WO2015020690A1 - Co-expression coordonnée de la thrombine - Google Patents

Co-expression coordonnée de la thrombine Download PDF

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Publication number
WO2015020690A1
WO2015020690A1 PCT/US2014/014968 US2014014968W WO2015020690A1 WO 2015020690 A1 WO2015020690 A1 WO 2015020690A1 US 2014014968 W US2014014968 W US 2014014968W WO 2015020690 A1 WO2015020690 A1 WO 2015020690A1
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thrombin
host cell
gene
expression
amino acid
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PCT/US2014/014968
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English (en)
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Andras Gruber
Sean Mcclain
Mark VALASEK
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Aronora, Inc.
Absci, Llc
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Priority claimed from PCT/US2013/053562 external-priority patent/WO2014025663A1/fr
Application filed by Aronora, Inc., Absci, Llc filed Critical Aronora, Inc.
Priority to US14/909,707 priority Critical patent/US10508270B2/en
Priority to EP14834543.2A priority patent/EP3030651A4/fr
Publication of WO2015020690A1 publication Critical patent/WO2015020690A1/fr
Priority to US14/740,475 priority patent/US20150353940A1/en
Priority to US15/261,984 priority patent/US11371048B2/en
Priority to US16/675,102 priority patent/US20200063116A1/en
Priority to US17/748,734 priority patent/US20220364104A1/en
Priority to US18/310,317 priority patent/US20230340501A1/en

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    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6424—Serine endopeptidases (3.4.21)
    • C12N9/6429—Thrombin (3.4.21.5)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
    • C12Y304/21—Serine endopeptidases (3.4.21)
    • C12Y304/21005—Thrombin (3.4.21.5)

Definitions

  • AbSci, LLC and Aronora, Inc. are parties to a joint research agreement.
  • 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, and specifically the technical field of expression of thrombin.
  • Thrombin is a serine protease that plays opposing functional roles in blood coagulation: it is both a procoagulant that converts fibrinogen into an insoluble fibrin clot, and an anticoagulant that activates protein C, and thrombin also has an extracellular protease-activated receptor (PAR) activation function (Di Cera, "Thrombin", Mol Aspects Med 2008 Aug; 29(4): 203-254, doi: 10.1016/ j.mam.2008.01.001, Epub 2008 Feb 1). Wild-type thrombin expressed in mammalian cells is often used for its procoagulant properties, particularly to address the problem of surgical bleeding.
  • PAR protease-activated receptor
  • Thrombin variants engineered for optimal activity toward protein C and minimal activity toward fibrinogen have shown remarkable anticoagulant and antithrombotic properties of therapeutic interest, both in vitro and in vivo (Cantwell and Di Cera, "Rational design of a potent anticoagulant thrombin", J Biol Chem 2000 Dec 22; 275(51): 39827-39830; Epub 2000 Nov 1 ; Gruber et al, "The thrombin mutant W215A/E217A shows safe and potent anticoagulant and antithrombotic effects in vivo", J Biol Chem 2002 Aug 2; 277(31): 27581-27584; Epub 2002 Jun 17; US Patent 6706512; US Patent 7223583).
  • antithrombotic effect of anticoagulant thrombin variants in non-human primates is more efficacious than the direct administration of activated protein C, and is safer to use than the administration of low molecular weight heparins (Gruber et al., "Relative antithrombotic and antihemostatic effects of protein C activator versus low-molecular-weight heparin in primates", Blood 2007 May 1 ; 109(9): 3733-3740; Epub 2007 Jan 16).
  • thrombin in vivo proceeds through a series of intermediate forms, including prothrombin, prethrombin-1, and prethrombin-2.
  • Each step of this process involves a highly regulated cleavage of the precursor form of thrombin, until mature thrombin is produced (DiBella et al., "Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin", J Biol Chem 1995 Jan 6; 270(1): 163-169).
  • Mature thrombin is formed of two polypeptide chains, the A chain and the B chain, with a disulfide bond between the A and the B chain, and can be produced by cleavage of prethrombin-2 at a specific site.
  • Thrombin including anticoagulant thrombin variants, has been expressed in cultured mammalian host cells such as BHK21 cells (Guinto et al, "Unexpected crucial role of residue 225 in serine proteases", Proc Natl Acad Sci U S A 1999 Mar 2; 96(5): 1852-1857; Cantwell and Di Cera 2000, supra), and in E. coli (US Patent Application Publication 2012/0164129 Al).
  • Thrombin prepared in this way is typically expressed as prethrombin-2, which needs to be converted to mature thrombin (or "activated") by treatment with an enzyme, for example by treatment with prothrombinase complex which contains activated factor X, or immobilized ecarin, an enzyme purified from snake venom, or other activators.
  • the present invention provides methods for producing thrombin using coordinated coexpression systems, and in particular those that use inducible coexpression systems capable of controlled induction of each gene product required for the production of thrombin, and the thrombin produced by these methods.
  • One embodiment of the invention is a host cell comprising at least one expression construct comprising a polynucleotide sequence encoding a polypeptide selected from the group consisting of (a) a polypeptide comprising the amino acid sequence of the thrombin A chain, wherein the host cell further comprises a polynucleotide sequence encoding the amino acid sequence of the thrombin B chain, and said polynucleotide sequence encoding the amino acid sequence of the thrombin A chain is not contiguous with said polynucleotide sequence encoding the amino acid sequence of the thrombin B chain; (b) a polypeptide comprising the amino acid sequence of the thrombin B chain, wherein the host cell further comprises a polynucleotide sequence encoding the amino acid sequence of the thrombin A chain, and said polynucleotide sequence encoding the amino acid sequence of the thrombin A chain is not contiguous with said polynucleotide sequence en
  • At least one expression construct of the host cell comprises an inducible promoter.
  • Another embodiment of the invention is a host cell comprising two or more types of expression constructs, wherein at least one type of expression construct comprises an inducible promoter and a polynucleotide sequence encoding a polypeptide to be transcribed from the inducible promoter; wherein said polypeptide is selected from the group consisting of: (a) preprothrombin; (b) prothrombin; (c) prethrombin-1 ; (d) prethrombin-2; and (e) fragments of any of (a) - (c) that comprise the amino acid sequences of the thrombin A chain and the thrombin B chain.
  • the expression construct of each type comprises an inducible promoter that is different from the inducible promoter of the expression construct of each other type, or an origin of replication that is different from the origin of replication of the expression construct of each other type.
  • the polypeptide encoded by the expression construct is a polypeptide comprising the amino acid sequence of the thrombin A chain and comprises an amino acid sequence selected from the group consisting of: SEQ ID NO:3 and SEQ ID NO:8, or a polypeptide comprising the amino acid sequence of the thrombin B chain and comprises an amino acid sequence selected from the group consisting of SEQ ID NO:4 and SEQ ID NO: 6.
  • the polypeptide comprises a thrombin amino acid sequence variation selected from the group consisting of: E14eA/D141A/E18A; G14mP; R93A/W215A/E217A; T172Y/W215A/E217A; SI 95 A; W215A; W215A/E217A; W215E; W215G; a deletion of residues E146-K149e; W215G/a deletion of residues E146-K149e; W215L; W215V; E217A; and E229K.
  • a thrombin amino acid sequence variation selected from the group consisting of: E14eA/D141A/E18A; G14mP; R93A/W215A/E217A; T172Y/W215A/E217A; SI 95 A; W215A; W215A/E217A; W215E; W215G; a deletion of residues E146-K
  • the polypeptide comprises the thrombin amino acid sequence variation W215A/E217A, which in certain instances is selected from the group consisting of: SEQ ID NO: 6 and SEQ ID NO: 10.
  • the invention also provides host cells wherein at least one expression construct comprises a polynucleotide sequence encoding a metalloprotease, which in some embodiments is an ecarin polypeptide, and further instances comprises the amino acid sequence of SEQ ID NO: 12.
  • the invention provides host cells with expression constructs wherein at least one inducible promoter is an L-arabinose-inducible promoter, or is a propionate-inducible promoter, or is selected from the group consisting of: the arciBAD promoter, the prpBCDE promoter, the rhaSR promoter, and the xlyA promoter.
  • each inducible promoter is not a lactose-inducible promoter.
  • At least one expression construct further comprises a polynucleotide sequence encoding a transcriptional regulator that binds to an inducible promoter; in certain instances the transcriptional regulator is selected from the group consisting of: AraC, PrpR, RhaR, and XylR.
  • the polynucleotide sequence encoding the transcriptional regulator and the inducible promoter to which said transcriptional regulator binds are in the same expression construct.
  • telomere sequence selected from the group consisting of: pBAD18, pBAD18-Cm, pBAD18-Kan, pBAD24, pBAD28, pBAD30, pBAD33, pPR018, pPR018-Cm, pPR018-Kan, pPR024, pPRO30, pPR033, and pPR043.
  • Host cells comprising two types of expression constructs are also provided, and in some embodiments, one type of expression construct is produced by a method comprising a step of inserting a polynucleotide sequence into a pBAD24 plasmid, and the other type of expression construct is produced by a method comprising a step of inserting a polynucleotide sequence into a plasmid selected from the group consisting of pPR033 and pPR043.
  • a further aspect of the invention are host cells having an alteration of gene function of at least one gene encoding a transporter protein for an inducer of at least one said inducible promoter, where the gene encoding a transporter protein in some instances is selected from the group consisting of arciE, arciF, arciG, arciH, rhaT, xylF, xylG, and xylH.
  • 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 said inducible promoter and in certain embodiments is selected from the group consisting of arciA, arciB, arciD, prpB, prpD, rhaA, rhaB, rhaD, xylA, and xylB.
  • Other instances of the invention are host cells having a reduced level of gene function of at least one gene encoding a protein involved in biosynthesis of an inducer of at least one said inducible promoter, wherein the gene encoding a protein involved in biosynthesis of an inducer of at least one said inducible promoter is, in particular embodiments, selected from the group consisting of scpA/sbm, argK/ygfl), scpB/ygfG, scpC/ygfH, rmlA, rmlB, rmlC, and rmlD.
  • Host cells are also provided which have an altered gene function of a gene that affects the reduction/oxidation environment of the host cell cytoplasm and which in certain instances is selected from the group consisting of gor and gshB.
  • Additional embodiments of the invention are host cells having a reduced level of gene function of a gene that encodes a reductase, such as trxB.
  • the invention provides host cells comprising at least one expression construct encoding at least one disulfide bond isomerase protein, which in some aspects is DsbC, and which in further embodiments is a form of DsbC lacking a signal peptide.
  • Other instances of the invention are host cells comprising at least one polynucleotide encoding Ervlp, and prokaryotic host cells, which in some embodiments are E. coli.
  • the invention also provides an E. coli host cell comprising two types of expression constructs, wherein one type of expression construct is produced by a method comprising a step of inserting a polynucleotide sequence into a pBAD24 plasmid, and the other type of expression construct is produced by a method comprising a step of inserting a polynucleotide sequence into a plasmid selected from the group consisting of pPR033 and pPR043; and further comprising two or more of the following: (a) a deletion of the arciBAD genes; (b) an altered gene function of the arciE gene; (c) an altered gene function of the arciFGH genes; (d) a lacY(AlllC) gene; (e) a reduced gene function of the prpB and prpD genes; (f) a reduced gene function of the sbm/scpA-ygfD/argK-ygfGH/scpBC genes without affecting expression of
  • the host cell has an altered gene function of the arciE gene which is the expression of arciE from a constitutive promoter, and in other aspects, the host cell comprises a reduced gene function of the sbm/scpA-ygfD/argK-ygfGH/scpBC genes without affecting expression of the ygfl gene; a reduced gene function of the gor and trxB genes; and a polynucleotide encoding a form of DsbC lacking a signal peptide, and in certain embodiments, such host cells further comprise at least one expression construct comprising a polynucleotide sequence encoding a gene product to be transcribed from an inducible promoter.
  • Additional aspects of the invention are methods of producing thrombin polypeptides, such methods comprising growing a culture of the host cell of claim 1 , or growing a culture of the host cell of claim 2 or 3 and adding an inducer of at least one inducible promoter to the culture, which in some instances is propionate.
  • the host cell is E. coli.
  • the invention also provides methods further comprising purifying the thrombin polypeptides, which in some aspects comprise a His tag and are purified using a nickel column, and in other embodiments are purified using polyphosphate.
  • Thrombin produced by the methods of the invention is provided, which is some instances is human thrombin, and in certain embodiments has an amino acid sequence comprising the W215A/E217A variation, which can in further instances comprise amino acids 2 through 37 of SEQ ID NO: 3 and amino acids 2 through 260 of SEQ ID NO: 6, or is aglycosylated.
  • thrombin polypeptides produced by the methods of the invention comprise an A chain that shares at least 80% amino acid identity across at least 30 amino acids of SEQ ID NO:3 and a B chain that shares at least 80% amino acid identity across at least 50 amino acids of SEQ ID NO:4 or SEQ ID NO:6; and in particular instances the resulting thrombin molecule has a discernable level of thrombin activity.
  • kits comprising host cells of the invention, and kits comprising thrombin produced by the methods of the invention.
  • Fig. 1 is a schematic illustration of the inducible coexpression system, which includes a host cell (1), with a genome that preferably contains genetic alterations (2), comprising two different inducible expression vectors (3) and (4), which express different gene products upon application of inducers (5), forming a coexpressed product such as thrombin (6).
  • Fig. 2 is a schematic illustration of use of the inducible coexpression system to produce thrombin, in which the E. coli host cell genome (2), among other genetic alterations, encodes a cytoplasmic form of the disulfide isomerase DsbC which lacks a signal peptide; the expression vector pBAD24 (3) provides L-arabinose- inducible expression of the thrombin B chain, and the expression vector pPR033 (4) provides propionate-inducible expression of the thrombin A chain; forming upon induction with L-arabinose and propionate (5) the thrombin product (6).
  • the E. coli host cell genome (2) encodes a cytoplasmic form of the disulfide isomerase DsbC which lacks a signal peptide
  • the expression vector pBAD24 (3) provides L-arabinose- inducible expression of the thrombin B chain
  • the expression vector pPR033 (4) provides propionate-inducible expression of
  • Fig. 3. is a schematic diagram of portions of various expression constructs useful for the coexpression of thrombin polypeptides. Each diagram indicates the location of an inducible promoter (rightward-pointing chevron, black: prpBCDE promoter, gray: arciBAD promoter), and the polypeptide expressed from that inducible promoter.
  • A thrombin A chain
  • B thrombin B chain
  • downward- pointing arrows ecarin cleavage site
  • asterisks location of the W215A and E217A mutations within the B chain.
  • Fig. 4 shows the result of coexpression of thrombin A and WE-B chains in bacterial cells.
  • MW Molecular weight markers; std: human thrombin; Lane 1 : uninduced, 0 % arabinose, 0 mM propionate; Lane 2: 0.00001 % arabinose, 5 mM propionate; Lane 3: 0.0001 % arabinose, 5 mM propionate; Lanes 4: 0.0001 % arabinose, 20 mM propionate; Lane 5: 0.001 % arabinose, 0.5 mM propionate; Lane 6: 0.001 % arabinose, 5 mM propionate; Lane 7: 0.001 % arabinose, 20 mM propionate; Lane 8: 0.01 % arabinose, 5 mM propionate; Lane 9: 0.01 % arabinose, 20 mM propionate; Lane 10: 0.1 % arabinose, 20 mM propionate. Arrow indicates a protein band (thrombin comprising A and WE-B chains) at 33.8 kDa; this band is present in the
  • Fig. 5 is a western blot showing the result of coexpression of thrombin A and WE- B chains, and of thrombin A chains with an added N-terminal ecarin cleavage site (thrombin A+) with thrombin WE-B chains in bacterial cells.
  • Cells containing the pPR033_A and pBAD24_WE-B inducible expression constructs, or the pPR033_A+ and pBAD24_WE-B inducible expression constructs were induced to coexpress thrombin with 0.001% L-arabinose and 20 mM propionate, and were induced either for 5 hours at 30 degrees C, or for 24 hours at 16 degrees C.
  • the induced cells were pelleted and lysed, and the culture medium from the induction was also saved for analysis.
  • Soluble proteins from the cell lysate and from the culture medium of the various samples were separated by gel electrophoresis under reducing conditions on a NuPAGE® Novex® 10% Bis-Tris gel, then transferred to a nitrocellulose membrane and visualized using a mouse monoclonal anti-thrombin antibody, and an alkaline phosphate conjugated anti-mouse secondary antibody.
  • the results show the presence of the thrombin WE-B chain (29.7 kDa) in both the cell lysates and the culture medium from the co-induced host cells.
  • MW MagicMarkTM XP protein standards; std: WE-thrombin standard; Lane 1 : A/WE-B thrombin, cell lysate, 5 hours, 30 degrees C; Lane 2: A/WE-B thrombin, culture medium, 5 hours, 30 degrees C; Lane 3: A+/WE-B thrombin, cell lysate, 5 hours, 30 degrees C; Lane 4: A+/WE-B thrombin, culture medium, 5 hours, 30 degrees C; Lane 5: A/WE-B thrombin, cell lysate, 24 hours, 16 degrees C; Lane 6: A/WE-B thrombin, culture medium, 24 hours, 16 degrees C; Lane 7: A+/WE-B thrombin, cell lysate, 24 hours, 16 degrees C; Lane 8: A+/WE-B thrombin, culture medium, 24 hours, 16 degrees C.
  • thrombin which does not require expensive or time- consuming additional procedures, such as expression of a thrombin precursor and activation of that precursor to produce thrombin, is addressed by development of the coordinated coexpression systems of the invention.
  • the embodiments of the invention involve coexpressing thrombin polypeptides, preferably the thrombin A chain and B chain as separate polypeptides, or thrombin precursor forms in conjunction with proteins such as chaperones or proteases, that are useful for the expression of active forms of thrombin.
  • the coordinated coexpression of polypeptides is the coexpression of two or more polypeptides in a way that allows for control of the relative levels of the polypeptides that are produced, and preferably for control of the relative levels of the polypeptides as they are being expressed in the cell.
  • bicistronic vectors can be used, in which the coding sequences for each polypeptide are expressed in tandem from a single promoter, with an internal ribosome entry site (IRES) or ribosome binding site (RBS) between the coding sequences allowing for translation of the downstream gene (Mazor et al, "Isolation of full-length IgG antibodies from combinatorial libraries expressed in Escherichia coli", Methods Mol Biol 2009; 525: 217-239, xiv; doi: 10.1007/978-1-59745-554-1 11).
  • IRS internal ribosome entry site
  • RBS ribosome binding site
  • bicistronic vectors are limited, however, in that both polypeptides are presumed to be expressed at the same level from the common transcript, in a 1 : 1 ratio, whether or not that is actually the most effective ratio for expression of the desired protein product. There is no easy way to 'fine-tune' the relative expression levels of the polypeptides when using a bicistronic vector approach.
  • the cellular concentration of the inducer must reach a threshold level to initiate the production of transport proteins, but once that threshold has been reached, an uncontrolled positive feedback loop can occur, with the result being a high level of inducer in the cell and correspondingly high levels of expression from inducible promoters: the "all-or-none" phenomenon.
  • Increasing the concentration of the inducer in the growth medium increases the proportion of cells in the population that are in high- expression mode.
  • lacYam lactose permease gene
  • the preferred coordinated coexpression methods for coexpressing thrombin polypeptides utilize compatible homogeneously inducible promoter systems that are, in some embodiments, activated by different inducers.
  • the advantages of this homogeneously inducible coexpression system include, without limitation: 1) improved compatibility of components within the inducible coexpression system; 2) improved control of thrombin polypeptide coexpression by independently titratible induction; 3) improved expression of thrombin proteins, which are difficult to express due to the requirement of disulfide bond formation, optionally coexpressed with other polypeptides; and 4) streamlined optimization of thrombin polypeptide coexpression.
  • thrombin polypeptides refers generally to mammalian thrombin polypeptides, with a particular subset of thrombin polypeptides being primate thrombin polypeptides, such as those having the following UniProtKB database accession numbers: F6QU78 ⁇ Callithrix jacchus, white-tufted-ear marmoset); G1RVB3 ⁇ Nomascus leucogenys ⁇ Hylobates leucogenys), northern white-cheeked gibbon); A0N064 (Macaca mulatta, rhesus macaque); G7PQA7 (Macaca fascicularis , crab-eating macaque (cynomolgus monkey)); G7NDG8 ⁇ Macaca mulatta, rhesus macaque); Q5R537 ⁇ Pongo abelii ⁇ Pongo pygmaeus abelii), Sumatran orangutan); G3QVP
  • Mammalian thrombin polypeptides share over 80% amino acid identity, and primate thrombin polypeptides over 90% amino acid identity, with the amino acid sequence of human thrombin.
  • the high degree of amino acid sequence conservation between these polypeptides makes it quite easy to determine the amino acid sequences of mammalian thrombin polypeptides that correspond to various forms and variants of human thrombin polypeptides. Therefore, examples and embodiments of the invention described with reference to the human thrombin polypeptide sequence are intended to be applicable to mammalian thrombin polypeptides generally, unless otherwise indicated.
  • the reference thrombin molecule is the human alpha-thrombin molecule as described by Bode et al.1989 ⁇ infra).
  • Human thrombin has a disulfide bond between the A and B chains, between residues C336 and C482 (amino acid numbering of human preprothrombin, SEQ ID NO: l), and three intrachain disulfide bonds connecting the following residues of the B chain: C391 with C407; C536 with C550; and C564 with C594.
  • thrombin refers to a mammalian thrombin molecule formed by two polypeptide chains, an A chain and a B chain, and includes variants that differ from the reference mammalian thrombin.
  • a chain and B chain refer to polypeptides that form thrombin, and thus include variants that differ from the reference mammalian thrombin A chain and/or B chain.
  • Thrombin molecules preferably have a discernable level of one or more activities such as, but not limited to, prothrombin- cleaving activity, protein C activation, fibrinogen-cleaving, and fibrin-clotting activity, as described further herein.
  • the amino acid sequence of human preprothrombin (from UniProtKB database accession number P00734) has been provided as SEQ ID NO: l .
  • the amino acids that are present in mature thrombin have been numbered according to a few different systems.
  • Table 1 shows part of the amino acid sequence of human preprothrombin (i.e. a portion of SEQ ID NO: l), and the numbering of those amino acids according to (1) their position in the A and B chains of mature thrombin, (2) the numbering system of Bode et a/.1989 ⁇ infra), and (3) human preprothrombin (SEQ ID NO: l).
  • the preprothrombin amino acid sequence represents the full thrombin polypeptide as it is initially expressed; amino acids 1 - 24 of SEQ ID NO: l are the signal peptide, and amino acids 25 - 43 of SEQ ID NO: 1 are a propeptide that is removed to form prothrombin (amino acids 44 - 622 of SEQ ID NO: l). Amino acids 44 - 198 of SEQ ID NO: l are removed when prothrombin is cleaved by thrombin to form prethrombin-1 (amino acids 199 - 622 of SEQ ID NO: l).
  • prethrombin-1 is cleaved by activated factor X (Xa) (or other enzymes) to form prethrombin-2 (amino acids 328 - 622 of SEQ ID NO: 1).
  • prethrombin-2 is cleaved by Xa to form the A chain (also called the light chain) (amino acids 328-363 of SEQ ID NO: l), and the B chain (also called the heavy chain) (amino acids 364-622 of SEQ ID NO: l) of mature thrombin.
  • a chain also called the light chain
  • B chain also called the heavy chain
  • SEQ ID NOs 3 and 4 are provided as SEQ ID NOs 3 and 4, respectively.
  • Thrombin polypeptides produced by the methods of the invention can include variations from the wild-type or reference thrombin amino acid sequence for a given species. These variations can be in the A chain or B chain of thrombin, or in both chains.
  • Example 4 Examples of assays for thrombin activity, including of thrombin- and fibrinogen-cleavage assays, are provided in Example 4.
  • Anticoagulant effects of thrombin variants can also be determined using in vivo assays in experimental animals (Gruber et al, "The thrombin mutant W215A/E217A shows safe and potent anticoagulant and antithrombotic effects in vivo", J Biol Chem 2002 Aug 2; 277(31): 27581-27584; Epub 2002 Jun 17).
  • Examples of thrombin polypeptide variants are described below; the amino acid positions of the variations in the thrombin amino acid sequences are given using the numbering system of Bode et al. (supra), unless otherwise indicated.
  • the double thrombin mutant referred to as W215A/E217A thrombin (or WE- thrombin) is constructed by combining the two single mutations W215A and E217A in the human thrombin molecule [Cantwell, (2000) J. Biol. Chem. 275:39827-39830].
  • W215A and E217A refer to amino acid residue positions in the thrombin amino acid residue sequence using the position numbers as described in Bode et al. (supra), that correspond to amino acids 590 and 592 of SEQ ID NO: l, respectively.
  • the human thrombin referred to as E-WE-thrombin is WE- thrombin that has been produced in E.
  • WE-thrombin has enhanced protein C activating properties, and reduced fibrinogen-cleaving activity, making it a highly anticoagulant thrombin.
  • the R93A/W215A/E217A triple mutant of human thrombin is a thrombin variant in which the heparin-binding R93 residue has been mutated, producing an RWE-thrombin which appears to have a longer half-life than WE- thrombin.
  • the T172Y/W215A/E217A triple mutant of human thrombin is another thrombin variant that can be produced using the methods of the invention.
  • Another type of anticoagulant thrombin is the E229K variant (Leung et al., "Regulation of tissue inflammation by thrombin-activatable carboxypeptidase B (or TAFI)", Adv Exp Med Biol 2008; 632: 61-69).
  • Some thrombin polypeptide variants comprise altered or novel cleavage sites that promote cleavage of prethrombin-2 by thrombin. Some of these variants comprise changes to the FXa cleavage site between the A and B chains (DGR, amino acids 361 through 363 of SEQ ID NO: l), to make it more like the thrombin cleavage site in preprothrombin (VPR or IPR); these variants all include the G14mP substitution (International Patent Application Publication WO2013/064542 Al ; US Patent Application Publication 2006/205041 Al).
  • the E14eA/D141A/E18A triple mutant causes prethrombin-2 to spontaneously convert to thrombin (Pozzi et al, "Crystal structures of prethrombin-2 reveal alternative conformations under identical solution conditions and the mechanism of zymogen activation", Biochemistry 2011 Nov 29; 50(47): 10195-10202; doi: 10.1021/bi2015019; Epub 2011 Nov 8; US Patent Application Publication 2013/0309753 Al).
  • thrombin polypeptide variants have been characterized by Marino et al, ("Engineering thrombin for selective specificity toward protein C and PARI ", J Biol Chem 2010 Jun 18; 285(25): 19145-19152; doi: 10.1074/jbc.Ml 10.119875; Epub 2010 Apr 19), including alanine-substitution mutants at 97 different positions within the thrombin molecule, 19 single- substitution mutants at the W215 position (particularly W215E, W215L, and W215V, and also W215A, W215C, W215D, W215F, etc.), and a double mutant including the W215G substitution mutant and a deletion of residues E146-K149e.
  • thrombin variants include Y225F, Y225I, Y225P (Guinto et al., "Unexpected crucial role of residue 225 in serine proteases", Proc Natl Acad Sci U S A 1999 Mar 2; 96(5): 1852-1857); R221aA, K224A (Dang et al, "Rational engineering of activity and specificity in a serine protease", Nat Biotechnol 1997 Feb; 15(2): 146-149); H57A, H57S, D102N, G193A, S195A, S 195G (US Patent Application Publication 2007/0282095 Al); and M84A (US Patent Application Publication 2013/017591 Al).
  • the thrombin variant S195A is completely inactive as a serine protease, because the active site serine has been changed to an alanine. This thrombin variant is useful for its anticoagulant properties, which are likely a result of competition with wild-type thrombin.
  • Thrombin polypeptides produced by the methods of the invention are not limited to the above examples, but can include any type of thrombin polypeptide variant; preferably, thrombin polypeptides produced by the methods of the invention comprise an A chain that shares at least 80% amino acid identity across at least 30 amino acids of SEQ ID NO: 3 and a B chain that shares at least 80% amino acid identity across at least 50 amino acids of SEQ ID NO:4 or of SEQ ID NO:6.
  • the resulting thrombin molecule has a discemable level of thrombin activity as assayed according to one of the methods described in Examples 2 and 4 below.
  • the methods of the invention are designed to produce thrombin polypeptides that have been correctly folded and/or assembled into functional products, and that have a desired number of disulfide bonds in the desired locations within such thrombin polypeptides (which can be determined by methods such as that of Example 8).
  • 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 desired functional product.
  • a light chain of a human IgG antibody typically has three disufide bonds (two intramolecular bonds and one intermolecular bond), and a heavy chain of a human IgG antibody typically has seven disufide bonds (four intramolecular bonds and three intermolecular bonds).
  • thrombin polypeptides are coexpressed with other gene products, such as chaperones, that are beneficial to the production of the thrombin polypeptides.
  • 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.
  • DnaK/DnaJ/GrpE, GroEL/GroES, and ClpB can function synergistically in assisting protein folding and therefore expression of these chaperones in combinations has been shown to be beneficial for protein expression (Makino et al., "Strain engineering for improved expression of recombinant proteins in bacteria", Microb Cell Fact 2011 May 14; 10: 32).
  • a eukaryotic chaperone protein such as protein disulfide isomerase (PDI) from the same or a related eukaryotic species, is coexpressed or inducibly coexpressed with the desired gene product in certain embodiments of the invention.
  • PDI protein disulfide isomerase
  • Signal peptides Thrombin polypeptides coexpressed by the methods of the invention can contain signal peptides or lack them, depending on whether it is desirable for the thrombin polypeptides to be exported from the host cell cytoplasm into the periplasm, or to be retained in the cytoplasm, respectively.
  • Signal peptides also termed signal sequences, leader sequences, or leader peptides
  • 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 methods such as that provided in Example 9.
  • Glycosylation Thrombin polypeptides coexpressed by the methods of the invention may be glycosylated or unglycosylated.
  • 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 coexpressed polypeptides are aglycosylated, and in a more specific embodiment, the aglycosylated polypeptides are coexpressed in prokaryotic cells such as E. coli.
  • thrombin polypeptides coexpressed by the methods of the invention may be covalently linked to other types of molecules.
  • Molecules that may be covalently linked to coexpressed thrombin polypeptides 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), carboxymethyl- cellulose, 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
  • thrombin polypeptides to be coexpressed by the methods of the invention can be designed to include molecular moieties that aid in the purification and/or detection of the thrombin polypeptides.
  • Many such moieties are known in the art; as one example, a thrombin polypeptide 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.
  • 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.
  • 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.
  • extrachromosomal polynucleotide such as a plasmid or artificial chromosome.
  • the following are descriptions of particular types of polynucleotide sequences that can be used in expression constructs for the coexpression of gene products.
  • Origins of replication must comprise an origin of replication, also called a replicon, in order to be maintained within the host cell as independently replicating polynucleotides. Different replicons that use the same mechanism for replication cannot be maintained together in a single host cell through repeated cell divisions. As a result, plasmids can be categorized into incompatibility groups depending on the origin of replication that they contain, as shown in Table 2.
  • CloDF13 [4] CloDF13 20-40 [4] pCDFDuetTM-l (EMD Millipore
  • RSF1030 [4] RSF1030 (also > 100 [4] pRSFDuetTM-l (EMD Millipore called NTP1) Catalog No. 71341-3)
  • Origins of replication can be selected for use in expression constructs on the basis of incompatibility group, copy number, and/or host range, among other criteria. As described above, if two or more different expression constructs are to be used in the same host cell for the coexpression of multiple gene products, it is best if the different expression constructs contain origins of replication from different incompatibility groups: a pMBl replicon in one expression construct and a pl5A replicon in another, for example. The average number of copies of an expression construct in the cell, relative to the number of host chromosome molecules, is determined by the origin of replication contained in that expression construct. Copy number can range from a few copies per cell to several hundred (Table 2).
  • different expression constructs are used which comprise inducible promoters that are activated by the same inducer, but which have different origins of replication.
  • origins of replication that maintain each different expression construct at a certain approximate copy number in the cell, it is possible to adjust the levels of overall production of a gene product expressed from one expression construct, relative to another gene product expressed from a different expression construct.
  • an expression construct is created which comprises the colEl replicon, the ara promoter, and a coding sequence for subunit A expressed from the ara promoter: 'colEl-P ara -A'.
  • Another expression construct is created comprising the pl5A replicon, the ara promoter, and a coding sequence for subunit B: 'pl5A-P ara -B'. These two expression constructs can be maintained together in the same host cells, and expression of both subunits A and B is induced by the addition of one inducer, arabinose, to the growth medium.
  • a new expression construct for subunit A could be created, having a modified pMBl replicon as is found in the origin of replication of the pUC9 plasmid ('pUC9ori'): pUC9ori-P ara -A.
  • Expressing subunit A from a high-copy-number expression construct such as pUC9ori-P ara -A should increase the amount of subunit A produced relative to expression of subunit B from pl5A-P ara -B.
  • an origin of replication that maintains expression constructs at a lower copy number, such as pSClOl, could reduce the overall level of a gene product expressed from that construct.
  • Selection of an origin of replication can also determine which host cells can maintain an expression construct comprising that replicon. For example, expression constructs comprising the colEl origin of replication have a relatively narrow range of available hosts, species within the Enterobacteriaceae family, while expression constructs comprising the RK2 replicon can be maintained in E.
  • an expression construct comprises the RK2 replicon and some regulator genes from the RK2 plasmid, it can be maintained in host cells as diverse as Sinorhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus , and Rhodobacter sphaeroides (Kues and Stahl, "Replication of plasmids in gram- negative bacteria", Microbiol Rev 1989 Dec; 53(4): 491-516).
  • the 2-micron circle plasmid of Saccharomyces cerevisiae is compatible with plasmids from other yeast strains, such as pSRl (ATCC Deposit Nos. 48233 and 66069; Araki et al, "Molecular and functional organization of yeast plasmid pSRl ", J Mol Biol 1985 Mar 20; 182(2): 191-203) and pKDl (ATCC Deposit No.
  • Selection genes usually comprise a selection gene, also termed a selectable marker, which encodes a protein necessary for the survival or growth of host cells in a selective culture medium. Host cells not containing the expression construct comprising the selection gene will not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics or other toxins, or that complement auxotrophic deficiencies of the host cell.
  • a selection scheme utilizes a drug such as an antibiotic to arrest growth of a host cell. Those cells that contain an expression construct comprising the selectable marker produce a protein conferring drug resistance and survive the selection regimen.
  • antibiotics that are commonly used for the selection of selectable markers (and abbreviations indicating genes that provide antibiotic resistance pheno types) are: ampicillin (Amp R ), chloramphenicol (Cml R or Cm R ), kanamycin (Kan R ), spectinomycin (Spc R ), streptomycin (Str R ), and tetracycline (Tet R ).
  • Many of the representative plasmids in Table 2 comprise selectable markers, such as pBR322 (Amp R , Tet R ); pMOB45 (Cm R , Tet R ); pACYClW (Amp R , Kan R ); and pGBMl (Spc R , Str R ).
  • the native promoter region for a selection gene is usually included, along with the coding sequence for its gene product, as part of a selectable marker portion of an expression construct. Alternatively, the coding sequence for the selection gene can be expressed from a constitutive promoter.
  • the nucleotide sequence of the region between the transcription initiation site and the initiation codon of the coding sequence of the gene product that is to be inducibly expressed corresponds to the 5' untranslated region ('UTR') of the mRNA for the polypeptide gene product.
  • the region of the expression construct that corresponds to the 5' UTR comprises a polynucleotide sequence similar to the consensus ribosome binding site (RBS, also called the Shine-Dalgarno sequence) that is found in the species of the host cell.
  • RBS consensus ribosome binding site
  • the RBS consensus sequence is GGAGG or GGAGGU, and in bacteria such as E.
  • the RBS consensus sequence is AGGAGG or AGGAGGU.
  • the RBS is typically separated from the initiation codon by 5 to 10 intervening nucleotides.
  • the RBS sequence is preferably at least 55% identical to the AGGAGGU consensus sequence, more preferably at least 70% identical, and most preferably at least 85% identical, and is separated from the initiation codon by 5 to 10 intervening nucleotides, more preferably by 6 to 9 intervening nucleotides, and most preferably by 6 or 7 intervening nucleotides.
  • the ability of a given RBS to produce a desirable translation initiation rate can be calculated at the website salis.psu.edu/software/RBSLibraryCalculatorSearchMode, using the RBS Calculator; the same tool can be used to optimize a synthetic RBS for a translation rate across a 100,000+ fold range (Salis, "The ribosome binding site calculator", Methods Enzymol 2011 ; 498: 19-42).
  • a multiple cloning site also called a polylmker, is a polynucleotide that contains multiple restriction sites in close proximity to or overlapping each other.
  • the restriction sites in the MCS typically occur once within the MCS sequence, and preferably do not occur within the rest of the plasmid or other polynucleotide construct, allowing restriction enzymes to cut the plasmid or other polynucleotide construct only within the MCS.
  • MCS sequences are those in the pBAD series of expression vectors, including pBAD18, pBAD18-Cm, pBAD18-Kan, pBAD24, pBAD28, pBAD30, and pBAD33 (Guzman et al, "Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter", J Bacteriol 1995 Jul; 177(14): 4121-4130); or those in the pPRO series of expression vectors derived from the pBAD vectors, such as pPR018, pPR018-Cm, pPR018-Kan, pPR024, pPRO30, and pPR033 (US Patent No.
  • a multiple cloning site can be used in the creation of an expression construct: by placing a multiple cloning site 3' to (or downstream of) a promoter sequence, the MCS can be used to insert the coding sequence for a gene product to be coexpressed into the construct, in the proper location relative to the promoter so that transcription of the coding sequence will occur.
  • restriction enzymes are used to cut within the MCS, there may be some part of the MCS sequence remaining within the expression construct after the coding sequence or other polynucleotide sequence is inserted into the expression construct. Any remaining MCS sequence can be upstream or, or downstream of, or on both sides of the inserted sequence.
  • a ribosome binding site can be placed upstream of the MCS, preferably immediately adjacent to or separated from the MCS by only a few nucleotides, in which case the RBS would be upstream of any coding sequence inserted into the MCS.
  • Another alternative is to include a ribosome binding site within the MCS, in which case the choice of restriction enzymes used to cut within the MCS will determine whether the RBS is retained, and in what relation to, the inserted sequences.
  • a further alternative is to include a RBS within the polynucleotide sequence that is to be inserted into the expression construct at the MCS, preferably in the proper relation to any coding sequences to stimulate initiation of translation from the transcribed messenger RNA.
  • inducible Promoters The following is a description of inducible promoters that can be used in expression constructs for coexpression 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, 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 3 lists the genomic locations, in E. coli MG1655, of the nucleotide sequences for these examples of inducible promoters and related genes.
  • E. coli Escherichia coli
  • MG1655 American Type Culture Collection deposit ATCC 700926
  • Table 3 lists the genomic locations, in E. coli MG1655, of the nucleotide sequences for these examples of inducible promoters and related genes.
  • Nucleotide and other genetic sequences referenced by genomic location as in Table 3, 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.
  • Inducible promoters that can be included in expression constructs as part of the inducible coexpression systems of the invention include those that preferably 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 3, 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 7).
  • 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.
  • 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 ara promoter' are typically used to designate the araBAD promoter.
  • Several additional terms have been used to indicate the E. coli araBAD promoter, such as P ara , ParaB, ParaBAD, and P RAD -
  • the use herein of ara 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 arciBAD 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 arciE, which encodes the low-affinity L- arabinose proton symporter, and the arciFGH operon, which encodes the subunits of an ABC superfamily high-affinity L-arabinose transporter.
  • LacY lactose permease the LacY(AlWC) and the LacY(A177V) proteins, having a cysteine or a valine amino acid instead of alanine at position 177, respectively
  • LacY(AlWC) and the LacY(A177V) 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 arciBAD promoter by use of a lactose transporter of relaxed specificity", Proc Natl Acad Sci U S A 2002 May 28; 99(11): 7373-7377).
  • arciE 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 arciE so that it is only transcribed from a constitutive promoter. Constitutive expression of arciE can be accomplished by eliminating or reducing the function of the native arciE 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 arciE 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(A177C) protein appears to be more effective in transporting arabinose into the cell, use of polynucleotides encoding the LacY(A177C) protein is preferred to the use of polynucleotides encoding the LacY(A177V) protein.
  • the 'propionate promoter' or prp promoter' is the promoter for the E. coli prpBCDE operon, and is also called P prp B- 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
  • propionic acid or 'propanoic acid'
  • propionic acid or 'propanoic acid'
  • H(CH 2 ) or 'propanoic acid'
  • 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-ygfDGH 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-ygfD-ygfG-ygfH-ygfl, introduced into E.
  • genes sbm-ygfDGH are transcribed as one operon, and ygfl is transcribed from the opposite strand.
  • Eliminating or reducing the function of a subset of the sbm-ygfDGH gene products such as YgfG (also called ScpB, methylmalonyl-CoA decarboxylase), or deleting the majority of the sbm-ygfDGH (or scpA-argK-scpBC) operon while leaving enough of the 3' end of the ygfH (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-ygfDGH or scpA-argK-scpBC
  • ygfH or scpC
  • Rhamnose promoter (As used herein, 'rhamnose' means L- rhamnose.)
  • 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
  • Wickstrum et ah "The AraC/XylS family activator RhaS negatively autoregulates rhaSR expression by preventing cyclic AMP receptor protein activation", J Bacteriol 2010 Jan; 192(1): 225-232).
  • 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 rfiBDACX) 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 P xy iA and P xy i F 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 superfamily high- affinity D-xylose transporter.
  • the xylE gene which encodes the E. coli low- affinity xylose-proton symporter, represents a separate operon, the expression of which is also inducible by xylose.
  • 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 xy i F 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 xy iA or the P xy i F 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).
  • inducible coexpression systems of the invention can comprise a lactose-inducible promoter such as the lacZYA promoter. In other embodiments, the inducible coexpression systems of the invention comprise one or more inducible promoters that are not lactose- inducible promoters. Table 3. Genomic Locations of E. coli Inducible Promoters and Related Genes [1]
  • prpR gene (ca. 347692) - Keasling [8] : inferred transcript start ca. 347692, prpR
  • scpA-argK-scpBC 3058872 - NCBI scpA ATG 3058872, end of TAA 3061016; (or sbm-ygfDGIt) 3064302 (plus argK ATG 3061009, end of TAA 3062004; scpB ATG operon strand) 3062015, end of TAA 3062800; scpC ATG 3062824, end of TAA 3064302
  • rhaT promoter [2] (ca. 4098690) Via [10] : CRP binding 4098690-4098675, RNA pol
  • xylAB operon 3728830 - Song and Park [1 1] transcript start 3728830, xylA ATG
  • xylFGHR operon 3729092 - Song and Park [1 1] transcript start 3729092, xylF ATG
  • All genomic sequence locations refer to the genomic sequence of E. coli K-12 substrain MG1655, provided by the National Center for Biotechnology Information (NCBI) as NCBI Reference Sequence NC 000913.2, l l-JAN-2012.
  • the promoter portion of an expression construct can have somewhat less sequence at its 5' end than the promoter sequences as indicated in the table, or it can have a nucleotide sequence that includes additional sequence from the region 5' (or 'upstream') of the promoter sequences as indicated in the table, as long as it retains the ability to promote transcription of a downstream coding sequence in an inducible fashion.
  • Expression constructs of the invention can also comprise coding sequences that are expressed from constitutive promoters. Unlike inducible promoters, constitutive promoters initiate continual gene product production under most growth conditions.
  • a constitutive promoter is that of the Tn3 bla gene, which encodes beta-lactamase and is responsible for the ampicillin-resistance (Amp R ) phenotype conferred on the host cell by many plasmids, including pBR322 (ATCC 31344), pACYClW (ATCC 37031), and pBAD24 (ATCC 87399).
  • Another constitutive promoter that can be used in expression constructs is the promoter for the E.
  • coli lipoprotein gene, Ipp which is located at positions 1755731-1755406 (plus strand) in E. coli K-12 substrain MG1655 (Inouye and Inouye, "Up-promoter mutations in the Ipp gene of Escherichia coli", Nucleic Acids Res 1985 May 10; 13(9): 3101-3110).
  • a further example of a constitutive promoter that has been used for heterologous gene expression in E. coli is the trpLEDCBA promoter, located at positions 1321169-1321133 (minus strand) in E.
  • Constitutive promoters can be used in expression constructs for the expression of selectable markers, as described herein, and also for the constitutive expression of other gene products useful for the coexpression of the desired product.
  • transcriptional regulators of the inducible promoters such as AraC, PrpR, RhaR, and XylR
  • transcriptional regulators of the inducible promoters can alternatively be expressed from a constitutive promoter, on either the same expression construct as the inducible promoter they regulate, or a different expression construct.
  • gene products useful for the production or transport of the inducer such as PrpEC, AraE, or Rha, or proteins that modify the reduction-oxidation environment of the cell, as a few examples, can be expressed from a constitutive promoter within an expression construct.
  • Gene products useful for the production of coexpressed gene products, and the resulting desired product also include chaperone proteins, cofactor transporters, etc.
  • the inducible coexpression systems of the invention are designed to express multiple gene products; in certain embodiments of the invention, the gene products are coexpressed in a host cell.
  • host cells are provided that allow for the efficient and cost-effective inducible coexpression of 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 expression constructs of the inducible coexpression systems of the invention can be used in cell-free systems, such as those based on wheat germ extracts or on bacterial cell extracts, such as a continuous-exchange cell-free (CECF) protein synthesis system using E.
  • CECF continuous-exchange cell-free
  • Prokaryotic host cells expression constructs designed for coexpression of gene products are provided in host cells, preferably 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, Azo
  • archaea
  • 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 Eukaryotic host cells. Many additional types of host cells can be used for the inducible coexpression 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 lipolytica); other fungi ⁇ Aspergillus nidulans, Aspergillus niger, Neurospora crassa, Penicillium, Tolypocladium, Trichoderma reesia); insect cell lines ⁇ Drosophila melanogaster Schneider 2 cells and Spodoptera frugiperda Sf9 cells); and mammalian cell lines including
  • 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 7.
  • 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.
  • CCR carbon catabolite repression
  • CCR Carbon catabolite repression
  • the CCR mechanism of the cell would therefore appear to make it more difficult to use two or more carbon-source inducers in an inducible coexpression system, as the presence of the inducer that is the preferred carbon source would be expected to inhibit induction by less-preferred carbon sources (Lee et al., "Catabolite repression of the propionate catabolic genes in Escherichia coli and Salmonella enterica: evidence for involvement of the cyclic AMP receptor protein", J Bacteriol 2005 Apr; 187(8): 2793-2800; Park et al., "The mechanism of sugar- mediated catabolite repression of the propionate catabolic genes in Escherichia coir, Gene 2012 Aug 1 ; 504(1): 116-121, Epub 2012 May 3).
  • EII A glc glucose-specific EII A
  • Another alteration in the genome of a host cell such as E. coli, which leads to increased transcription of the prp promoter, is to eliminate or reduce the gene function of the ascG gene, which encodes AscG.
  • AscG is the repressor of the beta-D-glucoside-utilization operon ascFB under normal growth conditions, and also represses transcription of the prp promoter; disruption of the AscG coding sequence has been shown to increase transcription from the prp promoter (Ishida et al., "Participation of regulator AscG of the beta- glucoside utilization operon in regulation of the propionate catabolism operon", J Bacteriol 2009 Oct; 191 (19): 6136-6144; Epub 2009 Jul 24).
  • a further alternative is to increase expression of the transcriptional regulator of promoters inducible by the less-preferred carbon-source inducer, by placing it either under the control of a strong constitutive promoter, or under the control of the more -preferred carbon- source inducer.
  • the transcriptional regulator of promoters inducible by the less-preferred carbon-source inducer by placing it either under the control of a strong constitutive promoter, or under the control of the more -preferred carbon- source inducer.
  • the coding sequence for XylR is placed into the E. coli arciBAD operon (Groff et al., "Supplementation of intracellular XylR leads to coutilization of hemicellulose sugars", Appl Environ Microbiol 2012 Apr; 78(7): 2221-2229, Epub 2012 Jan 27).
  • Host cells comprising inducible coexpression constructs therefore preferably include an increased level of gene function for transcriptional regulators of promoters inducible by the less-preferred carbon- source inducer(s), and an eliminated or reduced gene function for genes involved in the CCR system, such as err and/or ptsG and/or ascG.
  • Dsb system comprising DsbABCD and DsbG.
  • Cytoplasmic Dsb proteins such as 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 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.
  • ahpC* Suppressor mutations 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 igshA) and defective in trxB, to grow in the absence of DTT; these include AhpC V164G, AhpC S71F, AhpC E173/S71F, AhpC E171Ter, and AhpC dupl 62-169 (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. coif Microb Cell Fact 2011 Jan 7; 10: 1).
  • Host cells comprising inducible coexpression constructs preferably also express cDsbC and/or Ervlp, are deficient in trxB gene function, are also deficient in the gene function of either gor, gshB, or gshA, and express an appropriate mutant form of AhpC so that the host cells can be grown in the absence of DTT.
  • Host cells can have alterations in their ability to glycosylate polypeptides.
  • eukaryotic host cells can have eliminated or reduced gene function in glycosyltransferase and/or oligosaccharyltransferase 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.
  • Inducible Coexpression Systems of the Invention involve host cells comprising two or more expression constructs, where the expression constructs comprise inducible promoters directing the expression of gene products, and the host cells have altered gene functions that allow for homogeneous inducible expression of the gene products.
  • Fig. 1 shows a schematic representation of an inducible coexpression system of the invention, with the following components: (1) host cell, (2) host genome (including genetic alterations), (3) an expression vector 'X' comprising an inducible promoter directing expression of a gene product, (4) a different expression vector ⁇ ' comprising an inducible promoter directing expression of another gene product, (5) chemical inducers of expression, and (6) the multimeric coexpression product.
  • Fig. 2 shows a schematic representation of a particular example of an inducible coexpression system of the invention, utilizing the araBAD promoter on a pBAD24 expression vector in combination with a propionate-inducible promoter (prpBCDE promoter) on a pPR033 expression vector (US Patent No. 8178338 B2; May 15 2012; Keasling, Jay), in an E. coli host cell housing the appropriate genomic alterations which allow for homogenously inducible expression. In this manner, tight control and optimization of expression of each component of a multimeric product can be achieved for use in a number of coexpression applications.
  • the host cell (1) is the Gram-negative bacterium Escherichia coli, commonly used in the art for protein expression.
  • the host genome (2) is the genome of the host cell organism with mutations or other alterations that facilitate homogenously inducible protein coexpression, including expression of a cytoplasmic form of the disulfide isomerase DsbC which lacks a signal peptide.
  • the genomic alterations include both an araBAD operon knockout mutation, and either expression of araE and araFGH from constitutive promoters, or a point mutation in the /acF gene (A1 17C) in an araEFGH-dQficiQnt background, to facilitate homogenous induction of plasmid- based ara promoters with exogenously applied L-arabinose, and also an inactivated proprionate metabolism gene, prpD, to facilitate homogenous induction of plasmid-based propionate promoters with exogenously applied propionate, which is converted to 2-methylcitrate in vivo.
  • genomic alterations that are useful for the inducible coexpression system, and may be introduced into the host cell, include without limitation: targeted inactivation of the scpA-argK-scpBC operon, to reduce background expression from the prpBCDE promoter; expression of the transcriptional regulator (prpR) for the less- preferred carbon-source (propionate) from an L-arabinose-inducible promoter such as the araBAD promoter, and/or an eliminated or reduced gene function for genes involved in the CCR system, such as err and/or ptsG, to avoid suppression by the CCR system of induction by propionate in the presence of L-arabinose; reductions in the level of gene function for glutathione reductase (gor) or glutathione synthetase (gshB), together with thioredoxin reductase (trxB), and/or expression of yeast mitochondrial sulfhydryl oxidase Ervlp in the host cell
  • one expression vector contains an L-arabinose-induced promoter, and is similar or identical to pBAD or related plasmids in which an araBAD promoter drives expression of an inserted expression sequence cloned into the multiple cloning site (MCS).
  • the L-arabinose-induced expression vector also contains a coding sequence for an antibiotic-resistance gene (such as the Tn3 bla gene, which encodes beta-lactamase and confers resistance to ampicillin) to facilitate selection of host cells (bacterial colonies) which contain an intact expression vector.
  • the L-arabinose induced expression plasmid also contains a polynucleotide sequence encoding araC, a transcriptional regulator that allows for L-arabinose induction of the araBAD promotor and through transcriptional repression reduces 'leaky' background expression in the non-induced state.
  • the other expression vector ('propionate-induced expression vector') is similar or identical to pPRO or related plasmids, in which a propionate-induced promoter drives expression of an inserted expression sequence cloned into the multiple cloning site (MCS).
  • the plasmid also contains a coding sequence for an antibiotic-resistance gene (such as the cat gene, encoding chloramphenicol acetyltransferase, which confers resistance to chloramphenicol) to facilitate selection of host cells which contain an intact expression vector.
  • An origin of replication (ORI) is required for propagation of the plasmid within bacterial host cells.
  • the propionate-induced expresssion vector contains a polynucleotide sequence encoding prpR, a transcriptional regulator that allows for propionate (2-methylcitrate) induction of the prpBCDE promotor and reduces 'leaky' background expression in the non-induced state.
  • a pBAD24 or related expression vector containing an L- arabinose-inducible arciBAD promoter is combined in a host cell with a pPR033 or related expression vector (p 15 A ORI, Cm R ) containing a propionate-inducible prpBCDE promoter.
  • the expression vectors are co-propagated and maintained using growth medium supplemented with ampicillin and chloramphenicol.
  • one expression vector comprises a polynucleotide sequence encoding the A chain of thrombin
  • the other expression vector comprises a polynucleotide sequence encoding the B chain of thrombin, each coding sequence cloned in-frame into the MCS of the respective expression vector.
  • coding sequence optimization for the host organism including adjustment for codon bias and GC-content, among other considerations will determine the coding sequences to be inserted into the expression constructs of the coexpression system.
  • coexpression of the thrombin A and B chains is induced by inexpensive exogenously applied chemical metabolites, L-arabinose and propionate (5).
  • the level of induction of expression of each thrombin polypeptide is independently titrated with its own chemical inducer, thereby facilitating optimization of protein coexpression.
  • This is useful for expression of protein complexes and proteins that require a binding partner for stabilization, and may facilitate expression of otherwise difficult to express proteins, such as those with poor solubility or cellular toxicity.
  • thrombin A and B chains are each separately expressed, then the proteins join and form interchain disulfide bonds (within the cytoplasm of the bacterial host) which allows the formation and stabilization of thrombin.
  • Proteins can be directed to various compartments of the host organism.
  • E. coli the protein can be expressed in the cytoplasm, cell membrane, periplasm, or secreted into the medium. After an appropriate incubation time, cells and media are collected, and the total protein extracted, which includes the coexpressed product, thrombin (6).
  • the desired product can be purified using a number of methods well known in the art depending on the nature of the gene products produced in the coexpression system (for example liquid chromatography). In the example shown in Fig. 2, the desired product (thrombin) is purified.
  • the expression vectors used in these experiments are the pBAD24 vector, obtained from the American Type Culture Collection (ATCC) as ATCC 87399, which has the nucleotide sequence shown in GenBank Database Accession No. X81837.1 (25-OCT-1995); and the pPR033 vector, obtained from the University of California (Berkeley, California), which has the nucleotide sequence (an updated version) shown in SEQ ID NO: 13.
  • the human thrombin A chain, and the human thrombin B chain containing the W215A E217A mutations would be expressed from separate expression constructs, with expression of the A chain from the propionate-inducible prpBCDE promoter in the pPR033 expression vector, and expression of the WE-B chain from the L-arabinose-inducible arciBAD promoter in the pBAD24 expression vector.
  • the amino acid sequences of the thrombin A chain, B chain, and the WE-B chain, with N-terminal methionine residues are provided as SEQ ID NOs 3, 4, and 6, respectively.
  • Amino acids 2 through 37 of SEQ ID NO:3; amino acids 2 through 260 of SEQ ID NO:4; and amino acids 2 through 260 of SEQ ID NO: 6 are the amino acid sequences of the human thrombin A chain, B chain, and WE-B chain, respectively.
  • the second arrangement of coexpression constructs is similar to the first, with the thrombin A and WE-B chains separately coexpressed as described above, and in which the A chain further comprises an additional 14 amino acids (including an N- terminal methionine) at its N-terminal end.
  • This extended A chain is referred to as the A+ polypeptide', with its amino acid sequence provided as SEQ ID NO: 8.
  • amino acids 2-14 of SEQ ID NO: 8 generally correspond to a portion of the preprothrombin, prothrombin, and prethrombin-1 amino acid sequences (amino acids 315 to 327 of SEQ ID NO: l) that is N-terminal to the A chain, except that the Asn 325 and Pro 326 residues of SEQ ID NO: l have been altered to Asp and Gly, respectively, to create a DGR cleavage site that can be recognized by ecarin.
  • thrombin protein in which the N-terminal end of the A+ polypeptide can be cleaved by ecarin, either in vitro or through coexpression of ecarin in vivo, to create human thrombin where the A chain has its native N-terminal Thr residue.
  • the A+ and WE-B chains are expressed contiguously, from the arciBAD promoter in the pBAD24 expression vector, as part of one expression construct; the amino acid sequence of the thrombin A+/WE-B polypeptide is provided as SEQ ID NO: 10.
  • the mature form of the ecarin polypeptide (SEQ ID NO: 12) is coexpressed, from the prpBCDE promoter in the pPR033 expression vector, along with the thrombin A+/WE-B polypeptide.
  • thrombin A+/WE-B polypeptide which is similar to WE- prethrombin-2 but with the additional 14 amino acids at its N-terminal end, as described for the A+ polypeptide.
  • the thrombin A+/WE-B polypeptide can be cleaved by ecarin at two locations - the engineered ecarin cleavage site within the A+ sequence that releases the native A chain N-terminus, and the ecarin cleavage site between the A and B chains - with ecarin cleavage at both locations producing a human thrombin protein with the native N-terminal residues on both the A and B chain.
  • the coding sequences used in these expression constructs were optimized for expression in E. coli by DNA2.0 (Menlo Park, CA), according to the methods of Welch et al. ("Design parameters to control synthetic gene expression in Escherichia coli", PLoS One 2009 Sep 14; 4(9): e7002; doi: 10.1371/journal. pone.0007002), and were also synthesized by DNA2.0.
  • nucleotides 21 through 131 of SEQ ID NO:2 encode human thrombin A chain, SEQ ID NO:3; nucleotides 21 through 800 of SEQ ID NO:5 encode human thrombin WE-B chain, SEQ ID NO: 6; nucleotides 21 through 170 of SEQ ID NO:7 encode human thrombin A+ polypeptide, SEQ ID NO:8; nucleotides 21 through 947 of SEQ ID NO: 9 encode human thrombin A+/WE-B polypeptide, SEQ ID NO: 10; and nucleotides 21 through 1301 of SEQ ID NO: 11 encode mature Echis carinatus ecarin, SEQ ID NO: 12.
  • these nucleotide sequences all contain a ribosome binding site at nucleotides 7 through 13 of SEQ ID NOs 2, 5, 7, 8, and 11, utilize a TAA termination codon, and have a variety of restriction sites flanking the optimized coding sequences on both sides, including an Nhel site 5' to both the ribosome binding site and the coding sequence, and a Sail site 3' of the TAA termination codon.
  • the optimized coding sequences and the pPR033 and pBAD24 vectors were digested with Nhel and Sail.
  • the desired insert fragments and cut vectors were gel purified, then ligated using 100 ng vector DNA and a 1 :3 molar ratio of vector: insert.
  • pPR033_A, pPR033_A+, pBAD_WE-B, pBAD_A+/WE-B, and pPR033_ecarin expression constructs were transformed into BL21 cells using the heat-shock method: 2.5 microliters of ligated DNA were added to 25 microliters of thawed BL21 cells and incubated on ice for 30 minutes, then at 42 degrees C for 10 seconds, reincubated on ice for five minutes, then 0.475 mL SOC medium was added and the cells were grown for one hour at 37 degrees C with shaking at 250 RPM, then pelleted at 3800 x g, resuspended in 0.1 mL supernatant, and plated.
  • the BL21 cells transformed with the various expression constructs were used to prepare DNA of the expression constructs, using either miniprep or midiprep methods.
  • the expression constructs were generally digested with Nhel and Sail, except for pPR033_A and pPR033_A+; the inserts are small, so these constructs were digested with Xhol (which cuts 325 nucleotides upstream of the Nhel site) and Sail.
  • the nucleotide sequences of the expression constructs are provided as SEQ ID NO: 14 (pPR033_A), SEQ ID NO: 15 (pPR033_A+), SEQ ID NO: 16 (pBAD_WE-B), SEQ ID NO: 17 (pBAD_A+/WE-B), and SEQ ID NO: 18 (pPR033_ecarin).
  • telomeres are also used for expressing polypeptides of interest from the propionate- inducible prpBCDE promoter.
  • pPR043 the nucleotide sequence encoding the transcriptional activator prpR has been optimized for expression in E.
  • the pPR043 vector also has only one Hindlll restriction site, which is in the multiple cloning site (MCS), in contrast with pPR033 which has two Hindlll sites, one in the MCS and a second in the prpR coding sequence.
  • MCS multiple cloning site
  • the thrombin expression constructs were co-transformed into E. coli ASE(DGH) cells in the following three sets of pairs, to create host cells to be used in the coexpression of thrombin: (1) pPR033_A (SEQ ID NO: 14) and pBAD_WE-B (SEQ ID NO: 16); (2) pPR033_A+ (SEQ ID NO: 15) and pBAD_WE-B (SEQ ID NO: 16); and (3) pBAD_A+/WE-B (SEQ ID NO: 17) and pPR033_ecarin (SEQ ID NO: 18).
  • the expression constructs were transformed into E.
  • the E. coli ASE(DGH) cells were derived from E. coli SHuffle® Express cells, and their genotype can be expressed as: E. coli SHuffle® Express AaraBAD Asbm-ygfDGH AaraEp::J23 l04.
  • the E. coli ASE(DGH) cells were produced as follows: deletions and alterations were made in the E. coli SHuffle® Express host cell genome by Gene Bridges GmbH (Heidelberg, Germany) using a recombineering method, described as deletion by counterselection, that seamlessly removes genomic sequences.
  • a deletion of the host cell arciBAD operon was made to reduce arabinose catabolism by the host cell, so that more of the arabinose inducer will be available for induction of a coexpressed gene product from an expression construct comprising the arciBAD promoter.
  • This deletion removes 4269 basepairs of the arciBAD operon, corresponding to position 70, 135 through 65,867 (minus strand) of the E. coli genome (positions within genomic nucleotide sequences are all given as in Table 3), so that most of the native arciBAD promoter through all but a few codons of the AraD coding region are removed.
  • position 65,866) is: TTAT
  • Another deletion was made within the sbm- ygfDGH (also called scpA-argK-scpBC) operon, eliminating the function of genes involved in the biosynthesis of 2-methylcitrate, to increase sensitivity of the host cell's propionate-inducible promoter to exogenously supplied propionate.
  • the sbm-ygfDGH deletion removes 5542 basepairs (position 3,058,754 through 3,064,295 of the E.
  • nucleotide sequence (plus strand) around the deletion junction is: ACAA
  • coli SHuffle® Express host cell genome Gene Bridges GmbH introduced a point mutation in the genomic rpsL gene coding sequence, which extends on the minus strand from position 3,472,574 through 3,472,200, changing the A at position 3,472,447 to a G, altering the codon for Lys43 to a codon for Arg, which results in a streptomycin-resistant phenotype when the mutant r/3 ⁇ 4*Z-Arg43 gene is expressed.
  • Another alteration to the host cell genome allowing for more tightly controlled inducible expression as described above, is to make the arciE promoter constitutive rather than responsive to arabinose.
  • araE promoter Most of the native araE promoter, including CRP-cAMP and AraC binding sites, was removed by deleting 97 basepairs (position 2,980,335 through 2,980,239 (minus strand)) and replacing that sequence with the 35-basepair sequence of the constitutive J23104 promoter (SEQ ID NO:20; the nucleotide sequence of J23104 was obtained from the partsregistry.org website, parts.igem.org/Main_Page).
  • the resulting junction site sequences within the altered araE promoter are: TGAA
  • the inducible coexpression system was used to produce thrombin, and specifically WE-thrombin, in bacterial cells.
  • E. coli ASE(DGH) cells comprising the WE- thrombin pPR033_A and pBAD24_WE-B expression constructs were stabbed and cultured overnight in 6 mL LB +cam +amp +0.2% glucose at 37 degrees C with shaking at 250RPM.
  • cell suspensions from each sample were divided into 4 x 1.2 mL aliquots, and the cells were pelleted by centrifugation at 4300 RPM for 5 minutes at 25 degrees C, and stored dry in the freezer after removal of the super- natants.
  • Lysates were clarified by centrifugation at 20,000 x g for 30 minutes at 4 degrees C; the supernatant was retained for analysis by gel electrophoresis.
  • a NuPAGE® Novex® 10% Bis-Tris gel (Life Technologies, Grand Island, New York) was loaded with the samples as described above, and was run in SDS-MES at 220V, 45 minutes, under non-reducing conditions. The gel was stained using RAPID stainTM (G- Biosciences).
  • a protein band migrates at 33.8 kDa on the gel; this band is absent in the uninduced cells, and present in the induced cells, with the greatest band intensity shown in samples induced with intermediate concentrations of arabinose.
  • Lysates were spun at 20,000 x g for 30 minutes at 4 degrees C.
  • the samples shown in the following table were prepared using NuPAGE® lOx Reducing Agent (Life Technologies) and 4X XT Sample Buffer (Bio-Rad, Hercules CA), then incubated at 95 degrees C for five minutes prior to loading.
  • a NuPAGE® Novex® 10% Bis-Tris gel (Life Technologies) was loaded with the above samples and run under reducing conditions in SDS-MES running buffer, at 4 degrees C and 220V, then transferred onto nitrocellulose membrane at 30V, 1 hour in IX NuPAGE® Transfer Buffer with 10% MeOH.
  • the nitrocellulose membrane was blocked and immunodetection was done using Novex® reagents according to the manufacturer's protocol; the primary antibody was 1 :750 monoclonal mouse anti-thrombin (product number HYB 109-03-02, Pierce Antibodies/Thermo Fisher Scientific, Waltham, MA), and the secondary antibody was Novex® Alk-Phos. Conjugated Anti-Mouse Antibody solution (Life Technologies).
  • E. coli ASE(DGH) cells comprising sets of WE-thrombin expression constructs - either pPR033_A and pBAD24_WE-B, or pPR033_A+ and pBAD24_WE-B - were induced as described in the section above under 'Western blot'.
  • 50-mL aliquots were made of those cells, and the cell pellets and supernatants from those aliquots were stored at -80 degrees C.
  • a non- induced control sample was prepared by growing E.
  • coli ASE(DGH) cells comprising the pPR033_A and pBAD24_WE-B expression constructs in LB + 0.2% glucose +cam +amp, then pelleting them, removing the supernatant, and resuspending them in M9 + 0.2% glucose. After incubating the cells for 5 hours at 30 degrees C with shaking at 250 RPM, the cells were divided into 4 x 50-mL aliquots, and pelleted at 3800 x g for 5 minutes at 25 degrees C.
  • the cell samples that were lysed included one cell pellet (corresponding to 50 mL culture) for each combination of expression vectors (A/WE-B, A+/WE-B) with each induction condition (30 degrees C for 5 hours, 16 degrees C for 24 hours), plus a cell pellet from the no-induction control sample.
  • the cell pellets were each thawed in a 50-mL tube, to which was added 2.5 mL Formulation Buffer (20 mM NaCitrate, 150 mM NaCl, 0.1 % Polysorbate 80, pH 6.0, filter sterilized), including 1.0 mg/mL lysozyme and 150 units benzonase (product number 70746, Novagen/EMD Millapore, Billerica, MA).
  • the pellets were resuspended and lysis proceeded on ice for 30 minutes, then the lysates were clarified by centrifugation at 20,000 x g for 30 minutes at 4 degrees C.
  • the lysates were flash-frozen in an ethanol/dry ice bath and stored at -80 degrees C.
  • Enzyme capture assay capture of thrombin using the extracellular domains of thrombomodulin and chromogenic assay for thrombin activity
  • Thrombin binds readily to the N-terminal region of thrombomodulin, which is found in recombinant human soluble thrombomodulin (rhs-TM), also called ART- 123, which comprises the extracellular domain of thrombomodulin and is glycosylated (Kishida et al, "Study on complex formation between recombinant human thrombomodulin fragment and thrombin using surface plasmon resonance", Am J Hematol 2000 Mar; 63(3): 136-140). ART-123 has been found to readily adhere to plastic.
  • rhs-TM recombinant human soluble thrombomodulin
  • a method of purifying thrombin takes advantage of this property: in general, a soluble polypeptide comprising the thrombomodulin extracellular domain, such as rhs-TM/ART-123, is immobilized on a solid substrate such as plastic or glass beads in a column, and a solution containing thrombin is passed through the beads under conditions suitable for binding. After an optional wash step, the thrombin is eluted from the column.
  • the binding of thrombin by the extracellular domain of thrombomodulin also provides the basis for a sensitive assay of thrombin activity.
  • the wells of a 96- well, flat bottom, clear microplate (catalog number 12565501, Thermo Fisher Scientific, Waltham, MA) were coated with 100 microliters of 50 micrograms/mL soluble thrombomodulin (rhs-TM/ART-123) in carbonate/bicarbonate buffer pH 7.6 (catalog number 21851, Sigma-Aldrich, St. Louis, MO).
  • the plate was stored covered at 4 degrees C overnight for two nights.
  • the thrombomodulin solution was removed and the plates were blocked with 3% BSA-TBST20(TBS plus Tween 20) for 45 minutes at room temperature. The block was then removed and the wells were washed three times with 150 microliters TBS, flicking and blotting between each wash (this step may not be important).
  • a standard curve to calibrate the assay was prepared using serial dilutions of purified thrombin (e.g., wild-type human thrombin) in sample dilution buffer (e.g., TBS, 0.01% Tween 20). Incubation was at RT or 37°C (minutes to hours range; for very dilute standards incubation may proceed for up to a week ). The absorbed samples/enzymes were removed by flick and blot. The plate was then washed five times with 150 microliters of TBS, flicking and blotting between each wash to reduce enzymes and contaminants that were not specifically bound to wells.
  • sample dilution buffer e.g., TBS, 0.01% Tween 20
  • reversible enzyme inhibitors e.g., benzamidine, 1-20 mM
  • all samples e.g., added to samples in sample dilution buffer
  • reversible enzyme inhibitors e.g., benzamidine, 1-20 mM
  • the samples that were tested correspond to the samples run on the western blot described in Example 1 above and shown in Figure 5; abbreviated designators for these samples are L AB16C, L AB30C, L A+B16C, L A+B30C, M AB16C, M AB30C, M A+B16C, and M A+B30C, where 'L' indicates a cell lysate and ' ⁇ ' indicates the culture medium.
  • the culture medium samples M AB16C, M AB30C, M A+B16C, and M A+B30C were concentrated with a spin filter protein concentrator for a 20-ml swing bucket (MAP003C36 3K, Pall Corp., Port Washington, NY), according to the manufacturer's instructions.
  • the prepared samples were added to the wells of the thrombomodulin-coated plate, 100 microliters of sample/well. Although not done in this experiment, it may be useful to dilute the samples into a sample buffer (e.g., TBS, 0.01% Tween 20) for consistency in binding rates.
  • a sample buffer e.g., TBS, 0.01% Tween 20
  • the chromogenic substrate S-2366 (D-Phe-Pip-Arg-p-nitroanilide, Chromogenix, Milan, Italy) was diluted to 0.8 mM with 50 mM Tris-HCl pH 8.3, and 100 microliters of the substrate solution was added to each well carefully to avoid bubbles.
  • the concentration of S-2366 can vary in a broad range (e.g., 0.4 mM to 4 mM), and the S-2366 substrate solution may contain some azide (e.g., 0.05 %) and EDTA (20 mM) to prevent bacteria and fungi from growing in the wells.
  • the method for the determination of thrombin enzyme activity is based on the difference in absorbance (optical density) between the pNA formed between two readings.
  • the formation of pNA increases the color development (absorbance) over time at 405 nm, and is proportional to the captured enzyme activity.
  • Development of color can take minutes (as for wild-type thrombin at picomolar or higher concentration) to weeks (as for mutant thrombins, or thrombins at femtomolar or lower concentration).
  • the microplate is stored in a wet chamber, sealed to prevent evaporation and changes in substrate volume in the wells when long reading times are required.
  • the limit of detection for wild-type thrombin appears to be single digit low femtomolar (fM) or even lower (attomolar). There is no theoretical limit, as the assay, when well done, can detect a single enzyme molecule, provided the substrate has a lower autocatalytic rate.
  • the plate reader (Versa Max with SoftMax Pro 5.0, Molecular Devices, Sunnyvale, CA) was set to measure absorbance at 405 nm at 37 degrees C. Absorbance was measured at two time points, at 1 day and at 4 days of incubation.
  • a 6xHis-SUMO protein tag to the N-terminal end of the thrombin B-chain coding sequence can assist in purification of the coexpressed thrombin protein.
  • expression constructs are prepared, one which encodes a SUMO-thrombin A chain fusion polypeptide, and the other a 6xHis-SUMO- thrombin B chain fusion polypeptide. These expression constructs are coexpressed, the resulting thrombin protein is purified using the 6xHis tag on the B chain, and then the SUMO tags are cleaved from both the A and B chains by SUMO protease.
  • the SUMO protease polypeptides are also fusion proteins comprising 6xHis tags, allowing for a two-step purification: in the first step, the coexpressed 6xHis-tagged thrombin is purified by binding to a nickel column, followed by elution from the column.
  • the SUMO tags on the purified thrombin are cleaved by the 6xHis-tagged SUMO protease, and the SUMO protease-thrombin reaction mixture is run through a second nickel column, which retains the SUMO protease but allows the untagged thrombin to flow through.
  • Thrombin can be purified using standard size-exclusion chromatography methods. It is also possible to purify thrombin by ion-exchange chromatography, for example on a MonoS-FPLC column by using a linear gradient of 0.05- 0.5 M NaCl in 5 mM MES, pH 6 at room temperature. Alternatively, FPLC can be performed using Resource Q and S columns with a linear gradient from 0.05 to 0.5 M choline chloride, 5 mM MES, pH 6, at room temperature.
  • a 1-mL heparin column (GE HealthCare Life Sciences, Piscataway, NJ) is prepared by washing with 5 mL 20% EtOH, followed by 10 mL binding buffer (10 mM NaP04, pH 7.1).
  • a positive control sample, WE-thrombin is diluted in binding buffer to a concentration of 0.01 mg/mL. Samples are loaded onto the column at a flow rate of about 0.5 mL/min and flow-through is kept for analysis. Samples are taken and tested for protein using the Bradford colorimetric assay. The column is washed in 10 mL binding buffer and samples are taken periodically to determine whether the wash contains protein. The column is eluted using 2M NaCl in binding buffer. Fractions are taken in 0.25 mL volumes and the Bradford assay shows that most thrombin protein comes off between 0.75 and 1.0 mL of elution, with some following in the 1-1.25-mL fraction.
  • protein samples are diluted in binding buffer and loaded onto a 5-mL heparin column (GE Healthcare Life Sciences) at 3 mL/minute. Bound protein is extensively washed with 100 mM NaCl, 10 mM Tris pH 8 at 25 degrees C before elution with a linear gradient of 100 mM to 1 M NaCl in 10 mM Tris, pH 8 at 25 degrees C. Fractions with OD 280 greater than 0.1 are combined, concentrated and applied to a detoxi-gel column to remove endotoxins. Concentration of the protein are determined by taking the OD 280 . The extinction coefficient of recombinant E-WE thrombin at 280 nm is 1.83 M 1 cm -1 .
  • a solution containing thrombin (routinely 105 mL) is applied to a column (2.6 x 15 cm) of heparin-Sepharose 6 Fast Flow (GE HealthCare Life Sciences) previously equilibrated using 0.025 M citrate buffer plus 0.15 M NaCl, pH 7.3, at 4 degrees C.
  • the column is first washed exhaustively with the equilibration buffer and then with 0.025 M citrate buffer plus 0.45 M NaCl, pH 7.3. Elution is performed with a 0.025 M citrate buffer plus 2.5 M NaCl, pH 7.3.
  • polyphosphate to purify thrombin arises from its ability to bind with high affinity to thrombin, without binding to prothrombin (Mutch et al., "Polyphosphate binds with high affinity to exosite II of thrombin", J Thromb Haemost 2010 Mar; 8(3): 548-555; doi: 10.111 l/j. l538-7836.2009.03723.x; Epub 2009 Dec 11).
  • Polyphosphate polyP, sold under the name "sodium phosphate glass” and available from Sigma (St.
  • Binding Buffer 50 mM Tris-HCl pH 7.5, 50 mM NaCl, 0.1% BSA
  • thrombin recovery is quantified by measuring rates of chromogenic Spectrozyme TH ( /-d-hexahydrotyrosol-Ala-Arg-/?-nitroanilide) hydrolysis compared to a standard curve.
  • Salt sensitivities of thrombin-polyP interactions were investigated by modifying either the Binding or Elution Buffer to contain 50-1000 mM NaCl. Purification of a larger amount of thrombin using immobilized polyP is accomplished in a similar manner by standard methods, using larger columns and volumes.
  • This purification method takes advantage of the fact that some, but not all, forms of thrombin bind to fibrinogen.
  • Thrombin variants with a reduced affinity for fibrinogen such as the thrombin W215A E217A variant, are not good candidates for purification by this method.
  • Fibrinogen is conjugated to Sepharose 4B, using the method of Cuatrecasas ("Protein purification by affinity chromatography. Derivatizations of agarose and polyacrylamide beads", J Biol Chem 1970 Jun; 245(12): 3059-3065) with a final concentration of 2 mg of fibrinogen per 1 ml of Sepharose 4B.
  • the resin is packed in a plastic column (1.5 x 3 cm, bed volume 6 ml) and equilibrated with 0.02 M NaC 1/0.01 M Tris-HCl (pH 7.4).
  • Thrombin, and to a lesser extent prethrombin-2 binds to the fibrinogen-Sepharose column, while prothrombin and prethrombin-1 do not; the affinity of thrombin and prethrombin-2 for fibrinogen-Sepharose depends on ionic strength and reaches a maximum at 50 mM concentration at 22 degrees C (Kaczmarek et al., "Fibrinogen-sepharose interaction with prothrombin, prethrombin 1, prethrombin 2 and thrombin", Biochim Biophys Acta 1987 Aug 21 ; 914(3): 275-282.).
  • Thrombin is bound to the fibrinogen-Sepharose column in 50 mM NaC 1/5 mM Tris-HCl (pH 7.4) at 22 degrees C and after an optional wash step is eluted with 250 mM NaCl/5 mM Tris-HCl (pH 7.4).
  • This assay detects the presence of enzymatically active thrombin in the sample.
  • a biological fluid sample such as cell lysates or a concentrated sample of proteins released into the culture medium, is electrophoresed under gentle non-reducing, non-denaturing conditions to maintain the secondary structure of the protein. Following PAGE the proteins in the gel are transferred to nitrocellulose, and the nitrocellulose paper is developed with a suitable chromogenic substrate such as S-2238.
  • Sample buffer (2X) 62.5mM Tris-HCL pH 6.8; 25% glycerol (may contain 20 mM benzamidine).
  • Running Buffer 25mM Tris; 192mM glycine; pH 8.3.
  • Transfer Buffer 48 mM Tris, 39 mM glycine, 20% methanol.
  • Standard enzyme ladder purified thrombin, activated protein C, factor XIa, and possibly other purified and distinct (in terms of size and/or charge) serine proteases that cleave the substrate of choice (at approximately 0.1 micrograms/mL each, but could be more or less concentrated, depending on the expected concentration of the enzyme in the sample).
  • the assay is specifically performed as follows:
  • Active thrombin in the sample appears as a band at the thrombin standard position. A single band suggests that it is probably active thrombin.
  • Thrombin and most enzymatically active thrombin variants cleave soluble fibrinogen to insoluble fibrin.
  • Fibrinogen (lg lyophilized, Kabi Vitrum, Sweden), dissolved in water to a concentration of 4 mg/mL, is filtered through a 0.8um syringe filter (Acrodisc 4188, GelmanSciences).
  • 0.8um syringe filter Acrodisc 4188, GelmanSciences.
  • Significantly lower concentrations of fibrinogen e.g., 0.1 mg/mL will also form a clot and may be more suitable for this simple assay.
  • Samples (100 microliters) containing WT thrombin or thrombin analogs (E-WE thrombin) are added to 96 well, flat bottom, clear microplate wells (Fisher brand 12565501). Once in the wells, 100 microliters fibrinogen solution is added, and the appearance of clots in the wells are observed visually (increase in turbidity). In this simple and thrombin-specific assay, fibrin forms a gel (clot) over time, and the clot that is readily detectable by the naked eye.
  • E-WE thrombin thrombin analogs
  • This assay may require higher thrombin concentrations to be useful, as fibrin inhibits thrombin (1 : 1), thus their ratio will have an effect on the outcome.
  • the 1 : 1 stoichiometric ratio of fibrinogen to thrombin is about 10: 1 by weight. Buffering of the sample can be important, as pH, salt, and other factors all affect thrombin activity and fibrin polymerization.
  • the assay could be potentially improved by adding calcium (2-5 mM), and some purified factor XIII (maybe 1 nM), as FXIII, once activated by thrombin, cross-links the fibrin fibers, making it a more turbid or denser gel.
  • a chromogenic substrate for thrombin could also be added to the fibrinogen, which would be cleaved and increase the light absorbance of the clot, and would be cleaved by thrombin that is already bound to fibrin.
  • a specific small molecule thrombin inhibitor could be used for a negative control.
  • a small molecule fibrinolysis inhibitor could also be added to reduce lysis of the clot (fibrinogen preps may be contaminated with plasminogen and plasminogen activators).
  • this peptide can be measured in the supernatant (clots may have to be spun to get the soup) and will correlate with the thrombin concentration.
  • Prothrombin is cleaved by thrombin; chromogenic substrate peptides that are analogs of the cleavage site, such as S-2238 (D-Phe-Pip-Arg-p-nitroanilide), can be used to test for this activity.
  • the substrate H-D-Phe-Pro-Arg-p-nitroanilide can also be used to assay prothrombin-cleaving activity, and the substrate H-D-Asp- Arg-Arg-p-nitroanilide is specific for activated protein C (Cantwell and Di Cera, "Rational design of a potent anticoagulant thrombin", J Biol Chem 2000 Dec 22; 275(51): 39827-39830; Epub 2000 Nov 1).
  • kinetic constants are determined for the cleavage of S-2238.
  • the release of /?-nitroaniline resulting from the hydrolysis of S-2238 is followed by measuring the rate of increase in absorbance at 405 nm. All absorbance measurements are made on a modified Cary Model 14 spectrophotometer.
  • the assays are carried out in polymethacrylate cuvettes at 37 degrees C in assay buffer (50 mM Tris-HCl, 0.15 M NaCl, 0.1% PEG, pH 7.4). Both the 10 riM thrombin stock solution and the 2-30 micromolar S-2238 stock solutions are prepared in assay buffer.
  • the thrombin solution contains 1 mg/ml bovine serum albumin.
  • the absorbance at 342 nm of 990 microliters of S-2238 stock is recorded, then 10 microliters of thrombin stock (10 nM) is added, and the absorbance at 405 nm is monitored for 5-10 minutes at 37 degrees C.
  • the amount of product formed is calculated by using an extinction coefficient of 9920 M "1 cm "1 for /?-nitroaniline at 405 nm, and the concentration of S-2238 is determined by using an extinction coefficient of 8270 M "1 cm "1 at 342 nm.
  • K m and k cat are calculated from triplicate assays by least-squares fit to a straight line of a plot of the inverse of the rate of /?-nitroaniline release against the inverse of the concentration of S-2238, using the program LINFIT (Bevington, "Data Reduction and Error Analysis for the Physical Sciences", McGraw-Hill New York 1969; 92-1 18).
  • thrombin variants The kinetic constants for the hydrolysis of S-2238 by thrombin variants are compared with k cat (97 ⁇ 8 s "1 ) and K m (2.71 ⁇ 0.25 micromolar) determined for wild-type thrombin (DiBella et cil., "Expression and folding of recombinant bovine prethrombin-2 and its activation to thrombin", J Biol Chem 1995 Jan 6; 270(1): 163-169).
  • kinetic constants are determined for the release of fibrinogen peptide A (FpA) from fibrinogen.
  • FpA fibrinogen peptide A
  • Approximately 0.01 NIH unit/ml (3 ng/ml) of wild-type thrombin or variant thrombin are incubated with 3.5 micromolar fibrinogen at 37 degrees C in 50 mM H 3 P0 4 , 100 mM NaCl, 0.1% PEG 8000, pH 7.5.
  • an aliquot of the reaction is quenched by adding 0.3 M perchloric acid.
  • the pH is raised to ⁇ 2 by adding 0.14 M NaOH.
  • An infinity time point (30 minutes) for the FpA release assay is determined using wild-type thrombin or variant thrombin at a concentration of 0.05 NIH unit/ml.
  • An LKB HPLC system consisting of a 2152 LC controller, a 2151 pump, a 2141 variable wavelength detector, a 2221 integrator, and a 2157 auto- sampler is used for the detection of FpA.
  • Kinetic constants and standard deviations are determined by least-squares fit to a straight line of a plot of -ln ⁇ ([FpA] ⁇ - [FpA] t )/([FpA] ⁇ - [FpA] 0 ) ⁇ against time using the program LINFIT, where [FpA] 0 is a baseline correction.
  • [FpA] 0 is a baseline correction.
  • k c K m is determined for the fibrinogen-cleaving activity of the thrombin variant and compared to that determined for wild-type thrombin (16.7 ⁇ 2.0 micromola 1 s "1 , DiBella 1995 supra).
  • the coordinated coexpression in yeast host cells of thrombin expression constructs is achieved by the following method.
  • the pJ1231-03C and pJ1234-03C vectors were used as the backbone part of the yeast expression constructs, as they contain elements necessary for plasmid maintenance in either E. coli (pUC origin of replication) or Saccharomyces cerevisiae (2-micron circle origin of replication, along with selectable markers useful in either host: KanR (E. coli) and Leu2 (yeast) in pJ1231-03C; AmpR (E. coli) and Ura3 (yeast) in pJ1234-03C.
  • the nucleotide sequences of the pJ1231-03C and pJ1234-03C vectors are shown as SEQ ID NOs 21 and 22, respectively.
  • the pJ1231-03C and pJ1234-03C vectors are treated with BfuAl restriction endonuclease (NEB, Catalog No. R0701S); fragments of the vectors that were not retained after BfuAl digestion comprise an expression promoter, the DasherGFP coding sequence, and the CYC1 terminator sequence.
  • PCR is performed on nucleotide sequences containing the coding sequences for the polypeptides to be expressed (such as expression constructs comprising the coding sequences for thrombin polypeptides), to create sequences that are then ligated into the BfuAl-cut pJ1231-03C and pJ1234-03C vectors.
  • the PCR-amplified fragments should contain the coding sequences for the transcriptional activators of the inducible promoters, for example, PrpR or AraC. All PCR reactions are performed using Platinum® Pfic DNA Polymerase (Life Technologies, Grand Island, New York, Catalog No. 11708021), and the QIAEX II DNA purification kit (QIAGEN, Catalog No. 20051) is used for purification of PCR-product and vector fragments, according to the manufacturers' instructions.
  • the PCR- amplified fragments When the PCR- amplified fragments have been prepared and cut with the appropriate restriction enzymes, they are ligated into the BfuAl-cut pJ1231-03C vector or the BfuAl-cut pJ1234-03C vector using T4 DNA ligase, to create the expression constructs that will be used in the yeast cells.
  • the ligase mixtures are transformed into E. coli DH5alpha cells and plated on LB agar plates with sufficient amounts of kanamycin or ampicillin to maintain the plasmids in the DH5 alpha cells.
  • Preparation of plasmid DNA is performed according to standard methods. (Optionally but preferably, the prepared plasmid DNA is used in sequencing reactions to confirm the sequences of the plasmid inserts.)
  • Competent INVSc-1 S. cerevisiae cells are prepared using the S.c. EasyCompTM Transformation Kit (Life Technologies, Catalog No. K5050-01) according to the manufacturer's instructions.
  • the INVSc-1 S. cerevisiae strain has the following genotype (MATa his3Al leu2 trpl-289 ura3-52/MATa his3Al leu2 trpl-289 ura3-52) and phenotype: His “ , Leu " , Trp " , Ura " .
  • a single colony from the INVSc-1 strain is inoculated in 10 milliliters of YPD medium (contains, per liter: 10 g yeast extract, 20 g peptone, 20 g glucose) and grown overnight at 30 degrees C in a shaking incubator at 250 rpm.
  • the overnight culture is diluted in 10 milliliters fresh YPD medium to an OD600 of 0.3 and grown until OD600 reached 0.8.
  • the cells are collected by centrifugation at 1500 rpm for 5 minutes at room temperature. After that, the cells are resuspended in 10 milliliters of Solution 1 (wash solution) and collected by centrifugation at 1500 rpm for 5 minutes at room temperature.
  • SC minimal medium contains, per liter: 6.7 g yeast nitrogen base, 20 g glucose, 0.05 g aspartic acid, 0.05 g histidine, 0.05 g isoleucine, 0.05 g methionine, 0.05 g phenylalanine, 0.05 g proline, 0.05 g serine, 0.05 g tyrosine, 0.05 g valine, 0.1 g adenine, 0.1 g arginine, 0.1 g cysteine, 0.1 g leucine (omitted in -Leu selective media), 0.1 g lysine, 0.1 g threonine, 0.1 g tryptophan, and 0.1 g uracil (omitted in -Ura selective media).
  • INVSc-1 cells transformed with pJ1231- based expression constructs are selected on plates without leucine at 30 degrees C for 48-72 hours.
  • INVSc-1 cells transformed with pJ1234-based expression constructs are grown under the same conditions on SC minimal agar plates without uracil.
  • INVSc-1 cells co-transformed simultaneously with pJ1231- and pJ1234-based expression constructs are selected under the same conditions on SC minimal agar plates without leucine or uracil. Cells from all colonies from each transformation are scraped and resuspended in 4 milliliters of liquid minimal SC medium in absence of the appropriate selective reagent and carbon source. OD 60 o in each culture was measured and normalized to 0.4 optical units.
  • Protein expression is induced by addition of an appropriate amount, such as 1 - 2%, sterile filtered inducer(s) (Sigma-Aldrich, St. Louis, Missouri, Catalog No. A3256-25G).
  • the time course for protein expression is 24 hours, at 30 degrees C in a shaking incubator at 250 rpm.
  • Cells from 0.5 milliliters of each pre-induced culture are collected by centrifugation, washed with 1 milliliter of de-ionized water and stored at -80 degrees C.
  • Samples from post- induced cultures are prepared in the same way.
  • Total protein extracts are prepared from pre- and post-induced cultures, resolved in 4-20% SDS-PAGE, and transferred to a PDVF membrane.
  • Expression level of target proteins was analyzed by Western blotting using antibodies such as anti-6xHis tag (for His- tagged thrombin polypeptides) and anti-Human IgG secondary antibodies.
  • Host cells containing L-arabinose-inducible and propionate-inducible expression constructs are grown to the desired density (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.
  • the concentration of L-arabinose necessary to induce expression is typically less than 2%.
  • 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%), and 0.001%.
  • concentrations of L-rhamnose or D-xylose necessary to induce expression of L-rhamnose-inducible or D-xylose-inducible promoters are tested similarly, with the tested concentrations ranging from 5% to 0.01%).
  • titration experiments can start at a 'standard' combination of inducer concentrations, which is 0.002%> of any of L-arabinose, L-rhamnose, or D-xylose, and/or 50 mM propionate, and test new combinations of inducer concentrations that vary from that of the 'standard' combination. Similar titration experiments can be performed with any combination of inducers used in an inducible coexpression system of the invention, including but not limited to L-arabinose, propionate, L- rhamnose, and D-xylose.
  • the cells After growth in the presence of inducers for 6 hours, 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.
  • 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 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-Rad 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 (fluores- cence/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.
  • the number and location of disulfide bonds in coexpressed protein 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 ah, "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 201 1 Nov 22).
  • a protease such as trypsin
  • any free cysteine residues are first blocked by alkylation: the coexpressed 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, and then is separated by non-reducing SDS-PAGE using precast gels. Alternatively, the coexpressed protein is incubated in the gel after electrophoresis with iodoacetamide, or without as a control.
  • the alkylating agent iodoacetamide 5 mM
  • the coexpressed protein is incubated in the gel after electrophoresis with iodoacetamide, 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.
  • Peptides are injected onto a 1 mm x 8 mm trap column (Michrom BioResources, 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).
  • Peptides are analyzed using a LTQ Velos linear ion trap with an ETD source (Thermo Scientific, San Jose, CA). Electrospray ionization is performed using a Captive Spray source (Michrom Bioresources, Inc.). 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, and 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 s.
  • 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 Scientific) using ZSA charge state analysis. Matching of MS2 and MS3 scans to peptide sequences is performed by Sequest (V27, Rev 12, Thermo 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. 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 inducible coexpression 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.
  • Coexpressed 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 Ready-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. 4. Incubate for five minutes at room temperature. Ready-Lyse Lysozyme is optimally active at room temperature. Lysis at lower temperatures (0°C-4°C) requires additional incubation time; at such temperatures incubation times are extended 2- to 4-fold.
  • PeriPreps Periplasting Buffer 200 mM Tris-HCl pH 7.5, 20% sucrose, 1 mM EDTA, and 30 U/microliter Ready-Lyse
  • 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.
  • the supernatant containing the periplasmic fraction is transferred to a clean tube.
  • 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. The addition of EDTA to the lysate to inactivate OmniCleave Endonuclease, likewise, may interfere with subsequent assay or use of the purified protein.
  • nuclease is to be added, 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 KCl, 1 mM EDTA, and 0.1% deoxycholate) for each milliliter of Lysis Buffer needed in Step 10.
  • PeriPreps Lysis Buffer (10 mM Tris-HCl pH 7.5, 50 mM KCl, 1 mM EDTA, and 0.1% deoxycholate
  • 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.
  • 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, using the default parameter settings described at the blast.ncbi.nlm.nih.gov/Blast.cgi website.
  • 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 procédés de production de thrombine à l'aide de systèmes de co-expression coordonnée, et notamment à l'aide de systèmes de co-expression déclenchable permettant de déclencher de façon commandée l'expression de chaque produit génétique nécessaire pour la production de thrombine. L'invention concerne également la thrombine produite au moyen de ce procédé.
PCT/US2014/014968 2012-08-05 2014-02-05 Co-expression coordonnée de la thrombine WO2015020690A1 (fr)

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US14/909,707 US10508270B2 (en) 2012-08-05 2014-02-05 Coordinated coexpression of thrombin
EP14834543.2A EP3030651A4 (fr) 2013-08-05 2014-02-05 Co-expression coordonnée de la thrombine
US14/740,475 US20150353940A1 (en) 2013-08-05 2015-06-16 Vectors for use in an inducible coexpression system
US15/261,984 US11371048B2 (en) 2012-08-05 2016-09-11 Vectors for use in an inducible coexpression system
US16/675,102 US20200063116A1 (en) 2012-08-05 2019-11-05 Coordinated coexpression of thrombin
US17/748,734 US20220364104A1 (en) 2012-08-05 2022-05-19 Vectors for use in an inducible coexpression system
US18/310,317 US20230340501A1 (en) 2012-08-05 2023-05-01 Coordinated coexpression of thrombin

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US14/909,707 A-371-Of-International US10508270B2 (en) 2012-08-05 2014-02-05 Coordinated coexpression of thrombin
US14/740,475 Continuation-In-Part US20150353940A1 (en) 2012-08-05 2015-06-16 Vectors for use in an inducible coexpression system
US16/675,102 Continuation US20200063116A1 (en) 2012-08-05 2019-11-05 Coordinated coexpression of thrombin

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Cited By (11)

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US11371048B2 (en) 2012-08-05 2022-06-28 Absci Llc Vectors for use in an inducible coexpression system
WO2016205570A1 (fr) * 2015-06-16 2016-12-22 Absci, Llc Vecteurs destinés à être utilisés dans un système de co-expression inductible
CN107849572A (zh) * 2015-06-16 2018-03-27 Absci有限责任公司 用于可诱导共表达系统中的载体
JP2018518957A (ja) * 2015-06-16 2018-07-19 エイビーエスシーアイ・エルエルシー 誘導性共発現系における使用のためのベクター
EP3310919A4 (fr) * 2015-06-16 2019-01-09 Absci, LLC Vecteurs destinés à être utilisés dans un système de co-expression inductible
JP2021153600A (ja) * 2015-06-16 2021-10-07 エイビーエスシーアイ・エルエルシー 誘導性共発現系における使用のためのベクター
AU2016278242B2 (en) * 2015-06-16 2022-06-09 Absci Corporation Vectors for use in an inducible coexpression system
EP4039810A1 (fr) * 2015-06-16 2022-08-10 Absci, LLC Vecteurs destinés à être utilisés dans un système de co-expression inductible
IL256094B1 (en) * 2015-06-16 2023-11-01 Absci Llc Vectors for use in an inducible reciprocal expression system
IL256094B2 (en) * 2015-06-16 2024-03-01 Absci Llc Vectors for use in an inducible reciprocal expression system
CN115850517A (zh) * 2022-11-14 2023-03-28 沈阳药科大学 一种提高GLP-1 Fc融合蛋白产量的方法

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