CODON OPTIMIZED POLYNUCLEOTIDE FOR HIGH LEVEL EXPRESSION OF CRM197
20170306302 · 2017-10-26
Inventors
- Akshay Goel (Hyderabad, IN)
- Ravi Pratap Narayan Mishra (Hyderabad, IN)
- Narender Dev Mantena (Hyderabad, IN)
- Mahima Datla (Hyderabad, IN)
Cpc classification
C12N15/74
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C12P21/02
CHEMISTRY; METALLURGY
International classification
C12P21/02
CHEMISTRY; METALLURGY
C12N15/10
CHEMISTRY; METALLURGY
C12N15/74
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
Abstract
The present invention relates to high level expression of bacterial toxoid or toxin protein of pharmacological interest by means of an optimized novel polynucleotide sequence and host transformed with the said polynucleotide. Specifically, the invention provides a method for high production of polypeptide CRM.sub.197 wherein, the polynucleotide of the invention is used to transform a suitable host resulting in over-expression of corresponding proteins and a method for isolating the expressed polypeptide. More particularly, the present invention relates to high level expression of CRM.sub.197 in Escherichia coli and a method for the isolation and purification thereof.
Claims
1. An optimized polynucleotide sequence comprising SEQ ID NO. 2 or a variant thereof which is at least 70% homologous to SEQ ID NO. 2.
2. The optimized polynucleotide sequence of claim 1, wherein the variant thereof is at least 70 to 88% homologous to SEQ ID NO. 2.
3. The optimized polynucleotide sequence of claim 1, wherein the variant thereof is, selected from the group consisting of SEQ ID NO. 3, 4, 5, 6, 7, 8, 9, and 10.
4. A process for the production of polypeptide, comprising steps of: a) selecting an optimized polynucleotide sequence of claim 1, b) optionally ligating the polynucleotide sequence of step (a) into a suitable vector, c) inserting or transforming the polynucleotide sequence into an Escherichia coli host cell to generate a transformed host cell, d) culturing the transformed host cell in a culture media for high level expression of a CRM.sub.197 polypeptide from the polynucleotide sequence of step (a), e) maintaining an induction temperature between 10 to 40° C. to produce the CRM.sub.197 polypeptide, f) extracting the CRM.sub.197 polypeptide from the transformed host cell, and g) purifying the CRM.sub.197 polypeptide to obtain pure CRM.sub.197 polypeptide with high yield.
5. The process of claim 4, wherein the suitable vector is a plasmid vector selected from the group consisting of pET9a, pET3a, pET3b, pET3c, pET5a, pET5b, pET5c, pET9b, pET9c, pET12a, pTWINl, pTWIN2, pET12b, pET12c, and pET17b.
6. The process of claim 4, wherein the Escherichia coli host cell is a strain selected from the group consisting of BL21 (DE3), BL21 A 1, HMS174 (DE3), DH5ot, W31 10, B834, origami, Rosetta, NovaBlue (DE3), Lemo21 (DE3), T7, ER2566, and C43 (DE3).
7. The process of claim 4, wherein the yield of the CRM.sub.197 polypeptide 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, or about 5 g/L.
8. The process of claim 4, wherein at least a portion of the CRM.sub.197 polypeptide is localized to the periplasm by providing a suitable induction temperature without any heterologous sequence for directed transport into the periplasmic space.
9. The process of claim 8, wherein the induction temperature is from 10 to 40° C.
10. The process of claim 4, wherein the CRM.sub.197 polypeptide is a carrier protein.
11. The process of claim 10, wherein the carrier protein is Diphtheria toxin mutant CRM.sub.197 polypeptide.
12. The process of claim 4, wherein the CRM.sub.197 polypeptide is conjugated with polysaccharide molecules isolated from Salmonella typhi, Salmonella paratyphi, Pneumococcus, Haemophilus influenzae, Meningococcus, Streptococcus pneumoniae, or other pathogenetic bacteria.
13. A vaccine comprising a CRM.sub.197 polypeptide generated from the polynucleotide sequence of claim 1, wherein the CRM.sub.197 polypeptide is used as a conjugated carrier.
14. The vaccine of claim 13, wherein the vaccine is against Salmonella typhi, Salmonella paratyphi, Pneumococcus, Haemophilus influenzae, Meningococcus, Streptococcus pneumoniae or other pathogenetic bacteria.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0039] 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.
[0040] Accordingly, the main embodiment of the present invention provides an optimized polynucleotide sequence (SEQ ID NO. 2) and its structural variants.
[0041] 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 CRM.sub.197.
[0042] 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.
[0043] Cytoplasmic expression refers to the expression of protein in the cytoplasmic compartment of the cell, enclosed within cell membrane.
[0044] 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.
[0045] 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 CRM.sub.197 in a single amino acid substitution and lack of cellular toxicity on target host.
[0046] The polynucleotide sequence of the CRM.sub.197 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 CRM.sub.197 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.
[0047] In yet another embodiment there is provided a process for the intracellular expression of CRM.sub.197 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.
[0048] The present invention also provides high level production of CRM.sub.197, 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.
[0049] 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.
[0050] 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 can 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).
[0051] 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 CRM.sub.197 protein in Corynebacterium diphtheriae is also described in U.S. Pat. No. 5,614,382.
[0052] 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 CRM.sub.197 in Escherichia coli wherein the host strain is selected from BL21 (DE3), BL21 Al, HMS174 (DE3), DH5ct, W31 10, B834, origami, Rosetta, NovaBlue (DE3), Lemo21 (DE3), T7, ER2566 and C43 (DE3).
[0053] 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 are selected on petri dishes with added antibiotic.
[0054] Suitable vectors used in the present invention include but not limited to pET9a, pET3a, pET3b, pET3c, pET5a, pET5b, pET5c, pET9b, pET9c, pET12a, pTWTNl, 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.
[0055] In another embodiment, Escherichia coli cells are used to express the polynucleotide encoding CRM.sub.197. 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.Math.cm-.sup.1 to 25 kV.Math.cm-.sup.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 petriplates for 10 to 36 hours, 25 to 40° C. where the positive colonies containing the polynucleotides of invention are selected.
[0056] 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.
[0057] The polynucleotide encoding the full length CRM.sub.197 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
[0058] The parameters for culturing the host are optimized for high level expression of CRM.sub.197 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, but 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.
[0059] 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 (-).
[0060] Subsequently, the host cells are inoculated in 500 mL flasks cultures; and allowed to grow under optimal conditions, during which CRM̂ 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 7.5-8.5, NaCl 100-150 mM, detergent 0.5-1.5% and protease inhibitor 0.5-1.5%, with agitation.
[0061] In one embodiment induction temperatures for expression is carried out between 10 to 40° C. In one embodiment CRM.sub.197 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 CRM.sub.197 is present in soluble fraction. In another embodiment when the induction temperature is maintained between 25 to 40° C., more than 80% of expressed CRM.sub.197 obtained is in the insoluble fraction as cytoplasmic inclusion bodies, from which it is purified after a solubilisation step from the pooled cytoplasmic fraction.
[0062] 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.
[0063] In the specific embodiment, the yield of soluble CRM.sub.197 is about 0.1 g/1, 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.
[0064] In the specific embodiment, the yield of insoluble CRM.sub.197 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.
[0065] The expressed protein is purified using ion exchange chromatographic column followed by affinity chromatography.
[0066] The invention thus involves more than one subsequent purification steps, and also exploits pI value of CRM.sub.197 in an ion exchange chromatographic step, whereby it is separated from other contaminating proteins. Finally, the quantity of CRM.sub.197 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 CRM.sub.197 polypeptide readily lacked the first Methionine amino acid, whose presence is not desired in the final CRM.sub.197 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. CRM.sub.197 amino acid sequence was analyzed by Insilico/bioinformatics tools; showed about 38.4% hydrophobicity in the protein. The isoelectric point of CRM.sub.197 is found about 5.81. CRM.sub.197 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 (
[0067] In another embodiment the presence of correct disulphide linkage was confirmed and compared with commercially available CRM.sub.197 polypeptides (Sigma Aldrich) (
[0068] 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 CRM.sub.197.
[0069] 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 CRM.sub.197 in Escherichia coli cell.
[0070] In another preferred embodiment, the present invention provides high level expression of Diphtheria toxin or CRM.sub.197 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:
a) selecting the gene SEQ ID NO: 2 or its variant thereof, which encodes polypeptide CRM.sub.197,
b) sub cloning the gene SEQ ID NO: 2, into an expression vector,
c) transforming the host Escherichia coli cell with the expression vector of step b;
d) culturing the transformed host cell in a culture media suitable for the expression of the toxin protein;
e) inducing the expression of fusion protein by adding IPTG as inducing agent at temperature in the range of 30 to 40° C.,
f) extracting the bacterial toxoid in insoluble form from the host cell and
g) purifying the CRM.sub.197 in pure form with yield more than 0.5 mg/l.
[0071] 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 CRM.sub.197 is associated with tag fusion protein, affinity chromatography may be used to separate CRM.sub.197 from other proteins.
[0072] In another preferred embodiment, a simple step involving a shift in temperature and pH of the column conditions also facilitate the elution of CRM.sub.197 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 CRM.sub.197 from tag.
[0073] In other embodiments, the CRM.sub.197 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.
[0074] In another embodiment, the CRM.sub.197 prepared according to the present invention is used as a conjugated carrier for vaccines such as those against Salmonella typhi, Salmonella paratyphi, Pneumococcus, Haemophilus influenzae, Meningococcus, Streptococcus pneumoniae and other pathogenetic bacteria.
[0075] 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.
Example 1
Step (i): Synthesis of Novel CRM.SUB.197 .Gene:
[0076] Full length CRM.sub.197 gene was optimized according to Escherichia coli codon usage. The following parameters were used for CRM.sub.197 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).
[0077] Optimized CRM.sub.197 gene (SEQ ID NO. 2) was cloned at multiple cloning site of pUC57 plasmid vector using BamHl and Sapl restriction sites, generating pUC57_CRMw. The vectors containing CRM.sub.197 gene was transformed in Escherichia coli DH5a host and clones was selected on LB+Kanamycinr plate. The presence and correctness of CRM.sub.197 gene in pUC57 was confirmed by restriction digestion of pUC57_CRM.sub.197 plasmid by Age I (located in CRM.sub.197 gene) and Nde I (located in pUC57 plasmid). Further the sequence of CRM.sub.197 was confirmed by PCR and DNA sequencing.
Step (ii): Insertion of CRM.sub.197 into Expression Vector pTWI1
[0078] Escherichia coli DH5a carrying pUC57_CRM.sub.197 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.
[0079] CRM.sub.197 (SEQ ID NO. 2) from pUC57 was excised, 5 μg plasmid was digested with restriction endonucleases BamHI and Sapl. The digested plasmid was run on 1% agarose gel and band corresponding to CRM.sub.197 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 pTWINl was also digested with BamHI and Sapl to generate restriction sites in it that is compatible with CRM.sub.197 gene. The digested pTWINl was also purified from gel using Qiagen Gel extraction kit with manufacturer's instructions.
[0080] The digested CRM.sub.197 gene was ligated in pTWINl using T4 ligase based DNA ligation kit (Promega) using manufacturer's instructions. Vector (pTWINl) and Insert (CRM.sub.197) was mixed in 1:3, 1:4, 1:5 ratio in the presence of T4 DNA ligase and buffers in a 200 reaction volume. Ligation mixture was incubated overnight at 16° C. Next morning 50 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.
[0081] CRM.sub.197 expression BL21-DE3 transformants 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.
[0082] To verify the correctness of clone, 2 μg plasmid was digested with Agel and Apal restriction endonuclease, respectively. Agel site is present in CRM.sub.197 and Apal is in pTWIN 1. Therefore double digestion with both the enzymes used for confirmation of correct clone. The clone was designated as pTWINl_CRMw (BL21-DE3). Furthermore clones were confirmed by. PGR using CRM.sub.197 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 CRM.sub.197:
[0083] 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 CRM.sub.197 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 CRM.sub.197 was examined by SDS-PAGE (
[0084] 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 500 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 CRM.sub.197 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-CRM.sub.197 antibody. In the western blot CRMw appeared as single immunoreactive band at ˜58 Kd. No CRM.sub.197 specific band was observed in uninduced culture. This experiment confirms that the clone generated in the present study can express rCRM.sub.197 protein. These clones were further used for large scale production and purification of CRM.sub.197.
Step (iv): Fermentation and Purification of CRM.sub.197 from BL21 Escherichia coli
[0085] 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 CRM.sub.197) was isolated by centrifugation of cell lysates. Supernatant was discarded and pellet was retained which contains Inclusion body (IBs). Ms were homogenized by resuspending pellet in 8M urea and protein was purified by ion exchange chromatography. Quantification of CRM.sub.197 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 CRM.sub.197 which appeared as ˜58 KD band in SDS-PAGE (
[0086] Solubilization test of the CRM.sub.197 prepared above was carried out in Electrophoretic gel run (SDS PAGE 12%) showing test conducted on solubilization of CRM.sub.197, Lane 1: Urea solubilized fraction of CRM.sub.197; Lane 2: Supernatant; Lane 3: Sample from first pellet wash; Lane 4: Sample pooled from 2nd pellet wash (
[0087] Presence of CRM.sub.197 in the sample was determined by Size exclusion chromatography (SEC-HPLC) wherein major eluted peak shows the presence of CRMw in the sample (
[0088] Primary amino acid sequence of the CRM.sub.197 prepared above was determined by Peptide mass fingerprint (mass spectrometry) and had 100% sequence similarity with the reference CRM.sub.197 sequence (
A. Peptic digest of BE rCRM analyzed by LC-MS.
B. Sequence coverage in Trypsin, Glu-C and Asp-N digest CRM.sub.197 (identical to SEQ ID NO. 1 i.e. shows 100% homology)
[0089] N-Terminal sequence of rCRM.sub.197 prepared above was confirmed by Edman degradation. The 10 amino acid sequence GADDVVDSSK (N-Term acetyl) shows starting portion of purified polypeptide. The first amino acid is identified as G (
[0090] CD spectra of the CRM.sub.197 prepared above was overlapped with the reference CRM.sub.197. The result shows that recombinant CRM.sub.197 prepared above is structurally similar to the reference CRM.sub.197 (
[0091] Confirmation of disulphide bonds of CRM.sub.197 prepared by the above method was analyzed (
TABLE-US-00001 TABLE I Cystine Numbering Peptide sequence 186 GQDAMYEYMAQACAGNR (SEQ ID NO: 11) 201 SVGSSLSCINLDWDVIR (SEQ ID NO: 12) 461 CR (SEQ ID NO: 13) 471 AIDGDVTFCRPK (SEQ ID NO: 14)
TABLE-US-00002 TABLE Ii Tryptic peptides Theoretical Observed chain combination Mono m/z Mono m/z Cys 186-201 935.6706 935.6694 Cys 461-471 913.9558 913.9539
[0092] Antigenic similarity of CRM.sub.197 prepared by the above method with reference CRM.sub.197 was confirmed by CRM.sub.197 specific ELISA. All the CRMs coming from difference source showed similar recognition profile with monoclonal antibodies (