07-06 Absci Corporation Inactivation du gène ptsp augmentant l'expression du gène actif
WO2023133462A1 (fr) 2022-01-07 2023-07-13 Absci Corporation Conception de variants de séquence de biomolécule avec des attributs préalablement spécifiés
WO2024006269A1 (fr) 2022-06-29 2024-01-04 Absci Corporation Procédé de criblage par affinité
WO2024040020A1 (fr) 2022-08-15 2024-02-22 Absci Corporation Enrichissement de cellule spécifique à une activité d'affinité quantitative

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030104981A1 (en) * 1995-11-03 2003-06-05 Jelena Mandic Human insulin analogues
US20150353940A1 (en) * 2013-08-05 2015-12-10 Absci, Llc Vectors for use in an inducible coexpression system

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20030104981A1 (en) * 1995-11-03 2003-06-05 Jelena Mandic Human insulin analogues
US20150353940A1 (en) * 2013-08-05 2015-12-10 Absci, Llc Vectors for use in an inducible coexpression system

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
LOBSTEIN, J ET AL.: "SHuffle, a novel Escherichia coli protein expression strain capable of correctly folding disulfide bonded proteins in its cytoplasm", MICROBIAL CELL FACTORIES, vol. 11, no. 56, 2012, pages 1 - 16, XP021129014, DOI: doi:10.1186/1475-2859-11-56 *

Cited By (13)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2020069011A1 (fr) * 2018-09-25 2020-04-02 Absci, Llc Procédés de purification de protéines
CN113286810A (zh) * 2018-09-25 2021-08-20 Absci有限责任公司 蛋白质纯化方法
JP2022502039A (ja) * 2018-09-25 2022-01-11 エイビーエスシーアイ・エルエルシー タンパク質精製方法
US11584785B2 (en) 2018-09-25 2023-02-21 Absci, Llc C-peptides and proinsulin polypeptides comprising the same
CN113260626A (zh) * 2018-11-05 2021-08-13 豪夫迈·罗氏有限公司 在原核宿主细胞中产生双链蛋白质的方法
WO2023114905A1 (fr) 2021-12-16 2023-06-22 Absci Corporation Protéines de fusion associées à une membrane pour améliorer la compétence de cellules
WO2023114452A1 (fr) 2021-12-17 2023-06-22 Absci Corporation Criblage en phase solide pour des souches bactériennes haute performance
WO2023122567A1 (fr) 2021-12-21 2023-06-29 Absci Corporation Analyse de sélection de fola pour identifier des souches ayant une expression de protéine cible soluble accrue
WO2023122448A1 (fr) 2021-12-23 2023-06-29 Absci Corporation Produits et procédés d'expression hétérologue de protéines dans une cellule hôte
WO2023129881A1 (fr) 2021-12-30 2023-07-06 Absci Corporation Inactivation du gène ptsp augmentant l'expression du gène actif
WO2023133462A1 (fr) 2022-01-07 2023-07-13 Absci Corporation Conception de variants de séquence de biomolécule avec des attributs préalablement spécifiés
WO2024006269A1 (fr) 2022-06-29 2024-01-04 Absci Corporation Procédé de criblage par affinité
WO2024040020A1 (fr) 2022-08-15 2024-02-22 Absci Corporation Enrichissement de cellule spécifique à une activité d'affinité quantitative

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