Patent Grant
10280409
This application is a 371 U.S. National Stage Application of International Patent Application No. PCT/IN2015/000427 filed Nov. 17, 2015, which claims priority to Indian Patent Application Serial No. 4045/CHE/2014 filed on Nov. 20, 2014, the entire contents of which are incorporated herein by reference and relied upon.
The present invention relates to a high level expression of bacterial toxoid by means of an optimized novel polynucleotide sequence and host transformed with the said polynucleotide.
The invention also provides a method for high production of polypeptide CRM197 wherein, the polynucleotide of the present invention is used to transform a suitable host resulting in over-expression of corresponding proteins and a method for isolating the expressed polypeptide.
Diphtheria toxin (DT) is a protein exotoxin that is synthesized and secreted by Corynebacterium diphtheriae. The toxigenic strains of Corynebacterium diphtheriae contain a bacteriophage lysogen carrying the toxin gene. Mature form of DT is synthesized as a 535 amino-acid containing single polypeptide, which is derived from an initial 536 pro-peptide which undergoes proteolysis at positions 190, 192 and 193 to form the mature toxin. This splicing or proteolysis results into two subunits, A and B which are joined together by a disulfide bridge (Moskang et al Biol. Chem. 264: 15709-15713, 1989). Subunit A is catalytically active NAD-dependent ADP-ribosyl transferase portion. It is responsible for rendering Elongation Factor-2 (EF-2) inactive, and hence down regulates the protein synthesis in a target cell.
Diphtheria toxin is highly cytotoxic; a single molecule can be lethal to a cell, and a dose of 10 ng/kg can kill animals and humans. In course of providing an artificial immunity by injection of this toxoid, a process of detoxification should be included to render it safe for consumption by the recipient. Conventionally it was detoxified by chemical modification of the natural forms of DT, in such a manner that it still retains the required antigenicity required in a vaccine preparation.
Subsequently, a genetically detoxified form of Diphtheria Toxin known as Cross Reacting Material 197 or CRM197 was introduced; which essentially retains the immunological cross-reacting properties of DT.
CRM197 has been used in preparation of conjugate vaccines including Corynebacterium diphtheria, Hepatitis B, Bordetella pertussis, Clostridium tetani, Neisseria meningitides, Streptococcus pneumonia, Haemophilus influenza. It was generated by nitrosoguanidine mutagenesis of the toxigenic corynephage β, which was then used to infect Corynebacterium diphtheria. (Uchida et al Nature New Biology (1971) 233; 8-11, Nucleic Acids Res. 1984 May 25; 12(10):4063-9)
CRM197 has been studied for its potential use as a DT booster or vaccine antigen. The CRM197 protein has the same molecular weight as DT; but differs in a single base change in the A subunit i.e. a base change in the polynucleotide sequence of wild type DT, wherein a replacement of Guanine to Adenine results into an amino acid substitution at position 52, resulting into glutamic acid in CRM197 instead of glycine (Giannini G. et al., 1984). This point mutation results in a significant loss of toxicity and renders CRM197 safe for human use.
Production of significant quantities of diphtheria toxins such as CRM197 for use in vaccines has been hindered due to low level of expression in wild type bacteria. This problem has been addressed previously by expressing CRM197 in Escherichia coli by Bishai et al., (J. Baeteriol. 189:5140-5151). who describe the expression of a recombinant fusion protein containing diphtheria toxin (including the tox signal sequence), but this led to the production of degraded protein. The low yield in active form is also associated with degradation, improper folding, or both, depending on the specific characteristics, e.g., size and secondary structure, of the toxin. Hence as with most biopharmaceuticals, there is additional loss of expressed protein that occurs during the purification steps of CRM197, whereby maintaining a biologically active form of CRM197 poses a challenge. Therefore, there is need to achieve high level expression of bacterial toxoid CRM197 in an active form.
WO 2011/042516 discloses an improved process for making a bacterial toxin by periplasmic expression comprising the steps of a) growing a culture of the bacterial host cell containing an expression vector in which particular signal sequence are linked to the sequence of a bacterial toxin and b) inducing expression of the polypeptide containing particular signal sequence linked to a bacterial toxin such that a bacterial toxin is expressed periplasmically.
WO 2013/178974 A1 discloses a process for the intracellular expression of CRM197 in an Escherichia coli host, comprising expressing a vector comprising a gene encoding CRM197 operably linked to a Promoter and at feast one perfect Palindrome Operator sequence.
WO 2015/134402 A1 discloses a process for producing a recombinant CRM197 in a reduced genome of Escherichia coli host comprising incubating a reduced genome Escherichia coli comprising an expression vector comprising a nucleotide sequence encoding a CRM197 protein fused to a signal sequence that directs transfer of the CRM197 protein to the periplasm operably linked to an expression control sequence under conditions suitable for the expression of the recombinant CRM197 protein, whereby a yield of at least 1 gram per liter of soluble CRM197 is obtained and wherein the native parent Escherichia coli strain is a 12 strain, preferably K12 MG1655.
US 2012/0128727 A1 discloses an isolated nucleic acid molecule which encodes polypeptide CRM197, an expression vector comprising the isolated nucleic acid molecule and a method for recombinant production of a CRM197 tag fusion protein, comprising culturing the recombinant cell under conditions favoring production of said CRM197 tag fusion protein, and isolating said fusion protein.
US 2012/0289688 A1 discloses a process for periplasmic expression of a recombinant polypeptide by (A) Growing a culture of a gram-negative host cell; and (B) Inducing expression of a polypeptide such that a protein is expressed periplasmically; wherein one or more of the following steps is actioned during expression: (i) The pH of step a) is lower than the pH of step b); (ii). The temperature of step a) is higher than the temperature of step b); or (iii). The substrate feed rate of step a) is higher than the substrate feed rate of step b).
US 2014/0050758 A1 discloses a process for periplasmic expression of a bacterial toxoid comprising the steps of: a) growing a culture of a gram negative host cell in a fermentation medium, wherein the host cell is transformed with a polynucleotide, and wherein the polynucleotide encodes the bacterial toxoid and a periplasmic signal sequence; inducing expression of the bacterial toxoid;
U.S. Pat. No. 8,530,171 discloses a method for producing a recombinant toxin protein in a Pseudomonas host cell, said method comprising: ligating into an expression vector a nucleotide sequence encoding the toxin protein; transforming the Pseudomonas host cell with the expression vector; and culturing the transformed Pseudomonas host cell in a culture media suitable for the expression of the recombinant toxin protein; wherein the recombinant carrier protein is CRM197, and wherein the recombinant protein is produced at a yield of soluble or active CRM197 protein of about 0.2 grams per liter to about 12 grams per liter.
Conjugated polysaccharide vaccines that use CRM197 as a carrier protein have been approved for human use. These include: MENVEO® (Meningococcal (Groups A, C, Y, and W-135) Oligosaccharide Diphtheria CRM197 Conjugate Vaccine) (Novartis Vaccines and Diagnostics), a vaccine indicated for preventing invasive meningococcal disease caused by Neisseria meningitidis subgroups A, C, Y, and W-135; MENJUGATE® (Meningococcal Group C-CRM197 Conjugate Vaccine) (Novartis Vaccines and Diagnostics), a meningococcal group C conjugate vaccine; and PREVNAR® (Pneumococcal 7-valent Conjugate Vaccine (Diphtheria CRM197 Protein)) (Wyeth Pharmaceuticals, Inc.), a childhood pneumonia vaccine that targets thirteen serotypes of Streptococcus pneumoniae, and HIBTITER® (Haemophilus b Conjugate Vaccine (Diphtheria CRM197 Protein Conjugate)) (Wyeth), a Haemophilus influenzae type b vaccine. In addition, CRM197 has potential use as a boosting antigen for C. diphtheria vaccination and is being investigated as a carrier protein for use in other vaccines.
There has recently been a growing interest in CRM197 because of its potential antitumor action relating to its capacity to bind the soluble form of HB-EGF (Mekada et al, US Patent Publication NO. 2006/0270600A1). This antitumor function is attributable not only to CRM197, but also to other non-toxic derivatives of the DT toxin (e.g. the double mutant DT52E148K, or the fusion protein GST-DT). These mutants have been constructed by PGR, starting from the gene encoding CRM197. In said studies, however, the whole CRM197 was produced using cultures of C. diphtheria, grown at 35° C. for 16-17 hours. The CRMw was purified from the supernatant by means of an initial precipitation with ammonium sulphate, followed by three successive steps in ion exchange and hydrophobic chromatography (Mekada et al.).
Hence, there is an evident need for an alternative method for the production of CRM197 with high yield and cost-effective manner. Therefore, a method for economically producing CRM197 would greatly facilitate vaccine research, development and manufacturing.
The main objective of the present intention is to provide an optimized polynucleotide for high level expression of CRM197.
Yet another objective is to provide a tunable process for controlling the expression of polypeptide as to obtain CRM197.
Yet another objective is to provide a high level expression process for commercial production of CRM197 in pure form with high yield.
The present invention provides an optimized polynucleotide sequence comprising of SEQ ID NO. 2 and its variants which are at least 70% homologous to the said optimized polynucleotide sequence SEQ ID NO. 2.
In another embodiment, the present invention provides an optimized polynucleotide sequence (SEQ ID NO. 2) and its structural variants selected from but not limited to SEQ ID NO. 3, 4, 5, 6, 7, 8, 9 and 10 useful for high level expression of polypeptide
In yet another embodiment, the present invention provides an optimized polynucleotide sequence (SEQ ID NO. 2) and its variants like SEQ ID NO. 3, 4, 5, 6, 7, 8, 9 and 10 which are at least 70 to 88% homologous to the said optimized polynucleotide sequence SEQ ID NO. 2.
The present invention further provides a process for the production of polypeptide, comprising steps of:
The polypeptide obtained above is suitably used as a carrier protein for preparation of conjugated immunogenic preparations.
The polypeptide expression for use as pharmaceutical product or vaccines requires achieving high biomass and/or productivity of the host cell line. The efficiency of polypeptide production can be significantly diminished in absence of multiple factors, which includes use of an optimal polynucleotide sequence encoding that polypeptide. The genetic code is known to exhibit degeneracy, which amounts to the variance in the polynucleotide sequence encoding the same amino acid sequence. The rate of synthesis of amino acid chain is a determinant factor in the overall expression levels from an individual gene, which effect the design of the expression construct, is of high significance. Thus the construction of an optimal polynucleotide sequence is important in determining the overall expression levels of a polypeptide and has to be well regulated. It includes, but is not limited to the frequency with which the codons are preferred in an organism or in case of artificial vehicles or vectors, the nearest frequency desired. This in turn reflects tRNA abundance or the cognate cellular tRNA frequencies from which the synonymous codon choice patterns has to be carefully selected. Additional factors also include the potential for formation of secondary structures, mRNA levels and RNA stability, subsequent intended manipulations to be carried out, synthesis routes and so on. The occurrence of these structures has to be carefully regulated as the choice of these patterns differs with the optimizations for individual protein of interest and expression hosts.
Accordingly, the main embodiment of the present invention provides an optimized polynucleotide sequence (SEQ ID NO. 2) and its structural variants.
In another embodiment, the invention provides an optimized polynucleotide sequence (SEQ ID NO. 2) and its structural variants selected from but not limited to SEQ ID NO. 3, 4, 5, 6, 7, 8, 9 and 10 having equal to or more than 70% similarity useful for high level expression of polypeptide for CRM197.
Periplasmic expression refers to the secretion of the expressed product from the intended gene of interest (such as a bacterial toxoid or Diphtheria Toxoid) in the periplasmic space within a host cell.
Cytoplasmic expression refers to the expression of protein in the cytoplasmic compartment of the cell, enclosed within cell membrane.
Induction of expression refers to the step performed to induce the expression from the polynucleotide so that the product is obtained at an accelerated rate, this may involve addition of suitable inducing agent such as IPTG, arabinose. maltose and the like.
The optimized sequence of the present invention is applicable to Other variants of SEQ ID NO. 2 selected from but not limited to SEQ ID NO. 3, 4, 5, 6, 7, 8, 9, 10 and also include sequences in the production of derivatives of SEQ ID NO. 1, wild type Diphtheria toxin which retains the same inflammatory and immunostimulatory properties and is capable of binding to the cell receptor HB-EGF, but differs from CRM197 in a single amino acid substitution and lack of cellular toxicity on target host.
The polynucleotide sequence of the CRM197 may be derived from the sequence of Diphtheria Toxin (Greenfield, L. et al., 1983, Proc. Natl. Acad. Sci. USA 80:6853-6857), or by using the amino acid sequence of CRM197 given by Giannini G. et al, (1984) as reference. The wild type polynucleotide sequence thus obtained was optimized for high expression in the host cell, more preferably Gram negative cell, more preferably Escherichia coli as host cell. Such a polynucleotide sequence of the present invention can be prepared by chemical synthesis or by means of an assembly procedure.
In yet another embodiment there is provided a process for the intracellular expression of CRM197 in a host cell wherein an expression construct with regulatory sequence provides for the expression of the polynucleotide of the invention. This polynucleotide sequence may be associated with a signal sequence for directed transport of the encoded polypeptide. It may be operably linked to periplasmic signal sequence which provides the expression targeted to be secreted in the periplasmic space of the host.
The present invention also provides high level production of CRM197, wherein periplasmic expression is brought about by providing a suitable induction temperature for the expression of polynucleotide, without any heterologous sequence for directed transport into the periplasmic space.
Optionally the polynucleotide of the invention may also be associated with polynucleotides of tag polypeptides. The presence of a tag is also known to enhance the stability and solubility of the protein in the cytoplasm and for its subsequent purification.
These tags can be associated at 5′ terminus or 3′ terminus, singly or in combination pertinent to multi-tagging, with an oligonucleotide sequence that encodes a tag polypeptide to facilitate its cytoplasmic stability and/or subsequent purification using matrices and resins with a high affinity for the various tag peptides. Various tags which cap. be used according to the invention include HA Tags (hemagglutinin), MYC Tag, Strep II, FLAG, HPC (heavy chain of protein C), glutathione-S-transferase (GST), maltose-binding protein (MBP), cellulose-binding protein (CBD) and chitin-binding protein (CBP).
The polynucleotide of invention may also be incorporated in a vector construct comprising regulatory sequence, with molecular techniques well known in art (See Sambrook et al, Molecular Cloning, 2nd ed., (1989)). This includes but is not limited to, a suitable promoter, origin of replication, ribosomal binding site, transcription termination sequence, selectable markers and multiple cloning site. In particular, a plasmid with an efficient and specific construct is preferred; such as one including T7 Promoter specific for RNA polymerase enzyme of the phage T7. Such methods may be referred to but are not limited to one disclosed in U.S. Patent Application NO. 2012/0128727; U.S. Pat. No. 5,055,294; U.S. Pat. No. 5,128,130: U.S. Pat. No. 5,281,532; U.S. Pat. No. 4,695,455; U.S. Pat No. 4,861,595; U.S. Pat. No. 4,755,465 and U.S. Pat. No. 5,169,760. A plasmid system for producing CRM197 protein in Corynebacterium diphtheriae is also described in U.S. Pat. No. 5,614,382.
In one embodiment, the host cell is a gram negative host cell. Host cell expression systems like Escherichia coli, Bacillus sp., Pseudomonas sp., have been extensively discussed in the production of proteins. In one embodiment the polynucleotide of the invention is preferably used for the intracellular expression of CRM197 in Escherichia coli wherein the host strain is selected from BL21 (DE3), BL21 A1, HMS174 (DE3), DHSct, W31 10, B834, origami, Rosetta, NovaBlue (DE3), Lemo21 (DE3), 17, ER2566 and C43 (DE3).
In a preferred embodiment, the present invention provides a polynucleotide sequence (SEQ ID NO. 2) encoding bacterial toxoid which is optionally ligated into a vector, followed by its insertion in a host cell. The insertion into host cell may be performed by any of the methods known in the art. Such an insertion or transformation may be performed by a physical or a chemical method of transformation. Subsequently, the converted colonies arc selected on petri dishes with added antibiotic.
Suitable vectors used in the present invention include but not limited to pET9a, pET3a, pET3b, pET3c, pET5a, pET5b, pET5c, pET9b, pET9c, pET12a, pTWTN1, pTWTN2, pET12b, pET12c, pET17b and in general, all the vectors that have a strong phage T7 promoter (e.g. pRSETA, B and C [Invitrogen]) and pTYB1, pTYB2, pTYB3 and pTYB4.
In another embodiment, Escherichia coli cells are used to express the polynucleotide encoding CRM197. The inserted Polynucleotide is verified for proper orientation and position by sequencing. The resultant construct is used to transform host cells by any of the known chemical or physical methods. For example electroporating host cells with an electric field in range 6.5 kV.cm-1 to 25 kV.cm-1, a preferred chemical method here which is used to transform host. These cells are allowed to grow for 30 minutes to 120 minutes at 25 to 40° C. in a suitable medium as LB or SOC medium and then transferred to selection media petri plates for 10 to 36 hours, 25 to 40° C. where the positive colonies containing the polynucleotides of invention are selected.
The selection of positive colonies can be done with or without markers. Suitable markers which can be used are selected from, but not limited to, antibiotics such as ampicillin, kanamycin and the like.
The polynucleotide encoding the full length CRM197 protein is cloned adjacent to T7 lacI promoter that drives the expression of protein in T7 polymerase positive strains of Escherichia coli. The expression of polynucleotide is stringently controlled by T7 promoter which is induced ip the presence of IPTG or in auto-induction mediums
The parameters for culturing the host are optimized for high level expression of CRM197 protein. In one embodiment, the culture media components, culture conditions including growth temperature, concentration of inducers and induction time is optimized. The culture media used may be selected from, hut not limited to, chemically defined media, LB (Luria-Bertani), TB (Terrific Broth), SOB (Super Optimal Broth), SOC (Super Optimal broth with catabolic repressor), YT broth (Yeast Extract and Tryptone). Super broth, rich media, minimal media, mineral media and the like. The ingredients of media includes, but is not limited to, a carbon source such as, e.g., glucose, sucrose, or glycerol, organic nitrogen source, such as peptone, tryptone, amino acids, or a yeast extract, inorganic nitrogen source is used and this may be selected from among, e.g., ammonium salts, aqueous ammonia, and gaseous ammonia, supplements as supplemented with, e.g., low levels of amino acids, vitamins, peptones, or other ingredients. The culture media may be prepared using the methods known in the art.
The transformed host cells may be tested for expression on small volume such as 5-50 ml in LB, terrific broth or chemically defined medium. The expression may be subjected to different concentrations of inducers ranging from about 0.01 mM, about 0.05 mM, about 0.2 mM, about 0.3 mM, about 0.4 mM, about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM and about 1 mM. The polypeptide expression is determined in an electrophoretic set up, preferably in SDS PAGE electrophoresis and viewed as over-expressed bands stained with Coomassie Brilliant Blue (-).
Subsequently, the host cells are inoculated in 500 mL flasks cultures; and allowed to grow under optimal conditions, during which CRMA expression continued from 16 hours to 32 hours. After culturing in constant agitation and preferably under aerobic conditions, the cells are harvested, and lysed. Any of known methods may be applied to lyse cells, preferred method includes a lysis buffer containing a detergent at an appropriate concentration. After lysis, the protein component is pooled in one or more centrifugation steps. Lysis is carried out in a buffer containing Tris-HCl 20-50 mM pH 7.5-8.3, NaCl 100-150 mM, detergent 0.5-1.5% and protease inhibitor 0.5-1.5%, with agitation.
In one embodiment induction temperatures for expression is carried out between 10 to 40° C. In one embodiment CRM197 is derived at an induction temperature in a tunable manner, wherein when induction temperature is maintained between 10 to 20° C., more than 80% expressed CRM197 is present in soluble fraction,. In another embodiment when the induction temperature is maintained between 25 to 40° C., more than 80% of expressed CRM197 obtained is in the insoluble fraction as cytoplasmic inclusion bodies, from which it is purified after a solubilisation step from the pooled cytoplasmic fraction.
In one embodiment cytoplasmic inclusion bodies are solubilized with various concentration of Urea, per se 1M Urea, or 2M Urea, or 3 M Urea, or 4 M Urea, or 5 M Urea, or 6 M Urea, or 7 M Urea, or 8 M Urea, or 9 M Urea.
In the specific embodiment, the yield of soluble CRM197 is about 0.1 g/l, 0.25 g/L, 0.5 g/L, about 1 g/L, about 1.5 g/L, about 2 g/L, about 2.5 g/L, about 3 g/L, about 3.5 g L, about 4 g/L, about 4.5 g/L, about 4.5 g/L, about 5 g/L.
In the specific embodiment, the yield of insoluble CRM197 is about 0.25 g/L, 0.5 g/L, about 1 g/L, about 1.5 g/L, about 2 g/L, about 2.5 g/L, about 3 g/L, about 3.5 g/L, about 4 g/L, about 4.5 g/L, about 4.5 g/L, about 5 g/L.
The expressed protein is purified using ion exchange chromatographic column followed by affinity chromatography.
The invention thus involves more than one subsequent purification steps, and also exploits pI value of CRM197 in an ion exchange chromatographic step, whereby it is separated from other contaminating proteins. Finally, the quantity of CRM197 is quantified by BCA/Bradford/Lowry Assay and visualized in 10-12% acrylamide gel (SDS-PAGE). The identification of polypeptide is done by Western blot and similar immunoassays. The purity and integrity of purified polypeptide is measured by SDS-PAGE and HPLC methods. The yield of the protein thus expressed is 500-1000 mg/L of the culture medium and can be subsequently varied by modulating the culture additives and conditions, as well as purification steps. The method of the invention also provides an industrially applicable method of tuning the induction time and subsequently modulating the pH and temperature of the chromatographic steps provides simple, inexpensive, and is not laborious. It excludes need of extensive steps involving preparation of buffers or kit or working solution thereof. During the removal of tag there is no need to provide additional buffers or salts or enzymes or equipment. In particular embodiment, the purified CRM197 polypeptide readily lacked the first Methionine amino acid, whose presence is not desired in the final CRM197 protein and removal of which entails requirement For additional purification steps. The Polypeptide thus obtained is in active and native form; it readily lacks the undesired Methionine as first amino acid without the need of additional steps. CRM197 amino acid sequence was analyzed by Insilico/bioinformatics tools; showed about 38.4% hydrophobicity in the protein. The isoelectric point of CRM197 is found about 5.81. CRM197 protein contained 4 cysteine amino acid residue and 21 proline residues. The refolding of polypeptide is confirmed by functional assays by measuring endonuclease activity over DNA. Biophysical/secondary structure confirmation is done by Circular Dichroism (CD) analysis (
In another embodiment the presence of correct disulphide linkage was confirmed and compared with commercially available CRM197 polypeptides (Sigma Aldrich) (
In a preferred embodiment, the present invention provides an optimized polynucleotide sequence (SEQ ID NO. 2) and its structural variants having equal to or more than 70% similarity, preferably 85 to 99% similarity, useful for high level expression of polypeptide for CRM197.
In yet another preferred embodiment, the present invention provides an optimized polynucleotide sequence (SEQ ID NO. 2) useful for high level expression of polypeptide for CRM197 in Escherichia coli cell.
In another preferred embodiment, the present invention provides high level expression of Diphtheria toxin or CRM197 or variants thereof, using nucleic acid SEQ ID NO: 2 or a variant thereof in gram negative bacterial cell, preferably Escherichia coli comprising the steps of;
The purification is carried out using chromatography. The chromatography technique may be affinity chromatography, gel filtration, high pressure liquid chromatography (HPLC) or ion exchange chromatography or combination of two or more. Preferably, when CRM197 is associated with tag fusion protein, affinity chromatography may be used to separate CRM197 from other proteins.
In another preferred embodiment, a simple step involving a shift in temperature and pH of the column conditions also facilitate the elution of CRM197 from the associated tag. More particularly, a pH in the range of 6.5-8.5 and temperature in the range of 4° C.-30° C. may be used to separate CRM197 from tag.
In other embodiments, the CRM197 prepared according to the present invention is used to conjugate with polysaccharide molecules isolated from Salmonella typhi, Salmonella paratyphi, Pneumococcus, Haemophilus influenzae, Meningococcus, Streptococcus pneumoniae and other pathogenetic bacteria.
In another embodiment, the CRM197 prepared according to the present invention is used as a conjugated carrier for vaccines such as those against Salmonella typhi, Salmonella paratyphi, Pneomococcus, Haemophilus influenzae, Meningococcus, Streptococcus pneumoniae and other pathogenetic bacteria.
The present invention will be more specifically illustrated with reference to the following examples. However, it should be understood that the present invention is not limited by these examples in any manner but includes variations thereof within the parameters described herein, as can be known to those well-versed in the art.
Step (i): Synthesis of Novel CRM197 Gene
Full length CRM197 gene was optimized according to Escherichia coli codon usage. The following parameters were used for CRM197 gene optimization: Codon Usage Bias, GC content, mRNA Secondary Structure, Custom Desired Patterns, Custom Undesired Patterns, Repeat Sequences (direct repeat, inverted repeat, and dyad repeat). Restriction Enzyme Recognition Sites (deletion or insertion).
Optimized CRM197 gene (SEQ ID NO. 2) was cloned at multiple cloning site of pUC57 plasmid vector using BamHI and Sapi restriction sites, generating pUC57_CRMw. The vectors containing CRM197 gene was transformed in Escherichia coli DH5a host and clones was selected on LB+Kanamycin plate. The presence and correctness of CRM197 gene in pUC57 was confirmed by restriction digestion of pUC57_CRM197 plasmid by Age I (located in CRM197 gene) and Nde I (located in pUC57 plasmid). Further the sequence of CRM197 was confirmed by PCR and DNA sequencing.
Step (ii): Insertion of CRM197 into Expression Vector pTWI I
Escherichia coli DH5a carrying pUC57_CRM197 was grown over night in LB-Kanamycin in 50 ml volume. Bacteria were centrifuged and pellet was used for plasmid isolation, isolation of plasmid was done by using Qiagen plasmid mini-prep kit using manufacturer instructions. Isolated plasmid was quantified by nano-drop.
CRM197 (SEQ ID NO. 2) from pUC57 was excised, Sag plasmid was digested with restriction endonucleases BamHI and Sapl. The digested plasmid was run on 1% agarose gel and band corresponding to CRM197 gene (SEQ ID NO. 2, −1.6 kb) was purified by using Qiagen Gel extraction kit using manufacturer's instructions. Subsequently the 5 μg of expression plasmid pTWIN1 was also digested with BamHI and SapI to generate restriction sites in it that is compatible with CRM197 gene. The digested pTWIN1 was also purified from gel using Qiagen Gel extraction kit with manufacturer's instructions.
The digested CRM197 gene was ligated in pTWIN1 using T4 ligase based DNA ligation kit (Promega) using manufacturer's instructions. Vector (pTWIN1) and Insert (CRM197) was mixed in 1:3, 1:4, 1:5 ratio in the presence of T4 DNA ligase and buffers in a 20 μl reaction volume. Ligation mixture was incubated overnight at 16° C. Next morning 5 μl of ligation mixture was added/transformed in BL21-DE3 Escherichia coli expression host. BL21 was transformed by using chemical transformation protocol. The ligation+ BL21 cells were incubated in ice for 30 min. After incubation heat shock was given for 45 seconds at 42° C. Sample was cooled at room temperature and 500 μl SOC medium was added into it. The tube with transformants was incubated for 2 hours at 37° C. with 200 rpm. From which 100 μl mixture was plated on LB+Ampicillin plate for screening of transformants.
CRM197 expression BL21-DE3 iransformants were selected next morning from Luria Broth+Ampicillin plates. Of these 5 clones growing on Luria Broth+Ampicillin were selected and grown in 10 ml Luria Broth+Ampicillin media for overnight at 37 degrees, 200 rpm. Culture was centrifuged and plasmid was extracted from cell pellet using Qiagen plasmid extraction kit.
To verify the correctness of clone, 2 μg plasmid was digested with Agel and Apal restriction endonuclease, respectively. Agel site is present in CRM197 and Apal is in pTWIN 1. Therefore double digestion with both the enzymes used for confirmation of correct clone. The clone was designated as pTWIN1_CRMw (BL21-DE3). Furthermore clones were confirmed by. PGR using CRM197 gene specific primers and DNA sequencing. The glycerol stock of BL21 expressing CRMm was made by growing bacteria in 10 ml Luria Broth+Ampicillin overnight. Next morning 40% sterilized Glycerol was added into culture and 1 ml aliquot was dispensed into cryovial. Vials were stored at −80 degree for further use in expression analysis.
Step (iii): Confirmation of Expression of CRMw:
BL21 clone stored at −80 degrees was streaked on Luria Broth+Ampicillin plate. Plate was incubated overnight at 37 degrees. Single colony was picked up and inoculated in 50 ml Luria Broth+Ampicillin media in 150 ml flask. Flask was incubated at 37 degrees, 200 RPM until OD600=1. Once OD reaches to desired point, 5 ml culture was drawn which is used as uninduced culture. Uninduced culture was kept on ice until use. To induce the expression of CRM197 gene 0.5 mM IPTG was added to remaining 45 ml culture and flask was further incubated for additional 4 hours at 30 degree and 200 rpm rotation. Induced culture was harvested after 4 hours and expression of CRM197 was examined by SDS-PAGE (
For SDS-PAGE analysis 1 ml culture of induced and uninduced culture (both normalized for OD600=1) was taken into 1.5 ml Eppendorf tube. The tube was centrifuged and pellet was resuspended into 50 μl PBS. In this suspension 50 μl SDS-PAGE loading buffer with reducing agent (2×) was added. The mixture was boiled at 100 degree for 5 min. Sample was cooled at room temperature and 20 μl of uninduced and induced culture was loaded in the 4-12% Tris Glycine gel. The gel was run for 1.5 hours at 150 volts. Gels were taken out and incubated in Coomassie Brilliant Blue dye for 1 hour After staining gel was detained in destaining solution containing 40% methanol=10% acetic acid for 3 hours. The CRM197 expression was visualized as ˜58 KD protein that is only visible in induced culture. For western blot a separate set of gel was run in the same manner as SDS-PAGE and gel was blotted on PVDF (polyvinylidene difluoride membrane). The membrane was immunoblotted by anti- CRM197 antibody. In the western blot CRMw appeared as single immunoreactive band at ˜58 Kd. No CRM197 specific band was observed in uninduced culture. This experiment confirms that the done generated in the present study can express rCRM197 protein. These clones were further used for large scale production and purification of CRM197.
Step (iv): Fermentation and Purification of CRM197 from BL21 Escherichia coli
One ml vial of BL21 Escherichia coli cells was inoculated into 50 ml LB+Amp media and grown overnight at 37 degrees, 200 rpm. Fermentation was done at 20 L scale. Escherichia coli cells were inoculated to the fermenter and cultivated at 30 degrees centigrade. The culture was induced with 0.5 mM IPTG at OD600=20. After 12 hours post induction fermentation culture was harvested and cell pellet was prepared by centrifugation. Cell pellet was lysed mechanically in homogenizer. Inclusion body (which contains the desired protein CRM197) was isolated by centrifugation of cell lysates. Supernatant was discarded and pellet was retained which contains Inclusion body (IBs). IBs were homogenized by resuspending pellet in 8M urea and protein was purified by ion exchange chromatography. Quantification of CRM197 at fermentation level was measured. Whole cells lysates was run on SDS-PAGE along with the known amount of BSA as standard. The quantification of CRM197 which appeared as ˜58 KD band in SDS-PAGE (
Solubilization test of the CRM197 prepared above was carried out in Electrophoretic gel run (SDS PAGE 12%) showing test conducted on solubilization of CRM197, Lane 1: Urea solubilized fraction of CRM197; Lane 2: Supernatant; Lane 3: Sample from first pellet wash; Lane 4: Sample pooled from 2nd pellet wash (
Presence of CRM197 in the sample was determined by Size exclusion chromatography (SEC-HPLC) wherein major eluted peak shows the presence of CRMw in the sample (
Primary amino acid sequence of the CRM197 prepared above was determined by Peptide mass fingerprint (mass spectrometry) and had 100% sequence similarity with the reference CRM197 sequence (
A. Peptic digest of BE rCRM analyzed by LC-MS.
B. Sequence coverage in Trypsin, Glu-C and Asp-N digest CRM197 (identical to SEQ ID NO. 1 i.e. shows 100% homology)
N-Terminal sequence of rCRMm prepared above was confirmed by Edman degradation. The 10 amino acid sequence GADDVVDSSK (SEQ ID NO. 13) (N-Term acetyl) shows starting portion of purified polypeptide. The first amino acid is identified as G (
CD spectra of the CRM197 prepared above was overlapped with the reference CRM197. The result shows that recombinant CRM197 prepared above is structurally similar to the reference CRM197 (
Confirmation of disulphide bonds of CRM197 prepared by the above method was analyzed (
Antigenic similarity of CRM197 prepared by the above method with reference CRM197 was confirmed by CRM197 specific ELISA. All the CRMs coming from difference source showed similar recognition profile with monoclonal antibodies (
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|---|---|---|---|
| 4045/CHE/2014 | Nov 2014 | IN | national |
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|---|---|---|---|
| PCT/IN2015/000427 | 11/17/2015 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2016/079755 | 5/26/2016 | WO | A |
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