Patent Grant
10287330
The present invention provides novel methods of producing diphtheria toxin. In particular, the present invention provides novel methods of producing nontoxic forms of diphtheria toxin, e.g., CRM197. The present invention also provides novel compositions comprising diphtheria toxin or nontoxic forms of diphtheria toxin, e.g., CRM197.
The CRM197 protein is a safe and effective T-cell dependent carrier for saccharides and is currently being used in many different vaccine formulations called conjugate vaccines. Diphtheria toxin is a protein exotoxin produced by the bacterium Corynebacterium diphtheriae upon infection with the phage 0197. Both, Diphtheria toxin (“DT”) and CRM197 are components of many vaccines, like for example against Bordatella pertussis, Clostridium tetani, C. diphtheriae, hepatitis B virus, and Haemophilus influenza type B (WO 9324148, WO 9700697, WO 02055105). In addition there has been a growing interest in CRM197 because of its potential antitumor activity relating to its capacity to bind the soluble form of HB-EGF (US 2006/0270600A1).
CRM197 is produced by C. diphtheriae infected by the non-toxigenic phage β197tox. β197tox was created by nitrosoguanidine mutagenesis of the toxigenic corynephage β (Uchida, T. et al. 1971, Nature New Biology 233:8-11). The CRM197 protein is a nontoxic form of diphtheria toxin but is immunologically indistinguishable from the diphtheria toxin. DT has a mass of 58.350 kDa (CRM197=58.415 kDa) and consists of the N-terminal A and the C-terminal B domains (21 and 37 kDa) which are linked by a disulfide bridge connecting Cys186 and Cys201. The A fragment is toxic after being released from its disulfide-bonded partner, the B fragment. Nicking of the holotoxin by mild proteolysis at the connecting peptide at positions 191-3 is a prerequisite for the A fragment activation. The B fragment has no apparent enzymatic activity but is required for toxicity, probably due to targeting the holotoxin to the target cell membranes (Broker M, Costantino P, De Tora L, McIntosh E D, Rappuoli R: Biochemical and biological characteristics of cross-reacting material 197 (CRM197), a non-toxic mutant of diphtheria toxin: use as a conjugation protein in vaccines and other potential clinical applications. Biologicals, 2011, 39(4):195-204.)
Infected C. diphtheriae cultures secrete the CRM197 protein across the cytoplasmic membrane out of the cell into the culture medium. The CRM197 protein has about the same molecular weight as the diphtheria toxin but differs therefrom by a single base change (guanine to adenine) in the structural gene. This single base change causes an amino acid substitution (glutamic acid for glycine, G52E) in the mature protein and eliminates the toxic properties of diphtheria toxin (Giannini G, Rappuoli R, Ratti G: The amino-acid sequence of two non-toxic mutants of diphtheria toxin: CRM45 and CRM197. Nucleic Acids Res 1984, 12(10):4063-4069).
Methods of preparing DT and CRM197 are described in U.S. Pat. No. 4,709,017, U.S. Pat. No. 5,843,711, U.S. Pat. No. 5,601,827, and U.S. Pat. No. 5,917,017. There are currently three different systems used for industrial preparation of CRM197. Two systems are based on the use of phage infected C. diphtheriae cells. The most recent development constitutes a recombinant expression system in Pseudomonas fluorescens. The method employs a secretion approach to the periplasm in a genetically optimized P. fluorescens strain using a CRM197 gene equipped with a signal peptide for secretion into the periplasm (US20110287443).
For example, diphtheria toxin is isolated from cultures of C. diphtheriae strain C7 (B197) and/or C. diphtheriae strain C7 (B197) pPx350 grown in a casamino acids and yeast extract-based medium under aerobic conditions. Adjustment of media components were shown to improve yields (U.S. Pat. No. 4,925,792, WO 2006 100108). CRM197 or DT are harvested from the supernatant of the culture, and concentrated by ultrafiltration. Ammonium sulfate precipitation is a first, and anionic exchange chromatography a second purification step.
However, production of significant quantities of the CRM197 protein for use in vaccines has been hindered due to low protein abundance (WO 2006 100108).
Techniques have been developed to bolster the production of CRM proteins using double lysogens (Isolation and characterization of C. diphtheriae nontandem double lysogens hyperproducing CRM197. R Rappuoli, Appl. Environ, Microbiol. September 1983 46:560-564; U.S. Pat. No. 4,925,792 issued to R. Rappuoli; and Integration of corynebacteriophages beta tox+, omega tox+, and gamma tox− into two attachment sites on the C. diphtheriae chromosome. R Rappuoli, J L Michel, and JR Murphy; J. Bacteriol. March 1983 153:1202-4210) of the nontoxigenic corynephage β197. Rappuoli reports yields of CRM197 from double and triple lysogens up to three fold higher than from the single lysogens. The production levels of CRM197 by single lysogens are adequate but economically unsatisfactory for the production of vaccines which utilize CRM197 protein. It is important to note that the construction of double and triple lysogenic strains in order to increase expression efficiency in C. diphtheria e is a long process which requires a laborious screening phase.
Plasmids were developed for recombinant expression of CRM197 in C. diphtheriae (U.S. Pat. No. 5,614,382, 1995/5614382_1997). This makes it possible to increase the number of copies of the gene (up to 5-10 per cell) without having to select pluri-lysogenic bacterial strains.
As in the case of the Corynebacterium strains infected by the phage β197tox, CRM197 is expressed in special culture media with a low ferrous content. Despite a reduction in the amount of time required for the genetic handling of the bacterial strain, the output of CRM197 does not increase dramatically by comparison with the use of double lysogenes.
Alternative expression host cells for DT included a Salmonella typhi vaccine strain cvd 908-htra (Orr N, Galen J E, Levine M M: Expression and immunogenicity of a mutant diphtheria toxin molecule, CRM197, and its fragments in S. typhi vaccine strain CVD 908-htrA. Infect Immun 1999, 67(8):4290-4294). Salmonella is a Gram negative bacterium and similar expression host as E. coli. Expression levels from various constructs (with, without signal peptide) in cvd 908-htra were low and solubility and immunogenicity were poor. Utilizing the alternative, non-Sec dependent translocation system of the hemolysin operon improved expression of soluble DT, but levels were still low.
Reports for production of CRM197 in E. coli show low yields of soluble CRM197 and formation of insoluble product in inclusion bodies. Truncation approaches have been used in an attempt to enhance expression to higher levels. (Bishai W R, Miyanohara A, Murphy J R: Cloning and expression in E. coli of three fragments of diphtheria toxin truncated within fragment B. Journal of Bacteriology 1987, 169(4):1554-1563)
A single strand expression plasmid for CRM197 containing the mutated diphtheria toxin gene encoding CRM197 was used for expression in E. coli (Bishai W R, Rappuoli R, Murphy J R: High-level expression of a proteolytically sensitive diphtheria toxin fragment in Escherichia coli. Journal of Bacteriology 1987, 169(11):5140-5151; Bishai 1987). In this publication, transcription of CRM197 was controlled by the endogenous and constitutive Ptox promotor. In addition, DT C-terminally fused to the alpha melanocyte stimulating hormone (“ABM508”) was expressed by the heat inducible PLambda promoter or the Ptac promoter for expression.
Bishai 1987 speculated that jamming of the secretion apparatus due to high level protein induction caused a growth stop after induction of expression of periplasmic DT/CRM197 variants. This can be a general problem in periplasmic protein expression that has been observed and resulted in low volumetric yields of protein previously (Benson S A, Hall M N, Silhavy T J: Genetic analysis of protein export in E. coli K12. Annual Review of Biochemistry 1985, 54:101-134). Jamming of the secretion apparatus and formation of insoluble protein suggested an inability of the E. coli cells to provide a productive translocation and folding environment for CRM197 biogenesis.
As a consequence, Bishai 1987 reasoned that cytoplasmic expression would avoid the translocon jamming. Thus, Bishai 1987 removed the signal peptide for directing the expression into the cytoplasm. Only at low temperatures and when cytoplasmic proteases were deleted, did the cytoplasmic expression constructs yield soluble product. Production was inefficient and lead to aggregates at elevated temperatures, and when proteases were present.
Bishai 1987 failed to show production of high levels of soluble protein CRM197 fusion protein, i.e., with signal peptide for periplasmic targeting. In a Coomassie stained SDS polyacrylamide gel, extracts containing the ABM508 expression construct showed an intense protein band corresponding to ABM508, whereas cells expressing CRM197 expressed from the natural promoter with the wild type signal peptide do not show an obvious band for CRM197 at the expected size of 58 kDa.
Thus, periplasmic expression was not considered an efficient production strategy and to date there is no efficient E. coli periplasmic expression system established for production of soluble and correctly folded CRM197 or DT.
A production system which is based on the cytoplasmic expression of insoluble CRM197 in inclusion bodies followed by solubilization, purification and refolding of the protein is provided by the first E. coli based expression system for CRM197 (WO2010 150230). Wild-type CRM197 without additional amino acids can only be obtained with this system when an additional proteolysis step is applied.
Signal peptides induce protein secretion to the periplasm and have various effects on protein biogenesis. (Powers T, Walter P: Co-translational protein targeting catalyzed by the E. coli signal recognition particle and its receptor. The EMBO Journal 1997, 16(16):4880-4886.) (Schierle C F, Berkmen M, Huber D, Kumamoto C, Boyd D, Beckwith J: The DsbA signal sequence directs efficient, cotranslational export of passenger proteins to the E. coli periplasm via the signal recognition particle pathway. Journal of Bacteriology 2003, 185(19):5706-5713).
Provided herein is a method to produce soluble, folded, full length CRM197 at high yields (e.g., at least 0.5 mg/1) in E. coli expression strains. In particular, a signal peptide is used to direct secretion of the protein into the periplasmic space.
Provided herein are methods for producing CRM197 wherein the method comprises culturing an E. coli cell comprising a nucleic acid encoding CRM197 wherein CRM197 is fused to a heterologous signal peptide that targets CRM197 to the periplasm of the E. coli cell. In more specific embodiments, the wild type signal peptide of CRM197 has been deleted. In even more specific embodiments, the wild type signal peptide of CRM197 has been replaced by the heterologous signal peptide. The heterologous signal peptide can be selected from the group consisting of the signal peptide from E. coli heat-labile enterotoxin, E. coli outer membrane porin (OmpA), E. coli maltose binding protein (MalE), E. carotovorans pectate lyase (PelB), and Bacillus sp. endoxylanase (XynA). CRM197 can be produced at a concentration of at least 5, 10, 25, 50, 75, 100, 125, 125, 150, 175, 200, 225, 250, 300, 400, 500, 600, 700, 800, 900, or at least 1000 mg protein per liter culture medium. At least 50% of the produced protein is properly folded as determined by circular dichroism. At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, or at least 99.9% of the produced protein is properly folded. At least 50% of the produced protein is not present in aggregates. At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, or at least 99.9% of the produced protein is not present in aggregates. At least 50% of the produced protein is soluble. At least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99%, 99.5%, 99.8%, or at least 99.9% of the produced protein is soluble.
In certain embodiments, the heterologous nucleotide sequence encodes a cleavage site between the signal peptide that targets CRM197 to the periplasm and the CRM197 protein wherein the cleavage site comprises the amino acid sequence aa1-aa2-aa3-(cleavage site)-aa4-aa5-aa6-aa7-aa8, wherein
In certain specific embodiments, the heterologous nucleotide sequence encodes the protein of SEQ ID NO: 1 or 2. The heterologous nucleotide sequence can be operatively linked to a promoter selected from the group consisting of the 1-arabinose inducible araBAD promoter (PBAD), the lac promoter, the 1-rhamnose inducible rhaP BAD promoter, the T7 RNA polymerase promoter, the trc and tac promoter, the lambda phage promoter p L, and the anhydrotetracycline-inducible tetA promoter/operator.
In certain embodiments, the nucleic acid encoding CRM197 is inserted in a high copy expression plasmid. The high copy expression plasmid can be pEC415, pBR322, pBAD, pET series, pUC series, pACT3, pEXT22, pEXT20, pBLUESCRIPT series, pGEM series.
In certain embodiments, the expression of CRM197 can be induced at a culture density of OD600>0.3. Specifically, the expression of CRM197 can be induced at a culture density of OD600>0.5, >1, or >1.5.
CRM197 can be expressed at a temperature of 37° C. CRM197 can be expressed at a temperature of 20, 25, 30, 32, or 35° C.
In certain embodiments, at least 50% of CRM197 protein that has been produced in accordance with the methods provided herein have an N-terminus of ADDV (SEQ ID NO: 7), GADDV (SEQ ID NO: 9), or MGADDV (SEQ ID NO: 12). More specifically, at least 60%, 70%, 80%, 90%, 95%, 98%, 99%, at least 99.5% or 100% of the expressed CRM197 have an N-terminus of ADDV (SEQ ID NO: 7), GADDV (SEQ ID NO: 9), or MGADDV (SEQ ID NO: 12). In certain embodiments, at least 50% of the expressed CRM197 have a disulfide bond between Cys186 and Cys201. At least 60%, 70%, 80%, 90%, 95%, 98%, 99%, at least 99.5% or 100% of the expressed CRM197 have a disulfide bond between Cys186 and Cys201.
DT-Diphtheria Toxin
CRM197-Cross reactive material 197, DT with a mutation of glycine 52 to glutamate, G52E
AMB508-fusion protein consisting of alpha melanocyte-stimulating hormone sequence fused to the CRM197 sequence
β197tox-non-toxigenic corynephage 197
preprotein-protein including the signal peptide
mature protein, processed protein-protein after signal peptide cleavage
IPTG-Isopropyl-β-D-thiogalactopyranosid, inducer for lac, trc, and related promoters, to be added to the growth media at induction time
ParaBAD-promoter of the araBAD operon, inducible by the addition of L-arabinose to the growth media
Provided herein are methods for the expression of diphtheria toxin and nontoxic forms of diphtheria toxin, e.g., CRM197. More specifically, provided herein are methods for the expression of CRM197 and secretion of CRM197 into the periplasmic space of E. coli cells in a soluble and correctly folded form.
In certain embodiments, CRM197 is expressed using a heterologous signal peptide that targets CRM197 in the periplasmic space of the host cell. In certain more specific embodiments, the host cell is E. coli. An expression cassette containing the CRM197 gene and a heterologous signal peptide can be constructed using standard molecular biology techniques. Specifically, the wild type signal peptide of CRM197 is deleted and a heterologous signal peptide is introduced instead. On a nucleic acid level, care should be taken that the sequence encoding the signal peptide is cloned in frame with the nucleic acid encoding the remainder of CRM197. In certain specific embodiments, the heterologous signal peptide replaces the wild type signal peptide. In other embodiments, the wild type signal peptide is deleted or functionally inactivated and the heterologous signal peptide is introduced at a different location of the protein. In certain embodiments, a proteolytic cleavage site is introduced between the signal peptide and the remainder of the molecule. The proteolytic cleavage site can be recognized and cleaved in the periplasm of the host cell. In certain more specific embodiments, the signal peptidase is recombinantly expressed in the host cell.
In certain embodiments, the following parameters can affect the expression of the protein of interest. More detailed information on these various aspects are provided in the following sections.
The nucleic acid can encode the mature, secreted CRM197 (SEQ ID NO:6):
In general, a method provided herein is conducted as follows. First, an expression plasmid as described herein is introduced into a host cell (e.g., E. coli or Salmonella sp expression strain). The transformation mix can be plated on rich media supplemented with the antibiotic for which the expression plasmid carries a resistance marker. A single colony can be used to inoculate a small culture volume (e.g., 5 ml) consisting of, e.g., TB medium, or a similar rich medium containing glycerol as a carbon source and lacking or supplemented with the suitable antibiotic. The culture can then be incubated between 20-35° C. until stationary phase and then diluted to fresh medium of identical or similar composition, pre-warmed at 20-35° C., at a ratio of 1:50 to 1:100. The fresh culture can then be grown to exponential growth phase (OD600 of 0.6-1.2) and expression is induced by the addition of the appropriate inducer, which depends on the promoter used in the expression plasmid. Examples of inducers include arabinose or a different chemical of physical condition for high level protein induction. Then, expression is continued. In certain embodiments, expression is continued before harvesting for at least 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, or at least 30 hours.
Samples can be taken at any time. At all-time points the amount of soluble CRM197 formed is evaluated by analysis via SDS-PAGE and Coomassie staining of periplasmic extracts. A comparison to commercial CRM197 of known concentration allows estimating the yield.
In certain embodiments, the following control is used to determine that the expressed CRM197 protein is soluble. Without being bound by theory, such soluble protein is correctly folded. CRM197 detected at around 58 kDa in periplasmic extracts prepared by the sucrose-lysozyme method (Kowarik M, Young N M, Numao S, Schulz B L, Hug I, Callewaert N, Mills D C, Watson D C, Hernandez M, Kelly J F et al: Definition of the bacterial N-glycosylation site consensus sequence. The EMBO journal 2006, 25(9):1957-1966) can be used as a standard for soluble protein.
In certain embodiments, provided herein are methods that result in a concentration of properly folded CRM197 of at least 1 mg/l, 2 mg/l, 3 mg/l, 4 mg/l, 5 mg/l, 6 mg/l, 7 mg/l, 8 mg/l, 9 mg/l, 10 mg/l, 11 mg/l, 12 mg/l, 13 mg/l, 14 mg/l, 15 mg/l, 20 mg/l, 25 mg/l, 50 mg/l, 75 mg/l, or at least 100 mg/l.
In certain embodiments, depending on the signal peptide cleavage site sequence, the N terminus of CRM197 can be ADDV (SEQ ID NO: 7), or GADDV (SEQ ID NO: 9), or MGADDV (SEQ ID NO: 12).
In certain embodiments, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the resulting CRM197 proteins have a disulfide bond between Cys186 and Cys201 connecting A and B fragments. The presence of this disulfide bond can be demonstrated using a thiol assay (Hansen R E, Ostergaard H, Norgaard P, Winther J R: Quantification of protein thiols and dithiols in the picomolar range using sodium borohydride and 4,4′-dithiodipyridine. Anal Biochem 2007, 363(1):77-82).
In certain embodiments, at least 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, 99%, or 100% of the resulting CRM197 proteins are soluble and are not present in aggregates.
The present invention describes production methods for DT and CRM197. Illustrative protein sequences for CRM197 are provided as SEQ ID NOs: 3 and 4, and the corresponding full length expression plasmid DNA sequences are SEQ ID NOs: 1 and 2.
Various secretory pathways from the cytoplasm to the periplasm exist in E. coli. These pathways include the Sec pathway, the SRP dependent pathway, and the twin arginine pathway for secretion. The TatABC pore is thought to be responsible for the secretion of folded proteins. Without being bound by theory, the signal peptide can determine which secretory pathway is chosen by the cell. (Driessen A J, Nouwen N: Protein translocation across the bacterial cytoplasmic membrane. Annual review of biochemistry 2008, 77:643-667.)
In certain embodiments, the heterologous signal peptide for use with the present methods is an amino-terminal hydrophobic signal sequences that is cleaved during the translocation process. In certain embodiments, conditions for a method provided herein are chosen such that the protein does not fold into its stable three-dimensional structure in the cytoplasm of the host cell. Without being bound by theory, folding in the cytoplasm may prevent export. In certain other embodiments, the heterologous signal peptide encodes an arginine signature that targets the protein to the twin arginine pathway for secretion.
In certain embodiments, an unfolded protein may be maintained in an export competent state in several different ways: (i) the heterologous signal peptide can be chosen such that the protein may be translocated across a membrane simultaneously with translation of the protein, thus ensuring that not even its secondary structures are formed in the cytoplasm due to the absence of amino acid polymer; (ii) chaperones or antifolding factors that prevent folding in the cytoplasm (Randall L L, Topping T B, Smith V F, Diamond D L, Hardy S J: SecB: a chaperone from E. coli. Methods Enzymol 1998, 290:444-459.) can be provided; (iii) the heterologous signal sequences is chosen and/or inserted such that it acts as intrapolypeptide chaperones to prevent rapid folding; and/or (iv) the DT or CRM197 is modified such that it contains features in its final structure (e.g., disulfide bonds) that do not form in the environment of the cytoplasm so that the proteins cannot attain their final folded conformations in the cytoplasm.
5.2.1 Signal Peptides
Illustrative heterologous signal peptides that can be used with the methods provided herein are: the E. coli DsbA signal sequence, the MalE, OmpA, and PelB signal peptides. Without being bound by theory, the choice of signal peptide can determine the secretion route, e.g., SRP-dependent vs. SecB dependent route to the translocon. The optimal expression conditions may differ for different targeting pathways. There are reports claiming technologies that allow the identification of the targeted secretion pathway (Marrichi M, Camacho L, Russell D G, DeLisa M P: Genetic toggling of alkaline phosphatase folding reveals signal peptides for all major modes of transport across the inner membrane of bacteria. J Biol Chem 2008, 283(50):35223-35235).
The preferred signal peptides are selected from known and predicted, secreted proteins which are efficiently exported to the periplasm of E. coli via co-translational pathways. Among others, the signal peptides of E. coli heat-labile enterotoxin, E. coli outer membrane porin A (OmpA), E. coli maltose binding protein (MalE), E. carotovorans pectate lyase (PelB), or Bacillus sp. endoxylanase (XynA) can be used.
5.2.2 Cleavage Sites
Without being bound by theory, signal peptides are cleaved off the preprotein by a signal peptidase, and in E. coli there are SPaseI and II. SPaseI is cleaving most soluble and some membrane protein signal peptides, whereas SPaseII cleaves signal peptides from lipoproteins. SPaseI is the signal peptidase responsible in the presented invention. SPaseI usage could be determined (Paetzel M, Karla A, Strynadka N C, Dalbey R E: Signal peptidases. Chemical reviews 2002, 102(12):4549-4580).
Without being bound by theory, cleavage site locations are defined by i) the structure of the characteristic signal peptide organization with a hydrophobic core, a charged N terminus and a hydrophilic C terminus, and ii) by the primary sequence around the cleavage position (often A-X-A) (Heijne G: The distribution of positively charged residues in bacterial inner membrane proteins correlates with the trans-membrane topology. The EMBO journal 1986, 5(11):3021-3027.). Both parameters are well understood and prediction programs have a high accuracy (Petersen T N, Brunak S, von Heijne G, Nielsen H: SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods 2011, 8(10):785-786). The program SignalP 4.0 server provides a cleavage probability based on the sequence of the first 70 amino acids of the pre-protein. In certain embodiments, engineered cleavage sites for use with the methods provided herein have a Y score of at least 0.4, 0.5, 0.6, 0.7, 0.72. 0.75, 0.8, 0.85, 0.9, or at least 0.95.
In certain embodiments, the signal peptide cleavage site is designed such that the predicted N-terminus is the N-terminus of the naturally existing protein. In other embodiments, the signal peptides and N terminus of the CRM197 are designed such that the N terminus is as close to the natively found N terminus as possible.
The natural N terminus after signal peptide cleavage of the CRM197 protein is GADDV (SEQ ID NO: 9) . . . (Bell C E, Eisenberg D: Crystal structure of nucleotide-free diphtheria toxin. Biochemistry 1997, 36(3):481-488). In certain specific embodiments, the N-terminus of CRM197 expressed in E. coli using the DsbA signal peptide can be:
Other cleavage sequences, and signal peptide cleavage site combinations are set forth in Table 2 below.
A vast variety of expression vectors is known for recombinant expression in E. coli cells. In principle, any vector backbone can be used. Illustrative vectors are: pEC415 (Schulz H, Hennecke H, Thony-Meyer L: Prototype of a heme chaperone essential for cytochrome c maturation. Science 1998, 281(5380):1197-1200), pBR322 (Bolivar F, Rodriguez R L, Greene P J, Betlach M C, Heyneker H L, Boyer H W, Crosa J H, Falkow S: Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 1977, 2(2):95-113), pBAD (Invitrogen corporation, Carlsbad, Calif.), pET series (Invitrogen), pUC series (Lin-Chao S, Chen W T, Wong T T: High copy number of the pUC plasmid results from a Rom/Rop-suppressible point mutation in RNA II. Mol Microbiol 1992, 6(22):3385-3393), pACT3, pEXT22, pEXT20 (Dykxhoom D M, St Pierre R, Linn T: A set of compatible tac promoter expression vectors. Gene 1996, 177(1-2):133-136.), pBLUESCRIPT series (Stratagene, Agilent Technologies, Santa Clara, Calif.), pGEM series (Promega Corp., Madison, Wis.). All these vectors could be used for cloning the expression cassette of the preprotein under control of an inducible promoter.
Illustrative plasmids are provided as SEQ ID NOs: 1 and 2. The vector backbone is based on pBR322 containing a medium to high copy pMB1 origin of replication, an ampicillin resistance cassette which can be exchanged by a kanamycin cassette, the regulon of the araBAD operon encoding the AraC repressor and the araBAD promoter for high level protein expression induction.
In certain embodiments, a target protein, e.g., CRM197 or DT, is expressed from chromosomally integrated constructs. This strategy requires additional technologies which are well known to those skilled in the art and would result in a genome-integrated expression construct consisting of the same elements as an expression plasmid but not requiring the selection cassette (only for selection upon genomic integration) and the origin of replication.
Among well-known high expression inducible promoters, any can be used that is functional at the temperature for expression of the protein of interest. In certain embodiments, a promoter to be used with the methods provided herein is active below the temperature of 37° C., below 36° C., 35° C., 34° C., 33° C., 32° C., 31° C., or below 30° C. The following list contains illustrative bacterial expression promoters that can be used with the methods provided herein (Table 1):
E.
coli
E.
coli
E.
coli
E.
coli
E.
coli
E.
coli
E.
coli
E.
coli
E.
coli
E.
coli
E.
coli
E.
coli
E.
coli
E.
coli
Salmonella
Bacillus
Vitreoscilla
Culture medium for protein production can be any defined, semi-defined or complex medium suitable for over-expression of recombinant proteins in E. coli. A rich complex medium like terrific broth (TB) is preferred, but defined mineral salts media may also be used. Terrific broth is composed of 24 g/l yeast extract, 12 g/l tryptone or peptone (i.e. proteolytically digested casein, soy protein or other protein), and 4% (v/v) glycerol. In addition, the medium is buffered.
In certain specific embodiments, the concentration of Magnesium ions is at most 10 nM, 50 nM, 100 nM, 250 nM, 500 nM, 750 nM, or at most 1 mM. In certain specific embodiments, no Magnesium is added. In certain specific embodiments, no MgCl2 is added to the culture medium.
In certain specific embodiments, the pH of the culture medium is between 6 and 9. In certain specific conditions, yeast extract can be present in the culture medium at a concentration of between 10-30 g/l. In certain specific embodiments, the culture medium comprises glycerol from 2.5% to 10%. In certain other embodiments, the culture medium comprises glycerol at least 5%, 10%, 15%, or at least 20%.
Expression cultures before induction can be grown at different temperatures, for example, temperatures ranging from 4-35° C. or 18-37° C. In certain embodiments, expression cultures before induction are grown at a temperature within the range of 18-20° C., 20-22° C., 22-24° C., 24-26° C., 26-28° C., 28-30° C., 30-32° C., 32-34° C., or 34-36° C. In certain embodiments, expression cultures before induction are grown at a temperature of about 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., or 37° C.
Cultivation temperatures after induction can fall into certain ranges, for example, temperatures ranging from 4-35° C. or 18-37° C., and can be different from the before induction conditions. For example, a pre-induction culture can be grown at higher temperatures, e.g., a temperature described above, and then shifted to a lower temperature, e.g., a temperature in the range of 15-30° C., for production. In certain embodiments, cultures after induction are grown at a temperature within the range of 18-20° C., 20-22° C., 22-24° C., 24-26° C., 26-28° C., 28-30° C., 30-32° C., 32-34° C., or 34-36° C. In a specific embodiment, said temperature falls within a range that is lower than the range at which the pre-induction culture is grown. In certain embodiments, cultures after induction are grown at a temperature of about 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C., 25° C., 26° C., 27° C., 28° C., 29° C., 30° C., 31° C., 32° C., 33° C., 34° C., 35° C., 36° C., or 37° C. In a specific embodiment, said temperature falls within a range that is lower than the range at which the pre-induction culture is grown.
Depending on the construct, expression time can be from 2-20 hrs. Inducer concentrations are, dependent on the promoter, from 0.01 to 1% (w/v) arabinose (ParaBAD), or from 10 to 1000 μM IPTG. Induction can be done at OD600 values obtained during fermentation between 0.3 to 1.5 in shake flask cultures, and at OD600 between 5 to 200 in bioreactor fermentations. In certain specific embodiments, induction is done at an OD600 of between 5 and 50, 25 and 75, 50 and 100, 75 and 125, 100 and 150, 125 and 175, 150 and 200, or 175 and 200. In certain embodiments, induction is done at the beginning of the log phase in shake flask. Bioreactor fermentations may be done at constant pO2 values ranging from 0% to 40%. pO2 regulation may be done by regulating stirrer speed or aeration rate.
In certain embodiments, the promoter is inducible with arabinose; arabinose concentrations can be at least 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or at least 1% (w/v) arabinose. In certain embodiments, concentration of the inducer arabinose is at most 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or at most 1% (w/v) arabinose
In certain embodiments, the promoter is inducible with IPTG; IPTG concentrations can be at least 10, 25, 50, 75, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900 or at least 1000 μM IPTG. In certain embodiments, concentration of the inducer IPTG is at most 10, 25, 50, 75, 100, 200, 250, 300, 400, 500, 600, 700, 750, 800, 900 or at most 1000 μM IPTG.
In certain embodiments, expression is performed in shake flask cultures. OD600 values at the time of induction are at least 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, or at least 1.5 in shake flask cultures. In certain embodiments, OD600 values at the time of induction are at most 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, or at most 1.5 in shake flask cultures.
In certain embodiments, expression is performed in bioreactor fermentations. OD600 values at the time of induction are at least 5, 10, 15, 20, 25, 50, 75, or at least 100 in bioreactor fermentations. In certain embodiments, OD600 values at the time of induction are at most 5, 10, 15, 20, 25, 50, 75, or at most 100 in bioreactor fermentations.
Bioreactor fermentations can be performed at constant pO2 values of at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, or at least 40%. In certain embodiments, bioreactor fermentations can be performed at constant pO2 values of at most 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, or at most 40%.
Expression strains for recombinant production of the target protein can be but are not limited to E. coli K12 and B strains, like W3110, DB1, DH5a, BL21, BL21(DE3), C43, JM109, JM101, JM110, and derivatives thereof (Huang C J, Lin H, Yang X: Industrial production of recombinant therapeutics in E. coli and its recent advancements. Journal of Industrial Microbiology & Biotechnology 2012, 39(3):383-399). Host cells may be chromosomally modified to accommodate optimal expression of the CRM197 protein. For example, periplasmic proteases like DepP, Prc, Spr, and/or protease III may be deleted in production strains. Deletions may be useful alone or in combinations with other proteases. In addition, suppressor mutations like for example sprW148R (Chen C, Snedecor B, Nishihara J C, Joly J C, McFarland N, Andersen D C, Battersby J E, Champion K M: High-level accumulation of a recombinant antibody fragment in the periplasm of E. coli requires a triple-mutant (degP prc spr) host strain. Biotechnology and bioengineering 2004, 85(5):463-474.) may increase CRM197 protein yield.
Methods to characterize yield, purity, stability, nicking degree, toxicity, endotoxin content are well established and define the quality for use of CRM197 in a vaccine. Analysis of CRM197 is done by, e.g., high performance size exclusion chromatography, isoelectric focusing, SDS-PAGE and Western Blot, molecular weight determination by MS, N terminal sequencing, amino acid analysis, reverse phase liquid chromatography, electrospray mass spectroscopy, and peptide mapping by mass spectroscopy after tryptic digestion.
Analytical methods are described and parameters that define acceptable quality are well established for use in medicinal products. Detailed information and framework parameters are given e.g., in the guidelines released by the European medicinal agency, EMEA and can be found at EMEA's website, e.g., for the CRM197-containing vaccine Prevenar.
5.8.1 Concentration of Expression Product
Standard protein concentration technologies like the Lowry assay, BCA assay, and Bradford assays could be used, as well as determination of the UV absorption at 280 nm and quantification from Coomassie stained SDS-PAGE gels by densitometry or capillary gel electrophoresis by fluorescent dye intensity measurements.
5.8.2 Folding of Expression Product
Folding of the product can be analyzed directly by circular dichroism spectroscopy, protein NMR spectroscopy, and HPSEC. Indirect methods include solubility measurement, protease resistance, and activity assays for toxicity in the case of the DT A fragment, and binding assays for the CRM197 and DT B fragments.
5.8.3 Inclusion Bodies of Expression Product
Inclusion body formation is easily quantified by first homogenization of the harvested cells after fermentation, low spin centrifugation for sedimentation of the insoluble matter, and comparing pellet and supernatant side by side in an equivalent optical density manner. Intensity of the protein band allows estimation of the proportion in supernatant (soluble protein) and pellet (insoluble aggregates and inclusion bodies).
5.8.4 Solubility of Expression Product
Supernatant solution containing the protein can be centrifuged and sterile filtered. If the protein remains in solution and is not depleted from the filtrate and supernatants, the protein is soluble. A more sophisticated method is dynamic light scattering. It allows the determination of particle size, which is indicative of the oligomeric or micro aggregated state of the purified protein.
Solubility is inversely proportional to aggregate formation such that a finding of high solubility demonstrates no or low level of aggregate formation.
5.8.5 Periplasmic Localization of Expression Product
Periplasmic localization is measured by fractionation of cells and comparing specific protein yields observed in periplasm and spheroplast fractions. Fractionation is done by using the sucrose-lysozyme method (Kowarik M, Young N M, Numao S, Schulz B L, Hug I, Callewaert N, Mills D C, Watson D C, Hernandez M, Kelly J F et al: Definition of the bacterial N-glycosylation site consensus sequence. The EMBO journal 2006, 25(9):1957-1966), osmotic shock (Johansson H J, Jagersten C, Shiloach J: Large scale recovery and purification of periplasmic recombinant protein from E. coli using expanded bed adsorption chromatography followed by new ion exchange media. J Biotechnol 1996, 48(1-2):9-14.), or polymyxin (Schulz H, Hennecke H, Thony-Meyer L: Prototype of a heme chaperone essential for cytochrome c maturation. Science 1998, 281(5380):1197-1200). Fraction aliquots are normalized based on sample volumes and culture OD600 and analyzed by SDS PAGE and Western blotting.
5.8.6 Cleavage of Signal Sequence
Cleavage of signal peptides is assayed by i) gel shift analysis of fractionated cells as described in 5.8.5. In this analysis, unprocessed pre-protein may be accumulating in cytoplasmic aggregates or membranes, and processed protein will be present in soluble, periplasmic fractions. Different electrophoretic mobility will constitute a shift between processed and unprocessed pre-proteins by SDS-PAGE (and Western blot if necessary). Eventually, N-terminal amino acid sequencing can be used to determine the processed N-terminus and thereby define the cleavage site experimentally.
5.8.7 Potential Toxicity of CRM197
CRM197 can be tested for the presence of active toxin by measuring the ADP-ribosyl transferase activity. Additionally, other tests (cytotoxicity in HeLa cells or Vero cells in vitro, lethality in guinea pigs in vivo, abnormal toxicity test) can be used to demonstrate the nontoxicity of CRM197
Diphtheria toxin, nontoxic forms of diphtheria toxin, e.g., CRM197, produced in accordance with the methods provided herein can be further processed to immunogenic compositions or vaccines. For example, the protein can be conjugated to an oligosaccharide or a polysaccharide to yield an immunogenic composition or vaccine. In certain, specific embodiments, such an immunogenic composition or vaccine has improved immunogenic properties over prior art compositions. Without being bound by theory, the methods provided herein provide a more homogenous population of soluble CRM197 protein. As such any immunogenic composition or vaccine is more effective than prior art compositions.
In certain embodiments, provided herein is a composition comprising a diphtheria toxin, nontoxic forms of diphtheria toxin, e.g., CRM197, that has been produced in accordance with the methods provided herein. In certain more specific embodiments, such a composition is a pharmaceutical composition. Even more specifically, such a pharmaceutical composition further comprises and pharmaceutically acceptable carrier.
Different experimental setups were tested and the CRM197 yield was determined by Western blotting using anti diphtheria toxin antiserum for detection of CRM197.
A DNA open reading frame for CRM197 expression was synthesized by a commercial provider (Genescript, Piscataway, N.J.) in a codon optimized fashion containing the N-terminal signal peptide of the DsbA protein of E. coli instead of the natural signal peptide, and a C terminal hexa-histidine tag. The resulting protein sequence is SEQ ID 5. The open reading frame for ssDsbA-CRM197-his6 was inserted into the NdeI and XbaI sites of pEC415 (Schulz H, Hennecke H, Thony-Meyer L: Prototype of a heme chaperone essential for cytochrome c maturation. Science 1998, 281(5380):1197-1200).
From this plasmid, various mutants were made to analyze the differences of the CRM197 yields. Mutations were introduced at the expected signal peptide cleavage site by quick change mutagenesis as described by the manufacturer (Stratagene, Agilent Technologies, Santa Clara, Calif.). The resulting constructs are summarized in Table 1.
The mentioned plasmids were transformed into BL21 and W3110 cells to perform protein expression experiments. Transformed colonies were picked from an LB plate and used to inoculate LB medium liquid culture, which were grown over night at 37° C. The high density cultures were diluted to an OD600 of 0.05 into fresh LB medium and grown further until the OD reached a value of OD600=0.5. Then arabinose was added for induction of recombinant protein expression. Initial experiments using some of the mentioned constructs were performed under various conditions.
However, no CRM197 protein was detected in cellular extracts when compared to control cells expressing no protein or expressing EPA (Ihssen J, Kowarik M, Dilettoso S, Tanner C, Wacker M, Thony-Meyer L: Production of glycoprotein vaccines in E. coli. Microbial cell factories 2010, 9:61). Neither at 30 nor 37° C., using overnight induction times and LB medium supplemented with ampicillin for plasmid maintenance.
Subsequently, expression was conducted as follows. For the expression, high density cultures from overnight incubations were diluted into terrific broth for better cell viability. Cultures were grown until exponential phase and induced for 2 hours and overnight, and then cells were harvested and cellular extracts prepared by dissolving OD equivalent amounts of biomass in Lämmli sample buffer. The extracts were separated by SDS PAGE and electrotransferred to nitrocellulose membranes for subsequent immunodetection using anti DT and anti his tag antisera. Surprisingly, a protein signal at the expected electrophoretic mobility of CRM197 at about 60 kDa was detected after 2 hours of induction. Expression constructs p932, p934, and p722 led to detectable signals in anti DT and anti his tag antiserum immunoblots. p932 appeared to produce most, p934 less, and p722 even lesser CRM197 signals. A control extract from cells containing an expression plasmid lacking a signal peptide sequence showed CRM197 at the correct molecular weight range and confirmed the identity of the material in the other lanes.
These experiments showed that CRM197 could be expressed, but not whether it was soluble or folded. As indicated in
The order of efficiency for CRM197 production was p932>p934>p722. The methionine residue encoded in the cleavage site from the CRM197 expressed from p722 may interfere with productivity, and also the glycine residue has some influence. It appears, however, that formation of an N terminus of CRM197 with one amino acid less (ADDV . . . ; p932) than the natural N terminus in combination with the serine residue at the −2 position relative to the cleavage site leads to the optimal expression context when the DsbA signal peptide is used.
However, it was possible to detect CRM197 signals in expression experiments using different expression constructs and TB medium.
To analyze solubility and overall yield in shake flasks, CRM197 was purified from cell cultures using two different expression constructs. Expression strain was BL21, the expression plasmids p932 or p933. 5 liter shake flasks containing 1 l TB supplemented with ampicillin were inoculated with a pre-culture grown in LB supplemented with ampicillin and cultured at 30° C. At OD600 of 0.5, arabinose was added to 0.2% (w/v) and expression allowed for 2 (p932) or 4 hours (p933). Cells were then harvested by centrifugation, resuspended in buffer for periplasma extraction (20% w/v sucrose, 30 mM Tris HCl pH 8.0, 1 mM EDTA, 1 mg/ml lysozyme, 1 tablet/80 mL Complete protease inhibitor mix (Roche, Basel, Switzerland)) at a ratio of 20 OD per ml, incubated on ice for 30 min, and centrifuged for 15 min at 8000 rpm and 4° C. The supernatant was further treated with DNase (Fluka, Balgach, Switzerland), centrifuged at 4° C., and the supernatant sterile filtered. The filtrate was prepared for purification using Ni2+ affinity chromatography. Load, wash and elution were performed at specific imidazole concentrations (10, 20, 500 mM). Elution fractions were analyzed by SDS PAGE and Coomassie brilliant blue staining (
A major band corresponding to CRM197 was detected in elution fractions from the purification. Protein determination resulted in values of about 2 mg protein from construct p932, and about 4 mg from construct p933 per liter fermentation broth. N terminal sequencing and MALDI MSMS of excised protein bands from this SDS PAGE gel confirmed the N terminus of CRM197 in both cases (see Table 1) and that the protein is indeed CRM197.
The difference between protein expressed from p932 and p933 is the signal peptide sequence and the resulting mature N-terminus of CRM197. p933 produced the correct wild type N terminus; although the Y score for cleavage efficiency is lower than for p932. In
To analyze the productivity of different constructs in parallel, small scale shake flask expression experiments were performed, the periplasmic extracts were prepared and analyzed by SDS PAGE for the CRM197 band intensity by Coomassie staining (
DsbA, MalE, and PelB signal peptides resulted in the best yields in combination with optimized expression conditions. The expression conditions had a stronger influence on yields than the signal peptide cleavage site configurations. However, the importance of signal peptide cleavage site sequence is shown e.g., by the low yields obtained with the p722 expression plasmid (at 25° C.). Although p722 encodes the DsbA signal, the yield is low compared to other sequences (encoded in e.g., p932, p933, p934, or p936). Signal peptide cleavage site configurations can be classified according to their yield efficiencies: ASA-ADD (SEQ ID NO: 15) and AMA-GADD (SEQ ID NO: 16) appear better than ASA-GADD (SEQ ID NO: 17), and AMG-ADD (SEQ ID NO: 18) being the least efficient site. Y scores do not correlate with expression levels.
All tested constructs containing the PelB signal resulted in high yields at 30° C. expression temperature. Differences in the signal peptide cleavage site sequence did not drastically influence yields. However, the differences in signal peptide cleavage site sequence were small in this set of constructs.
This application is filed pursuant to 35 U.S.C. § 371 as a United States National Phase Application of International Application No. PCT/EP2013/077968 filed 24 Dec. 2013, which claims priority to U.S. Provisional Ser. No. 61/746,366 filed 27 Dec. 2012. The content of the electronically submitted sequence listing on ASCII text file (Name: LB65508US Sub Seq Lstg ST25; 45,456 bytes; and Date of Creation: Mar. 7, 2017) is incorporated herein by reference in its entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/EP2013/077968 | 12/24/2013 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2014/102265 | 7/3/2014 | WO | A |
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| 5846711 | Moore et al. | Dec 1998 | A |
| 5917017 | Collier et al. | Jun 1999 | A |
| 6455673 | Collier | Sep 2002 | B1 |
| 9346861 | Dehottay et al. | May 2016 | B2 |
| 9422345 | Blais et al. | Aug 2016 | B2 |
| 9580719 | Retallack et al. | Feb 2017 | B2 |
| 9994622 | Blais et al. | Jun 2018 | B2 |
| 20030157093 | Neville, Jr. et al. | Aug 2003 | A1 |
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| 0208375 | Apr 1989 | EP |
| 0161188 | Apr 1991 | EP |
| D477508 | Apr 1992 | EP |
| 1762246 | Mar 2007 | EP |
| 9010015 | Sep 1990 | WO |
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| 20150376245 A1 | Dec 2015 | US |
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| 61746366 | Dec 2012 | US |
