Claims:1. An isolated nucleotide sequence of SEQ ID NO 2.
2. The isolated nucleotide sequence as claimed in claim, wherein said isolated nucleotide sequence is included in an expression vector or plasmid meant for expression of heterologous proteins in a bacterial cell.
3. The isolated nucleotide sequence as claimed in claim 1, wherein said isolated nucleotide sequence encodes for a leader peptide of SEQ ID NO 3.
4. The isolated nucleotide sequence as claimed in claim 1, wherein said isolated nucleotide sequence is ligated with sequence of a heterologous protein.
5. The isolated nucleotide sequence as claimed in claim 2, wherein said heterologous protein is granulocyte colony stimulating factor (GCSF).
6. The isolated nucleotide sequence as claimed in claim 2, wherein said isolated nucleotide sequence is ligated with sequence of a heterologous protein via a restriction endonuclease [RE] site.
7. The isolated nucleotide sequence as claimed in claim 1, wherein said isolated nucleotide sequence includes DNA sequence encoding for Methionine in its N-terminal end.
8. The isolated nucleotide sequence as claimed in claim 1, wherein said leader peptide of SEQ ID NO 3 is neutral.
9. A leader peptide of SEQ ID NO 3.
10. The leader peptide as claimed in claim 9, wherein said leader peptide is expressed as fused with a heterologous protein via an RE site.

, 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

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 universal plasmid vectors which lead to high yield of heterologous proteins through simple purification processes. The embodiments herein provide a plasmid vector having nucleotide sequence listed under SEQ ID NO. 1.

The expression construct of Figure 1 includes a DNA sequence, of SEQ ID NO 2 encoding for the leader peptide of SEQ ID NO. 3. 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 protein-leader peptide fusion.

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 formation of stable inclusion bodies when the expression construct of Figure 1 is expressed in the bacterial cells.

The DNA sequence for the protein of interest 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 synthesise 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.

The protein of interest may include filgrastim, interferon, human growth hormone, trypsin, carboxypeptidase, transferrin and various such recombinant proteins and peptides of therapeutic and non-therapeutic significance. A cleavage site may be included between the leader peptide and the protein of interest to cleave off the leader peptide and purify recombinant protein from the inclusion bodies. The expression vector of the embodiments herein has a sequence of SEQ ID No 1. The gene of interest may be inserted in any of the cleavage sites in the MCS.

The embodiments above are further explained through way of examples as follows:

Examples
Example 1: Construction of vector
The nucleotide sequence coding the leader peptide and the multiple cloning sites (MCS) were custom synthesized as single stranded oligonucleotides. The single stranded oligonucleotides were utilized for the synthesis of double stranded DNA fragment by overlapping PCR method. The restriction enzyme (RE) sites were incorporated at 5’ end and 3’ end of the synthesized DNA fragment. The Promoter/Operator region, Ribosome binding site (RBS), origin of replication and antibiotic resistant gene were cleaved and ligated with the PCR amplified leader peptide sequence in MCS region containing unique restriction enzyme sites. The DNA fragment was cloned downstream of RBS between the Nco I and Xho I restriction site. Thereafter, the positive clones were screened by PCR method and the nucleotide sequence of the cloned Leader sequence and MCS were confirmed by DNA sequencing for the correctness of nucleotides. The construction of vector employs standard techniques, reagents and/or kits.

Example 2: SDS PAGE analysis of GCSF expressed from the vector described herein
The sequence encoding for GCSF was incorporated in the MCS of the expression vector described herein along with in a control vector devoid of any leader sequence. In the vector described herein, an enzymatic site for Enterokinase is inserted between the leader sequence and the GCSF sequence. The expression vector was cloned in bacterial cells and the GCSF inclusion bodies were obtained. The leader peptide was cleaved off by enzymatic and/or chemical means and the expression of GCSF from both the vectors was analysed on SDS PAGE as shown in Figure 2a. Lane 1 shows Medium molecule weight marker, Lane 2 shows expression sample GCSF from control vector, Lane 3 shows GCSF expression sample from pBG-BactX vector. As may be observed, there is negligible expression of GCSF from the control vector. Figure 2b illustrates gel densitometry data comparison for expression of GCSF in control vector and in the vector as described herein. Lane 1 shows Medium molecule weight marker, Lane 2 shows densitometry data for GCSF expression in the control vector and Lane 3 shows densitometry data for GCSF expression in the vector as described herein.

Example 3: Comparison of expression levels of GCSF by MALDI TOF analysis
Figure 3a and 3b illustrates comparison of GCSF expression in a control vector with GCSF expression in vector of Figure 1, according to an embodiment herein. The expression level in control vector is negligible whereas the expression level in the vector described herein has expression level of 3o%.