Claims:An expression vector for expression of Insulin or Insulin analogues in a host cell comprising of:
a nucleotide sequence encoding leader peptide of SEQ ID NO 3;
a nucleotide sequence encoding an affinity tag linked to C-terminal end or N terminal end of nucleotide sequence of said leader peptide;
and a nucleotide sequence encoding for a cleavage site ligated to nucleotide sequence of said leader peptide through nucleotide sequence encoding said affinity tag;
a nucleotide sequence encoding a multiple cloning site (MCS) in upstream region of said leader peptide;
a nucleotide sequence encoding ribosome binding site (RBS) ligated to N-terminus or C-terminus of said leader peptide;
a nucleotide sequence encoding a promoter or operator in the downstream of said ribosome binding site;
and a nucleotide sequence encoding an antibiotic selection marker in upstream region of said promoter/operator sequence.
2. The expression vector of claim 1 wherein, said leader peptide is expressed as a fusion protein; said fusion protein comprising fusion of said leader peptide of SEQ ID NO 3 and Insulin or Insulin analogues.
3. The expression vector of claim 1 wherein, said host cell is bacteria, preferably E. coli.
4. Expression vector claimed in claim 1 or 2 wherein, said leader peptide has Methionine at N-terminus, followed by glycine to impart stability to fusion of said fusion protein and said leader peptide.
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5. The expression of claim 1 wherein, said affinity tag is his-tag.
6. The expression vector of claim 1 wherein, said leader peptide is linked to proinsulin via said cleavage site.
7. The expression vector of claim 1 or 6 wherein, said cleavage site is arginine.
8. The expression vector of claim 1 or claim 7 wherein, , Description:As required, detailed embodiments are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary, which can be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed structure. Further, the terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the invention.
The terms “a” or “an”, as used herein, are defined as one or more than one. The term “plurality”, as used herein, is defined as two or more than two. The term “another”, as used herein, is defined as at least a second or more. The terms “including” and/or “having”, as used herein, are defined as comprising (i.e., open language).
Vector Deposition
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The vector pBGBactX is deposited for the patent purposes under Budapest Treaty at the MTCC (Microbial Type of Culture Collection) Chandigarh, India. The deposit was made on March 21, 2013 and accorded deposit number as MTCC 5818. The sequence was characterised using DNA sequencer.
As mentioned, there is a need for plasmid vectors which lead to high yield of insulin and other heterologous proteins through simple purification processes. The embodiments herein provide a plasmid vector having nucleotide sequence listed under SEQ ID NO. 1.
Figure 1 illustrates an expression construct having a leader peptide, for production of insulin in bacterial cells, according to an embodiment herein. The expression construct includes a DNA sequence, of SEQ ID NO 2 encoding for the leader peptide of SEQ ID NO. 3. The expression construct further includes a DNA sequence encoding an affinity tag in the C-terminal end of the DNA sequence of the leader peptide. In one embodiment, the affinity tag is his-tag or a sequence with 6 histidines in succession. In a preferred embodiment, the DNA sequence encoding the affinity tag is ligated to the N-terminal end of the DNA sequence of the leader peptide.
Further, the leader peptide DNA sequence with his-tag is ligated to DNA sequence encoding B-chain (for B-C-A conformation) or to DNA sequence encoding A-Chain (A-C-B
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conformation) of proinsulin via a DNA sequence encoding for arginine. In a preferred embodiment, the DNA sequence encoding for arginine is ligated to the DNA sequence of the leader peptide through the DNA sequence encoding the affinity tag.
The leader peptide of SEQ ID NO. 2 includes DNA sequence encoding for Methionine in its N-terminal end. The DNA sequence for Methionine is followed up by addition of DNA sequence encoding for glycine. The addition of glycine provides stability to the proinsulin – protein fusion. The proinsulin and leader peptide assembly enables single step digestion using Trypsin to separate insulin molecule from leader peptide and C-chain. Furthermore, there is no arginine in the leader peptide sequence.
The leader peptide of SEQ ID NO 2 is a neutral peptide with nearly as many hydrophobic amino acids as hydrophilic amino acids. In one embodiment, the leader peptide has 49% amino acids as hydrophobic. The neutrality of the leader peptide enables increase in formation of stable proinsulin inclusion bodies when the expression construct of Figure 1 is expressed in the bacterial cells. Further, inclusion of arginine as the cleavage site for removal of the leader peptide of SEQ ID NO 2 ensures that a single step is required to cleave off the C-chain and the leader peptide from the proinsulin fusion to obtain active insulin.
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The DNA sequence for the protein of interest i.e. Insulin or its analogue is inserted in the Multiple Cloning Site (MCS) of the expression vector as shown in Figure 1. Multiple cloning site or polylinker constitutes a short segment of DNA which contains a number of (generally up to 20) Restriction Enzyme (RE) sites - a standard feature of engineered plasmids.
In a preferred embodiment, the leader peptide and the MCS are custom synthesised as single stranded oligonucleotides, which are used for synthesis of double stranded DNA fragment by PCR. In one embodiment, the overlapping PCR method is used to synthesis double stranded DNA. Optionally, the leader peptide and the MCS may be directly synthesised as double stranded DNA fragments. Further, the RE sites were incorporated at 5’ end and the 3’ end of the synthesised DNA fragment. Furthermore, a Promoter/Operator region, a Ribosome Binding Site (RBS), an origin of replication and a antibiotic resistant gene were ligated with the PCR amplified DNA sequence coding for leader peptide, followed by MCS containing unique restriction enzyme sites. In one embodiment, the leader peptide is cloned downstream of the RBS, between Nco1 and EcoR1 restriction sites in the MCS.
Accordingly, the cleavage site, to cleave off the leader peptide and elicit a recombinant peptide/protein of interest, may be customised according to the recombinant
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peptide/protein of interest. The heterologous protein or the protein of interest may be cloned between any of the two RE sites in the MCS.
In an embodiment, the expression construct of Figure 1 encodes a compound of Formula (I)
A-L-X-P
in which, L is the leader peptide of SEQ ID NO 3, A is the affinity tag, X is the cleavage site and P is a heterologous protein. In another embodiment, the expression construct of Figure encodes a compound of Formula (II)
L-A-X-P
In another embodiment, the expression construct of Figure 1 encodes a compound of formula (III):
A-L-Arg-B-C-A
Or a compound of formula (IV):
L-A-Arg-B-C-A
in which, L is the leader peptide, A is a his-tag, acting as the affinity tag with six consecutive histidine residues, arginine is the cleavage site that links the leader peptide via the his-tag in its C-terminal end to the B chain of Proinsulin, whereas C is the C chain of Pronsulin and A is the A chain of Proinsulin. In one
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embodiment, the C-chain of Proinsulin includes an arginine residue only.
In another embodiment, the expression construct of Figure 1 encodes a compound of formula (V):
L-A-Arg-A-C-B
Or a compound of formula (VI):
A-L-Arg-A-C-B
in which arginine, the cleavage site links the leader peptide via the his-tag in its C-terminal end to the A chain of Proinsulin.
In one embodiment, the leader peptide of SEQ ID NO 2 has first amino acid residue as methionine and the second amino acid residue as glycine, which imparts stability to the leader peptide. The advantage of having the arginine residue as the cleavage site to cleave off the leader peptide post-expression in the bacterial cells is that it enables single step, double reaction based enzymatic digestion of the compounds of formula I, II, III, IV, V or VI.
The embodiments above are further explained through way of examples as follows:
Examples:
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Example 1: Construction of Vector
The oligonucleotides for the human proinsulin (hPI) gene were custom synthesized (Sigma Aldrich). The single stranded oligonucleotides were reconstituted in 10 mM TE buffer (pH - 8.0). The 0.5 uM of each forward and reverse oligonucleotide was used for PCR reaction to form double stranded DNA. The cycling conditions used for the PCR were: one cycle of 95°C for 5 min for initial denaturation, followed by 35 cycles comprising of denaturation at 95°C for 20 sec, annealing at 65°C for 20 sec and elongation at 72°C for 30 sec. The final extensions of 5 min at 72°C were included for the complete synthesis of the gene. The series of sequential PCR reactions were carried out to synthesize the complete hPI gene. The EcoRI and XhoI restriction enzyme sites were incorporated at the 5’ end and the 3’ end of the hPI gene respectively in the final PCR amplification. The sequence ID of the vector synthesized herein is SEQ ID No 1.
Example 2: Purification of hPI gene
The hPI (human proinsulin) gene was purified using phenol chloroform iso-amyl alcohol (25:24:1 ratio) extraction method and precipitated using ethanol. The pellet obtained was washed with 70% ethanol, air dried and reconstituted in 10 mM Tris buffer (pH 8.0).
Example 3: Cloning hPI gene in the vector
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10 ug of the plasmid DNA described herein and purified hPI gene were digested in 50 µl of reaction volume containing 1 X restriction buffer with 10 Units each of EcoR I and Xho I (MBI Fermentas). The reaction was incubated for 30 min at 37°C in the water bath. The digested plasmid DNA and hPI gene were purified using Qiagen gel Extraction Kit and the purified samples were eluted in 30 µl of elution buffer. The 10 µl ligation reactions were set using different vector to insert ratio and 4 Weiss units of T4 DNA ligase (MBI Fermentas). The ligation reaction were incubated at 4°C for 16 hours and then transformed into DH5a strain of E. coli. The transformants were selected on Luria agar containing 75µg/ml of Kanamycin. The sequence identity of the desired hPI gene is confirmed by nucleotide sequencing using automated DNA sequencer (CEQ 8000, Beckman Coulter).
Example 4: Transforming E. coli cells
The vector-hPI DNA was transformed into E. coli expression host BL21 (DE3) and was allowed to grow in standard culture conditions. After the fermentation was completed, the inclusion bodies were isolated after lysing of cells. The inclusion bodies contained human pro-insulin in unfolded form.
Example 5: Isolation and purification of Refolded Insulin from human proinsulin
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The inclusion bodies having human proinsulin were further reduced and subjected to refolding using conventional methods in the presence of cysteine and cystine. The cysteine to cystine ratio was used in the ratio of 1: 10. The refolding was performed at alkaline pH in the range of 8 – 10.5, preferably 9.5. The refolding reaction was incubated for 24 h at 4oC. The refolded Proinsulin was converted to mature insulin by proteolysis using trypsin and Carboxypeptidase b with a ration of Proinsulin to enzyme of 300:1 and 600:1 (w/w), respectively. Digestion was performed in 0.1 M Tris/HCl, 1 mM MgCl2, pH 7.5 at ambient temperature for 30 min. Figure 2 illustrates MALDI-TOF spectrum obtained for Human Insulin and leader peptide obtained post enzymatic digestion of human Proinsulin, in accordance with the embodiments described herein. The peak of 5.8 kDa corresponds to Human Insulin and mass of 4.75 kDa corresponds to leader peptide.Hence, proving a single step digestion using the expression vector as described herein.
Example 6: Expression analysis
SDS PAGE analysis of Human Insulin and Insulin analogues expressed from control vector and the vector described herein was performed. The reaction was run on 15 % SDS-PAGE and stained with Coomassie brilliant blue.
Figure 3 illustrates SDS PAGE analysis of insulin and insulin analogues expressed in a control vector and in
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the vector of Figure 1. Lane 1 shows medium molecule weight marker, Lane2 shows Human Insulin uninduced sample from control vector, Lane 3 shows Human Insulin expressed from control vector, Lane 4 shows Human Insulin uninduced sample from the vector described herein, Lane 5 shows Human Insulin expressed from the vector described herein, Lane 6 shows Insulin Aspart uninduced sample from control vector, Lane 7 shows Insulin Aspart expressed from control vector, Lane 8 shows Insulin Aspart uninduced sample from the vector described herein, Lane 9 shows Insulin Aspart expressed from the vector described herein, Lane 10 shows Insulin Lispro uninduced sample from control vector, Lane 11 shows Insulin Lispro expressed from control vector, Lane 12 shows Insulin Lispro uninduced sample from the vector described herein, Lane 13 shows Insulin Lispro expressed from the vector described herein.