DESC:TECHNICAL FIELD
The present disclosure describes a method for production of recombinant peptides and proteins. In particular, the present disclosure relates to a method for the production of insulin and insulin analogs by expression in host cell including bacteria, yeast, or a combination thereof. The expression construct described in the present disclosure includes the usage of fusion tag/leader sequence for the expression and accumulation of recombinant protein in the host cell.

BACKGROUND OF THE DISCLOSURE
Controlled expression of the desired polypeptide or protein is accomplished by coupling the gene encoding the protein through recombinant DNA techniques downstream of a promoter, the activity of which can be regulated by external factors. This expression construct is carried on a vector, most often a plasmid. Introduction of the plasmid carrying the expression construct into a host bacterium or yeast followed by culturing that microorganism in the presence of compounds that activate the promoter results in high levels of expression of the desired protein. In this way, large quantities of the desired protein can be produced.

E. coli is the most commonly used prokaryote for protein production. There is an increasing worldwide demand for human insulin and its analogs, requiring highly productive expression systems. Eli Lily made the first recombinant insulin available from an E. coli expression system in 1982 (humulin). E. coli continues to be used for recombinant insulin and insulin analogs production (e.g. Eli Lily-Humalog, Sanofi Aventis-Lantus, Sanofi Aventis- Aprida). Human Insulin is a polypeptide consisting of two separate chains which are A chain (21 amino acids) and the B – chain (30 amino acids) linked by a characteristic pattern of disulfide bridges. Formation of native insulin from Proinsulin follows two steps, the first step involves folding and formation of disulphide bridges and the second step involves proteolytic cleavage with subsequent release of C peptide.

One of the major obstacles experienced in the production of (pro) insulin is the rapid intracellular degradation of the recombinant protein. Pro insulin is in general produced as a fusion partner responsible for directing the recombinant gene product towards the formation of inclusion bodies. Further, different approaches have been tried for the expression of human insulin and its analogs in E.coli. For example, one approach is based on expression of proinsulin without any fusion partner but with N- terminal methionine which can be later removed by Cyanogen bromide or proteolytic cleavage. However, the aforesaid prior art approaches suffer from various limitations/drawbacks such as low expression, low yield, stability and purity of the recombinant protein/peptide.

Peptides employed as tags for production of proteins are found to be either too specific to certain heterologous proteins or are too large. The use of higher molecular weight peptides often results in yields that are deceptive when used as fusion tags to small molecular weight proteins. The ratio of the protein of interest to the fusion tag has always been known to tilt towards an increased amount of the fusion tag thus rendering the entire exercise futile.

Hence, the present disclosure aims at overcoming the drawbacks of prior art by providing an improved method for enhanced expression of recombinant protein/peptides.

STATEMENT OF DICLOSURE
The present disclosure relates to method for obtaining recombinant insulin or analogue thereof, said method comprising -
a) designing an expression construct comprising leader sequence - B – C – A, wherein the ‘leader sequence’ is selected from tryptophan operon leader (TrpLE) or fragment derived from thioredoxin protein; ‘B’ represents B chain of the insulin or analog thereof, ‘A’ represents A chain of the insulin or analog thereof; and ‘C’ represents connecting peptide, and wherein the leader sequence is fused to N-terminal of the B-chain,
b) cloning the expression construct into a vector,
c) transforming a host cell with the vector, and
d) culturing the host cell to obtain the insulin or analog thereof.

BRIEF DESCRIPTION OF ACCOMPANYING FIGURES
In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figure together with a detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, in accordance with the present disclosure wherein:

Figure 1 shows schematic representation of TrpLE leader peptide fused to B chain which is connected to A chain through C peptide.
Figure 2 shows Vector map of pET41b (+).
Figure 3 shows Vector map of pET41b (+) cloned with TrpLE DDDDK Insulin at NdeI and BamHI.
Figure 4 shows Vector map of pET41b (+) cloned with TrpLE KR Insulin at NdeI and BamHI.
Figure 5 shows Vector map of pET41b (+) cloned with TrpLE KR Glargine at NdeI and BamHI.
Figure 6 shows Vector map of pET41b (+) cloned with TrpLE DDDDK Glargine at NdeI and BamHI.
Figure 7 shows Vector map of pET41b (+) cloned with TrpLE DDDDK Lispro at NdeI and BamHI.
Figure 8 shows Vector map of pET41b (+) cloned with TrpLE DDDDK KCH Lispro at NdeI and BamHI.
Figure 9 shows TrpLE DDDDK Insulin expression in E.coli BL21 DE3 cells transformed with TrpLE DDDDK Insulin/ pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: Clone 5 Uninduced, 2: Clone 5 Induced, 3: Clone 6 Uninduced, 4: Clone 6 Induced, 5: See blue prestained ladder, 6: Clone 7 Uninduced, 7: Clone 7 Induced, 8: Clone 8 Uninduced, 9: Clone 8 Induced samples.
Figure 10 shows TrpLE DDDDK Insulin (with R as the C peptide) expression in E.coli BL21 DE3 cells transformed with TrpLE DDDDK Insulin/ pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: See blue prestained ladder, 2: Clone 1 Uninduced, 3: Clone 1 Induced, 4: Clone 2 Uninduced, 5: Clone 2 Induced, 6: Clone 3 Uninduced, 7: Clone 3 Induced, 8: Clone 4 Uninduced, 9: Clone 4 Induced samples.
Figure 11 shows TrpLE DDDDK Insulin (with DDDDK as the C peptide) expression in E.coli BL21 DE3 cells transformed with TrpLE DDDDK Insulin/ pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: Clone 1 Uninduced, 2: See blue prestained ladder, Lanes 3, 4, 5, 6, 7, 8, 9 and 10: Clones 1 to 8 Induced samples.
Figure 12 shows TrpLE KR Insulin expression in E.coli BL21 DE3 cells transformed with TrpLE KR Insulin /pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: See Blue Prestained ladder, 2: Clone 1 Uninduced, Lanes 3, 4, 5, 6, 7, 8, 9, and 10: Clones 1 to 8 Induced samples.
Figure 13 shows TrpLE DDDDK Glargine expression in E.coli BL21 DE3 cells transformed with TrpLE DDDDK Glargine/pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: Clone 1 Uninduced, 2: Clone 1 Induced, 3: See Blue Prestained Ladder, Lanes 4, 5, 6, 7 and 8: Clones 1 to 6 Induced samples.
Figure 14 shows TrpLE KR Glargine expression in E.coli BL21 DE3 cells transformed with TrpLE KR Glargine/pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: Clone 1 Uninduced, 2: See Blue Prestained Ladder, Lanes 3, 4, 5, 6, 7 and 8: Clones 1 to 6 Induced samples.
Figure 15 shows comparison of Insulin and TrpLE – Insulin expression along with Glargine and TrpLE Glargine expression on Tricine SDS PAGE. Lane 1: See Blue Prestained Ladder, Lanes 2 and 3: Insulin Uninduced and Induced, Lanes 4 and 5: TrpLE Insulin Uninduced and Induced, Lanes 6 and 7: Glargine Uninduced and Induced, Lanes 8 and 9: TrpLE Glargine Uninduced and Induced.
Figure 16 shows TrpLE DDDDK Lispro expression in E.coli BL21 DE3 cells transformed with TrpLE DDDDK Lispro/pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: Clone 1 Uninduced, 2: See Blue Prestained Ladder, Lanes 3, 4, 5, 6, 7, 8, 9 and 10: Clones 1 to 8 Induced samples.
Figure 17 shows TrpLE DDDDK KCH Lispro expression in E.coli BL21 DE3 cells transformed with TrpLE DDDDK Lispro/pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: Clone 1 Uninduced, 2: See Blue Prestained Ladder, 3, 4, 5, 6, 7, 8, 9, and 10: Clones 1 to 8 Induced samples.
Figure 18 shows Thioredoxin Insulin (TRXINS) expression in E.coli BL21 DE3 cells transformed with TRXINS/pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: See blue prestained ladder 2: Clone 1 Uninduced, 3: Clone 1 Induced, 4: Clone 2 Uninduced, 5: Clone 2 Induced, 6: Clone 3 Uninduced, 7: Clone 3 Induced, 8: Clone 4 Uninduced, 9: Clone 4 Induced samples.
Figure 19 shows schematic representation of thioredoxin tag (124 amino acids) sectioned into 4 different fragments of ~30 amino acids each.
Figure 20 shows sSchematic representation of truncations in the thioredoxin tag from N terminus and its fusion with Gene of Interest (GOI) for expression in E.coli.
Figure 21: shows schematic representation of truncations in the thioredoxin tag from C terminus and its fusion with Gene of Interest (GOI) for expression in E.coli.
Figure 22 shows ANTRX Insulin (ANTRXINS) expression in E.coli BL21 DE3 cells transformed with ANTRXINS/pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: See Blue Prestained Ladder, Lane 2: Clone 1 Uninduced, Lanes 3, 4, 5, 6, 7, 8 and 9: Clones 1 to 7 Induced samples.
Figure 23 shows BNTRX Insulin (BNTRXINS) expression in E.coli BL21 DE3 cells transformed with BNTRXINS/pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: Clone 1 Uninduced, Lane 2: Clone 1 Induced, Lane 3: See Blue Prestained Ladder, Lanes 4, 5, 6, 7, 8, 9 and 10: Clones 1 to 8 Induced samples.
Figure 24 shows CNTRX Insulin (CNTRXINS) expression in E.coli BL21 DE3 cells transformed with CNTRXINS/pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: Clone 1 Uninduced, Lane 2: See Blue Prestained Ladder, Lane 3: Clone 1 Induced, Lanes 4, 5, 6, 7, 8, 9 and 10: Clones 1 to 8 Induced samples.
Figure 25 shows DCTRX Insulin (DCTRXINS) expression in E.coli BL21 DE3 cells transformed with DCTRXINS/pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: See Blue Prestained Ladder, Lanes 2 and 3: Clone 1 and 2 Uninduced, Lanes 4, 5, 6, 7, 8, 9 and 10: Clones 1 to 8 Induced samples.
Figure 26 shows ECTRX Insulin (ECTRXINS) expression in E.coli BL21 DE3 cells transformed with ECTRXINS/pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: See Blue Prestained Ladder, Lane 2 Clone 1 Uninduced, Lanes 3, 4, 5, 6 and 7: Clones 1 to 5 Induced samples.
Figure 27 shows FCTRX Insulin (FCTRXINS) expression in E.coli BL21 DE3 cells transformed with FCTRXINS/pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: See Blue Prestained Ladder, Lane 2 Clone 1 Uninduced, Lanes 3, 4, 5, 6, 7, 8, 9 and 10: Clones 1 to 8 Induced samples.
Figure 28 shows FCTRXINS (1 – 15), FCTRXINS (10 - 24), ANTRXINS (19 – 30) expression studies were carried out E.coli BL21 DE3 cells transformed with respective plasmids and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: See Blue Prestained Ladder, Lane 2: Clone 1 FCTRXINS (1 – 15) Uninduced, Lanes 3, 4, 5, 6 and 7 Clones 1 to 5 FCTRXINS (1 – 15) Induced samples, Lane 8: Clone 1 FCTRXINS (10 – 24) Uninduced, Lanes 9, 10, 11, 12 and 13: Clones 1 to 5 FCTRXINS (10 – 24) Induced samples, Lane 14: See Blue Prestained Ladder, Lane 15: Clone 1 ANTRXINS (19 – 30) Uninduced, Lanes 16, 17, 18, 19 and 20: Clones 1 to 5 ANTRXINS (19 – 30) Induced samples.
Figure 29 shows FCTRXINS (1 – 10) expression in E.coli BL21 DE3 cells transformed with FCTRXINS (1 - 10) /pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: See Blue Prestained Ladder, Lane 2 Clone 1 Uninduced, Lanes 3, 4, 5, 6, 7, 8, 9 and 10: Clones 1 to 8 Induced samples.
Figure 30 shows FCTRXINS (5 – 20) expression in E.coli BL21 DE3 cells transformed with FCTRXINS (5 - 20) /pET41b (+) plasmid and induced with 1mM IPTG overnight. The samples are normalized to 0.5 OD, pelleted, re-suspended, solubilized using sample solubilizing gel buffer and loaded on Tricine SDS PAGE. Lane 1: Clone 1 Uninduced Lane 2: Clone 1 Induced, Lane 3: See Blue Prestained Ladder, Lanes 4, 5, 6, 7, 8, 9 and 10: Clones 1 to 8 Induced samples.
Figure 31 shows Vector map of pADB9
Figure 32 shows vector map of TrpLE Aspart pADB9
Figure 33 shows TrpLE Aspart expression in Saccharomyces cerevisiae. Lane 1: Saccharomyces host control; Lane 2: Aspart standard; Lane 3: see Blue Prestained ladder; Lane 4: TrpLE Aspart clone 1 without Galactose (Host Control); Lane 5: TrpLE Aspart clone 1 induced with 2% Galactose.
Figure 34 shows vector map of TrpLE Aspart PEP4 pADB11
Figure 35 shows TrpLE Aspart expression in PEP4 knocked out Saccharomyces cerevisiae clones. Lane 1: Host control; Lane M : See blue prestained ladder; Lanes 3, 4, 5, 6, 7 and 8 – TrpLE Aspart expressing PEP4 knock out clones.
Figure 36 analytical RP-HPLC profile of inclusion bodies.
Figure 37 shows RP-HPLC profile of inclusion bodies (fusion insulin) after solubilization -
Figure 38 shows RP-HPLC profile of inclusion bodies (fusion Glargine) after solubilization..
Figure 39 shows RP-HPLC profile of inclusion bodies (fusion Lispro) after solubilization..
Figure 40 shows RP-HPLC profile of refolded fusion insulin) after refolding..
Figure 41 shows RP-HPLC profile of refolded fusion Glargine after refolding..
Figure 42 shows RP-HPLC profile of refolded fusion Lispro after refolding.
Figure 43 shows RP-HPLC profile of proinsulin after Enterokinase digestion.
Figure 44 shows RP-HPLC profile of proglargine after Enterokinase digestion.
Figure 45 shows RP-HPLC profile of prolispro after Enterokinase digestion.
Figure 46 shows RP-HPLC profile of purified proinsulin after Enterokinase digestion.
Figure 47 shows RP-HPLC profile of purified proglargine after Enterokinase digestion.
Figure 48 shows RP-HPLC profile of purified prolispro after Enterokinase digestion.
Figure 49 shows RP-HPLC profile of insulin conversion from pro insulin by trypsin and carboxypeptidase digestion.
Figure 50 shows RP-HPLC profile of glargine conversion from pro-glargine by trypsin and carboxypeptidase digestion.
Figure 51 shows RP-HPLC profile of lispro conversion from pro-lispro by trypsin and carboxypeptidase digestion.
Figure 52 shows RP-HPLC profile of partial purified insulin after first preparative chromatography.
Figure 53 shows RP-HPLC profile of partial purified glargine after first preparative chromatography.
Figure 54 shows RP-HPLC profile of partial purified lispro after first preparative chromatography.
Figure 55 shows RP-HPLC profile (EP)of insulin reference standard ( innovator product)
Figure 56 shows RP-HPLC profile (EP) of purified in-house insulin sample after second preparative chromatography
Figure 57 shows overlay RP-HPLC profile (EP) of reference standard and purified in-house insulin sample after second preparative chromatography.
Figure 58 shows SE-HPLC profile (EP) of insulin reference standard (innovator product)
Figure 59 shows SE-HPLC profile (EP) of purified in-house insulin sample after second preparative chromatography
Figure 60 shows overlay SE-HPLC profile (EP) of reference standard and purified in-house insulin sample after second preparative chromatography.
Figure 61 shows intact mass of insulin (reference standard/ innovator product)
Figure 62 shows intact mass of purified in-house insulin
Figure 63 shows SDS PAGE (silver stained) under reducing condition of insulin ( reference standard ) and purified in-house insulin sample. Lane 1: molecular weight marker, Lane 2: reference standard (10 µg), Lane 3 and 4: in-house sample (10 µg) and (2 µg).
Figure 64 shows SDS PAGE (silver stained) under non-reducing condition of insulin (reference standard) and purified in-house insulin sample. Lane 1: molecular weight marker, Lane 2: reference standard (10 µg), Lane 3 and 4: in-house sample (10 µg) and (2 µg).
Figure 65 shows RP-HPLC profile (EP) of Lispro reference standard (innovator product)
Figure 66 shows RP-HPLC profile (EP) of purified in-house Lispro sample after second preparative chromatography
Figure 67 shows overlay RP-HPLC profile (EP) of reference standard (lispro) and purified in-house Lispro sample after second preparative chromatography.
Figure 68 shows SE-HPLC profile (EP) of Lispro reference standard (innovator product)
Figure 69 shows SE-HPLC profile (EP) of purified in-house Lispro sample after second preparative chromatography
Figure 70 shows overlay SE-HPLC profile (EP) of reference standard (Lispro) and purified in-house Lispro sample after second preparative chromatography.
Figure 71 shows RP-HPLC profile (EP) of Glargine reference standard (innovator product)
Figure 72 shows RP-HPLC profile (EP) of purified in-house glargine sample after second preparative chromatography
Figure 73 shows overlay RP-HPLC profile (EP) of reference standard ( glargine) and purified in-house glargine sample after second preparative chromatography.
Figure 74 shows SE-HPLC profile (EP) of glargine reference standard (innovator product)
Figure 75 shows SE-HPLC profile (EP) of purified in-house glargine sample after second preparative chromatography
Figure 76 shows overlay SE-HPLC profile (EP) of reference standard (glargine) and purified in-house glargine sample after second preparative chromatography.
Figure 77 shows SDS PAGE (silver stained) under non reducing condition of glargine (reference standard ) and purified in-house glargine sample. Lane 1: molecular weight marker, Lane 2: reference standard (10 µg), Lane 3, 4 and 5: in-house sample (2 µg), (5 µg) and (10 µg) respectively.
Figure 78 shows SDS PAGE (silver stained) under reducing condition of glargine (reference standard) and purified in-house glargine sample. Lane 1: molecular weight marker, Lane 2: reference standard (10 µg), Lane 3, 4 and 5: in-house sample (2 µg), (5 µg) and (10 µg) respectively.

DETAILED DESCRIPTION OF THE DISCLOSURE
The present disclosure relates to method for obtaining recombinant insulin or analogue thereof, said method comprising -
a) designing an expression construct comprising leader sequence - B – C – A, wherein the ‘leader sequence’ is selected from tryptophan operon leader (TrpLE) or fragment derived from thioredoxin protein; ‘B’ represents B chain of the insulin or analog thereof, ‘A’ represents A chain of the insulin or analog thereof; and ‘C’ represents connecting peptide, and wherein the leader sequence is fused to N-terminal of the B-chain,
b) cloning the expression construct into a vector,
c) transforming a host cell with the vector, and
d) culturing the host cell to obtain the insulin or analog thereof.

In an embodiment of the present disclosure, the expression construct comprises promoter selected from inducible promotor or constitutive promoter; and wherein said promoter is not a tryptophan (Trp) promoter.

In another embodiment of the present disclosure, the promoter is selected from a group comprising T5, T7, AOX, GAP, GAL, TPI, TEF, CUP1, PL, PR, TDH and ILV and PHOA, or any combination thereof.

In yet embodiment of the present disclosure, the insulin analog is selected from a group comprising glargine, aspart, lispro, glulisine and insulin detemir, or any combination thereof.

In still embodiment of the present disclosure, the leader sequence comprises a cleavage site selected from a group comprising nucleotide sequence corresponding to DDDDK, KR, KK, RR, RK, K and R, or any combination thereof; and wherein the leader sequence is conjugated to N-terminal of the B-chain via said cleavage site.

In still embodiment of the present disclosure, the leader sequence comprising cleavage site is selected from a group comprising SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 14 and combinations thereof.

In still embodiment of the present disclosure, the connecting peptide comprises nucleotide sequence corresponding to any one of DDDDK, KR, KK, RR, RK, K and R, or any combination thereof.
In still embodiment of the present disclosure, the leader - B – C – A sequence is selected from a group comprising SEQ ID No. 29 to 35.

In still embodiment of the present disclosure, the expression construct is cloned into the vector in tandem repeats in copies ranging from about 2 to about 20, preferably about 12.

In still embodiment of the present disclosure, wherein the vector is selected from a group comprising pET series, TA vector and combinations thereof.

In still embodiment of the present disclosure, the host cell is a prokaryote selected from a group comprising E.coli K12, E.coli BL21DE3, E.coli K12 DE3 and combinations thereof, or a eukaryote selected from a group comprising Pichia pastoris, Saccharomyces cerevisiae, Hansenula sp., Yarrowia sp., Kluveromyce sp. and combinations thereof.

In still embodiment of the present disclosure, the transformation of vector into host cell is carried out by technique selected from a group comprising heat shock, electroporation, spheroplast and combinations thereof.

In still embodiment of the present disclosure, the culturing of the host cell yields precursor of insulin or analog thereof; and said precursor is converted to functional insulin or analog thereof by – a) cleavage of the leader peptide and the connecting peptide, and cleavage of C-terminal extension of the B-chain.

In still embodiment of the present disclosure, the expressed precursor of insulin or analog thereof accumulate as inclusion bodies in the cytoplasm of host cell.

In still embodiment of the present disclosure, the leader peptide and the connecting peptide is cleaved by protease enzyme selected from a group comprising kexin, trypsin and a combination thereof; and the C-terminal extension of the B-chain is cleaved by a protease enzyme carboxypeptidase B.

In still embodiment of the present disclosure, the protease enzymes are employed in modes selected from a group comprising batch, fed-batch, continuous, immobilized mode or any combination thereof.

In still embodiment of the present disclosure, the functional insulin or analog thereof obtained is purified by a technique selected from a group comprising ion exchange chromatography, hydrophobic chromatography, multiple high pressure reverse phase chromatography or any combination thereof.

In still embodiment of the present disclosure, purity of the obtained functional insulin or analog thereof ranges from about 95% to about 100%, preferably about 97% to about 99.8%.

To overcome the non-limited drawbacks of the prior art and to provide for improved production of proteins and peptides, the present disclosure relates to the use of fusion tags for recombinant proteins and peptides.

The present disclosure relates to an expression construct for insulin or its analogs comprising leader peptide, inducible or constitutive promoter and gene of interest.

In an embodiment, the present disclosure provides expression construct involving fusion tags for the expression and accumulation of recombinant protein in the cell. In one embodiment, the leader peptide in the expression construct for insulin or its analogs is TrpLE leader peptide of tryptophan operon which acts as a fusion partner for the expression of pro insulin or its analogs. TrpLE sequence is a fragment of trp leader polypeptide fused to a portion of trpE polypeptide. TrpLE as fusion partner or leader peptide promotes the accumulation of expressed proteins as inclusion bodies in the cytoplasm due to its hydrophobic nature.

In embodiments of the present disclosure, the term leader peptide, leader sequence, leader tag, fusion tag, fusion protein, fusion partner and fusion fragment have the same scope and meaning and are used interchangeably.

In the present disclosure, the fusion tag may be a single peptide or a combination of 2 or more peptides from different sources. The term fusion tag denotes a peptide ranging from about 5 amino acids to 124 amino acids in length, which is fused to the gene of the protein of interest to increase its expression.

In embodiments of the present disclosure, the fusion tag is attached to a heterologous / homologous protein.

In yet another exemplary embodiment of the present disclosure, regions varying from as long as but not limited to 90 amino acids to as short as 10 amino acids are used as fusion partner, that promote the accumulation of expressed proteins as inclusion bodies in the cytoplasm.

In another embodiment, said Trp LE leader peptide is a codon optimized sequence with its cleavage site. In an embodiment, the Trp LE leader peptide is selected from a group comprising SEQ ID NO 1 and SEQ ID NO 2 having cleavage sites DDDDK [asp-asp-asp-asp-lys] (in SEQ ID NO: 1) and KR [ lysine-Arginine] (in SEQ ID NO: 2) respectively. In other embodiments, Trp LE leader peptide comprises KK, RR or RK as cleavage sites.

In an exemplary embodiment, the present disclosure employs use of fragments derived from the full length Thioredoxin protein as a fusion tag to heterologous proteins expressed in microbial expression systems or cell free expression systems that employ components of a microbial expression system.

In another exemplary embodiment, the fragments of Thioredoxin are employed either in isolation or in combination with fragments or other fusion proteins.

In another embodiment, the inducible / constitutive promoter in the expression construct for insulin or its analogs is selected from a group comprising T5, T7, AOX, GAP, GAL, TPI, TEF, CUP1, PL, PR, PHOA, TDH, ILV and combinations thereof. In an exemplary embodiment, the promoter is not Trp promoter.

In yet another embodiment, the gene of interest in the expression construct is insulin or insulin analog. In an embodiment, the insulin analog is selected from a group comprising glargine, aspart, lispro, glulisine and insulin detemir. In an embodiment of the present disclosure, the gene of interest is a codon optimized nucleotide sequence selected from a group comprising SEQ ID NO 3 (insulin), SEQ ID NO 6 (glargine), SEQ ID NO 9 (lispro) and SEQ ID NO 12 (aspart).

In an embodiment, the expression construct/vector is represented in Figures 3, 4, 5, 6, 7 and 8 respectively.

The present disclosure relates to a method of obtaining an expression construct for insulin or its analogs as described above, said method comprising:
a. cloning gene of insulin or insulin analog with TrpLE leader peptide in TrpLE - B – C – A fashion; and
b. cloning TrpLE - B – C – A gene into a vector to obtain an expression construct/recombinant vector comprising inducible/ constitutive promoter and TrpLE - B – C – A gene.

In an embodiment, the aforesaid step of cloning TrpLE - B – C – A sequence into a vector comprises introducing the said sequence in one or more copies in tandem repeats.

In another embodiment of the aforesaid method, fragments of thioredoxin or fragments of thioredoxin in combination with other fragments or other fusion proteins is employed as the leader peptide.

The present disclosure further relates to an expression system for insulin or its analogs, wherein said expression system is a recombinant host cell comprising expression construct/recombinant vector having leader peptide, inducible promoter and gene of interest.

In an embodiment, the leader peptide in the expression system for insulin or its analogs is TrpLE leader peptide of tryptophan operon which acts as a fusion partner for the expression of pro insulin or its analogs.
In an exemplary embodiment, the present disclosure relates to designing of fusion tags, expression of recombinant proteins as well as purification of obtained proteins.

In the present disclosure, the low protein yields are overcome by employing peptides as fusion tags which when fused to the heterologous protein greatly increase the yield. Small peptides are developed as fusion tags that not only render stability to the protein but also increase the final yield of the heterologous protein produced in expression hosts such as E. coli.

Specific domains that facilitate the accumulation of recombinant proteins as insoluble inclusion bodies in high yields are identified and further characterized. These domains are then used in different permutations and combinations as a tag to one or more recombinant proteins of therapeutic interest to indicate its use commercially.

Recombinant proteins are produced with a fusion partner responsible for directing the recombinant gene product towards the formation of inclusion bodies.

In the present disclosure, domains are identified, which function along with domains of other proteins which have a similar property of increasing the expression levels of recombinant proteins in microbial systems such as but not limited to E.coli.

The present disclosure provides for various sequences derived from protein such as a fusion partner or leader peptide for the expression of proteins or peptides including insulin or its analogs.

In embodiments of the present disclosure, short fragments derived from other well- known proteins which can serve in itself as a fusion tag and enhance the production of heterologous proteins in microbial cells including, but not limited to E.coli are provided.

Some of the non-limiting advantages offered by the method and fusion tags of the present disclosure are as follows:
• Increased yield of recombinant proteins,
• Formation of inclusion bodies with the proteins to prevent rapid intracellular degradation of the desired protein.

The present disclosure provides a method of obtaining an expression system for insulin or its analogs as described above, said method comprising:
a. cloning gene of insulin or insulin analog with TrpLE leader peptide in TrpLE - B – C – A fashion;
b. cloning TrpLE - B – C – A sequence into a vector to obtain an expression construct/recombinant vector comprising inducible promoter and TrpLE - B – C – A gene; and
c. transforming a host cell with the recombinant vector to obtain the expression system.

In an embodiment, the aforesaid step of cloning TrpLE - B – C – A sequence, or said sequence containing fragments of thioredoxin or fragments of thioredoxin in combination with other fragments or other fusion proteins as the leader peptide into a vector comprises introducing the said sequence in one or more copies in tandem repeats.

In another embodiment of the aforesaid method, fragments of thioredoxin or fragments of thioredoxin in combination with other fragments or other fusion proteins is employed as the leader peptide.

In an embodiment of the aforesaid method, fragments of thioredoxin may be employed in combination with other fragments obtained from proteins including but not limiting to glucagon, glucagon like peptides, GCSF, IFN alpha, IFN beta, or combinations thereof.

In an embodiment, the transformation step is carried out by a process selected from a group comprising heat shock, electroporation, spheroplast and combinations thereof.

The present disclosure relates to a method of enhanced expression of insulin or its analogs in a host cell by enzymatic conversion of proinsulin or its analogs to their active/functional forms.

In an embodiment, the method for expression of insulin or its analogs comprises steps of:
a. designing gene of insulin or insulin analog with TrpLE leader peptide in TrpLE - B – C – A fashion, or employing fragments of thioredoxin or fragments of thioredoxin in combination with other fragments or other fusion proteins as the leader peptide;
b. cloning the sequence obtained above into a vector to obtain an expression construct/recombinant vector comprising inducible promoter and said sequence comprising leader peptide and gene of insulin or its analog;
c. transforming a host cell with the recombinant vector; and
d. culturing the host cell followed by purification of the expression product, to obtain insulin or its analogs.

In an embodiment, the aforesaid step of cloning TrpLE - B – C – A sequence, or said sequence containing fragments of thioredoxin or fragments of thioredoxin in combination with other fragments or other fusion proteins as the leader peptide into a vector comprises introducing the said sequence in one or more copies in tandem repeats.

In another embodiment, promoters other than Trp promoter but not limited to T5, T7, Aox, Gap, Gal, TPI, TEF, CUP1, PL, PR, PHOA, TDH and ILV are employed for the expression of insulin or insulin analogs.

In an exemplary embodiment of the present disclosure, the method for expression of recombinant insulin or insulin analog comprises steps of:
a) designing insulin or its analogs in Trp LE-B-C-A format;
b) cloning Trp LE -B-C-A sequence into a vector;
c) transforming a host cell with the vector;
d) culturing host cell and expressing the respective insulin precursor in host cell;
e) cleaving of Trp LE leader peptide and C-peptide by protease;
f) removing C-terminal extension of B-chain by protease; and
g) purifying the functional insulin or its analog.

In an embodiment of the above process, the leader peptide (Trp LE) is a codon optimized sequence with its cleavage site selected from a group comprising SEQ ID NO 1 and SEQ ID NO 2 having cleavage sites corresponding to DDDDK (in SEQ ID NO: 1) and KR (in SEQ ID NO: 2) respectively.

In another exemplary embodiment of the present disclosure, the method for expression of recombinant insulin or insulin analog comprises steps of:
a) designing insulin or its analogs in leader-B-C-A format wherein the leader is fragment of thioredoxin or fragment of thioredoxin in combination with other fragments or other fusion proteins;
b) cloning leader-B-C-A sequence into a vector;
c) transforming a host cell with the vector;
d) culturing host cell and expressing the respective insulin precursor in host cell;
e) cleaving of leader peptide and C-peptide by protease;
f) removing C-terminal extension of B-chain by protease; and
g) purifying the functional insulin or its analog.

In another embodiment of the above process, the insulin or its analog (gene of interest) is a codon optimized sequence selected from a group comprising SEQ ID NO 3, SEQ ID NO 6, SEQ ID NO 9 and SEQ ID NO 12.

In yet another embodiment of the present disclosure, the host cell is prokaryotic. In an embodiment, the prokaryotic host cell is selected from a group comprising E.coli K12, E.coli BL21DE3, E.coli K12 DE3 and combinations thereof. In another embodiment of the present disclosure, the expression construct/vector carrying gene of interest in the prokaryotic host cell is inducible.

In yet another embodiment of present disclosure, the host cell is eukaryotic. In an embodiment, the eukaryotic host cell is selected from a group comprising P. pastoris, Saccharomyces cerevisiae, Hansenula, Yarrowia, Kluveromyces and combinations thereof, preferably P. pastoris. In another embodiment of the present disclosure, the expression construct/vector carrying gene of interest in the eukaryotic host cell is inducible or constitutive.

In still an embodiment of the present disclosure, the method of expression of recombinant insulin or insulin analog of the present disclosure results in high cell density expression of pro - insulin and its analogs.

The present disclosure further relates to the design of precursor molecule (insulin/insulin analogs) by optimizing the amino acid sequence of leader peptide and connecting peptide (C – peptide), mainly focusing on its susceptibility to cleavage by either Trypsin or Kexin and conversion to a functional protein by action of carboxypeptidase B. The disclosure also describes the cloning methodology, effect on expression level of insulin(s) precursor, refolding, as well as preparation of Insulin and its analogs from their precursor forms by enzymatic reaction(s) involving the simultaneous or sequential use of optimal quantities of proteases such as but not limited to Trypsin and/or Kexin and Carboxypeptidase B that works in conjunction directing the reaction in a controlled environment to avoid non-specific interactions and production of undesired byproducts followed by purification by chromatography to obtain functional insulin/insulin analogs.

The present disclosure also relates to the use of TrpLE leader sequence of Tryptophan operon as a fusion partner or leader peptide for the expression of pro insulin and its analogs. TrpLE sequence is a fragment of trp leader polypeptide fused to a portion of trpE polypeptide. TrpLE as fusion partner promotes the accumulation of expressed proteins as inclusion bodies in the cytoplasm due to its hydrophobic nature. The present disclosure also relates to the use of fragment of thioredoxin or fragment of thioredoxin in combination with other fragments or other fusion leader sequence as a fusion partner or leader peptide for the expression of pro insulin and its analogs.

The present disclosure further relates to the use of proteases selected from a group comprising Trypsin, Kexin, Carboxypeptidase B and combinations thereof for the simultaneous or sequential removal of leader peptide and C-terminal extension of B – chain of proinsulin. In an embodiment of the present disclosure, insulin or its analogs are expressed in leader - B – C – A format; B (1-29) – A (1-21), where B (1-29) is the ‘B’ chain peptide amino acid 1 to 29 and A (1-21) is the ‘A’ chain peptide amino acid 1 to 21 wherein A and B chain peptides are connected by means of a peptide bond C. Leader is connected to the N terminus of B – chain either by Enterokinase Cleavage site (such as DDDDK) or Dibasic amino acids (such as lysine, arginine, lysine-arginine, lysine-lysine or arginine-arginine) which is cleavable by protease such as Kexin or Trypsin. Further, the connecting peptide (C peptide) has at least two amino acids which permits its excision from A and B chains by Kexin or Trypsin. In other words, the leader peptide and connecting peptide are removed by incorporating a common cleavage site susceptible to Trypsin or Kexin. Post cleavage of leader peptide and connecting peptide, the C terminal extension of B-chain is removed by Carboxypeptidase B.

The present disclosure also relates to the use of DDDDK / Dibasic amino acids [including but not limiting to lys-arg (KR), arg-arg (RR), lysine (K), arginine (R) and lysine-lysine (KK)] to cleave the TrpLE leader peptide from prepro-insulin and simultaneously/sequentially cleaving the connecting peptide (C-peptide), by the protease Trypsin or Kexin.

The method of the present disclosure shows that an increased expression of insulin or its analogs is achieved when a leader sequence (optimized leader sequence) is used as a fusion peptide instead of untagged/unfused insulin or its analogs.

The present disclosure also relates to the use of ‘non-tryptophan promoter’ based expression of insulin or its analogs.

The present disclosure relates to optimal usage of proteases selected from a group comprising trypsin, kexin and carboxypeptidase-B in order to convert precursor to the active protein, and its purification. In an embodiment of the present disclosure, optimizing enzymatic reaction by using cleavage sequences specific to proteases selected from a group comprising trypsin, kexin and carboxypeptidase-B facilitates simultaneous and/or sequential:

Removal of Leader peptide;
Removal of C peptide;
Removal of C – terminal extension of B chain.

In yet another embodiment of the present disclosure, the above proteases are used in mode selected from a group comprising batch, fed-batch, continuous, immobilized mode and combinations thereof.

In an embodiment of the present disclosure, the insulin or their analogs are purified using techniques including but not limiting to ion exchange, hydrophobic chromatography, multiple high pressure reverse phase chromatography and combinations thereof. In another embodiment, the purification of insulin or its analog is carried out in a series of chromatographic steps including anion exchange chromatography at a pH range of about 6.5 to 8.5 and cation exchange chromatography at a pH range of about 3.5 to 5.5. Polishing is done by reverse phase chromatography at a pH range of about 1.5 to 4.0.

In an embodiment, purity of the functional insulin or analog thereof ranges from about 95% to about 100%, preferably about 97% to about 99.8%.
In another embodiment, the yield of the obtained functional insulin or analog thereof ranges from about 10 to about 30%, preferably about 20% to about 25%.

The present disclosure thus describes the efficient expression of recombinant proteins and peptides such as insulin or its analogs using B-chain’s N-terminal fusion tag of TrpLE leader peptide or fragment of thioredoxin or fragment of thioredoxin in combination with other fragments or other fusion leader sequence as a leader peptide.

In an embodiment of the present disclosure, the fusion gene leader- B – C – A of the present invention is introduced into the cell in one or more vector copies and/or in tandem repeats in a single vector.

An embodiment of the present disclosure relates to high cell density cultivation of prokaryotes and eukaryotes. In an exemplary embodiment, high cell density cultivation of bacteria including but not limiting to E.coli and yeast strain including but not limiting to Pichia pastoris and Saccaromyces cerevisiae cells is described.

An embodiment of the present disclosure also relates to refolding of E.coli expressed insulin or its analog precursor to obtain three dimensional structures thereby enabling conversion from inactive to active form enzymatically.

Sequence Information:
SEQ ID NO 1:
Nucleotide Sequence of TrpLE leader peptide with DDDDK:
ATGAAAGCCATCTTTGTGCTGAAAGGTAGCCTGGATCGTGATCCGGAATTTGATGATGATGATAAA

SEQ ID NO 2:
Nucleotide Sequence of TrpLE leader peptide with KR:
ATGAAAGCAATTTTTGTTCTGAAAGGTAGCCTGGATCGTGATCCGGAATTTAAACGT

SEQ ID NO 3:
Nucleotide Sequence of Insulin:
TTTGTTAATCAGCATCTGTGTGGTAGCCATCTGGTTGAAGCACTGTATCTGGTTTGTGGTGAACGTGGTTTTTTTTATACCCCGAAAACCAAACGTGTATTGTTGAACAGTGTTGTACCAGCATTTGTAGCCTGTATCAGCTGGAAAATTATTGTAATTAA

SEQ ID NO 4:
Amino acid sequence of TrpLE DDDDK Insulin:
MKAIFVLKGSLDRDPEFDDDDKFVNQHLCGSHLVEALYLVCGERGFFYTPKTKRGIVEQCCTSICSLYQLENYCN*

SEQ ID NO 5:
Amino acid sequence of TrpLE KR Insulin:
MKAIFVLKGSLDRDPEFKRFVNQHLCGSHLVEALYLVCGERGFFYTPKTKRGIVEQCCTSICSLYQLENYCN*

SEQ ID NO 6:
Nucleotide Sequence of Glargine:
TTTGTTAATCAGCATCTGTGTGGTAGCCATCTGGTTGAAGCACTGTATCTGGTTTGTGGTGAACGTGGTTTTTTTTATACCCCGAAAACCCGTCGTGGTATTGTTGAACAGTGTTGTACCAGCATTTGTAGCCTGTATCAGCTGGAAAATTATTGTGGTTAA

SEQ ID NO 7:
Amino acid sequence of TrpLE DDDDK Glargine:
MKAIFVLKGSLDRDPEFDDDDKFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRGIVEQCCTSICSLYQLENYCG*

SEQ ID NO 8:
Amino acid sequence of TrpLE KR Glargine:
MKAIFVLKGSLDRDPEFKRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRGIVEQCCTSICSLYQLENYCG*

SEQ ID NO 9:
Nucleotide Sequence of Lispro:
TTTGTGAACCAGCATCTGTGTGGTAGCCATCTGGTTGAAGCACTGTATCTGGTTTGTGGTGAACGTGGTTTTTTCTATACCAAACCGACCAAACGTGGTATTGTTGAACAGTGTTGTACCAGCATTTGTAGCCTGTATCAGCTGGAAAACTATTGCAATTAA

SEQ ID NO 10:
Amino acid sequence of TrpLE DDDDK Lispro:
MKAIFVLKGSLDRDPEFDDDDKFVNQHLCGSHLVEALYLVCGERGFFYTKPTKRGIVEQCCTSICSLYQLENYCN*

SEQ ID NO 11:
Amino acid sequence of TrpLE KCH Lispro:
MKAIFVLKGSLDRDPEFDDDDKFVNQHLCGSHLVEALYLVCGERGFFYTKPTKGIVEQCCTSICSLYQLENYCN*

SEQ ID NO 12:
Nucleotide sequence of Aspart:
TTTGTTAATCAACATTTGTGTGGTTCTCATTTGGTTGAAGCTTTGTATTTGGTTTGTGGTGAAAGAGGTTTTTTTTATACTGATAAAACTAAAAGAGGTATTGTTGAACAATGTTGTACTTCTATTTGTTCTTTGTATCAATTGGAAAATTATTGTAATTAA

SEQ ID NO 13:
Amino acid sequence of TrpLEDDDDK Aspart:
MKAIFVLKGSLDRDPEFDDDDKFVNQHLCGSHLVEALYLVCGERGFFYTDKTKRGIVEQCCTSICSLYQLENYCN*

SEQ ID NO 14:
Nucleotide sequence of Thioredoxin:
ATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCGATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCTGACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATATGGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGGCAACCAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTTCTGGTTCTCATCACCATCACCATCAC

SEQ ID NO 15:
Amino acid sequence of Thioredoxin
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSHHHHHHDDDDK

SEQ ID NO 16:
Amino acid sequence of ANTRX
MSDKIIHLTDDSFDTDVLKADGAILVDFWADDDDK

SEQ ID NO 17:
Amino Acid sequence of BNTRX
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNDDDDK

SEQ ID NO 18:
Amino Acid sequence of CNTRX
MSDKIIHLTDDSFDTDVLKADGAILVDFWAEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNDDDDK

SEQ ID NO 19:
Amino Acid sequence of DCTRX
MEWCGPCKMIAPILDEIADEYQGKLTVAKLNIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSHHHHHHDDDDK

SEQ ID NO 20:
Amino Acid sequence of ECTRX
MIDQNPGTAPKYGIRGIPTLLLFKNGEVAATKVGALSKGQLKEFLDANLAGSGSHHHHHHDDDDK

SEQ ID NO 21:
Amino Acid sequence of FCTRX
MKVGALSKGQLKEFLDANLAGSGSHHHHHHDDDDK

SEQ ID NO 22:
Amino Acid sequence of ANTRX (19 - 30)
MKADGAILVDFWADDDDK

SEQ ID NO 23:
Amino Acid sequence of FCTRX (1 - 15)
MKVGALSKGQLKEFLDDDDK

SEQ ID NO 24:
Amino Acid sequence of FCTRX (5 - 20)
MALSKGQLKEFLDANLADDDDK

SEQ ID NO 25:
Amino Acid sequence of FCTRX (10 - 24)
MQLKEFLDANLAGSGSDDDDK

SEQ ID NO 26:
Amino Acid sequence of FCTRX (1 - 10)
MKVGALSKGQDDDDK

SEQ ID NO 27:
Amino Acid Sequence of TrpLE leader peptide with DDDDK:
MKAIFVLKGSLDRDPEFDDDDK

SEQ ID NO 28:
Amino Acid Sequence of TrpLE leader peptide with KR:
MKAIFVLKGSLDRDPEFKR

SEQ ID NO 29:
Nucleotide sequence of TrpLE DDDDK Insulin:
ATGAAAGCCATCTTTGTGCTGAAAGGTAGCCTGGATCGTGATCCGGAATTTGATGATGATGATAAA TTTGTTAATCAGCATCTGTGTGGTAGCCATCTGGTTGAAGCACTGTATCTGGTTTGTGGTGAACGTGGTTTTTTTTATACCCCGAAAACCAAACGTGTATTGTTGAACAGTGTTGTACCAGCATTTGTAGCCTGTATCAGCTGGAAAATTATTGTAATTAA

SEQ ID NO 30:
Nucleotide sequence of TrpLE KR Insulin:
ATGAAAGCAATTTTTGTTCTGAAAGGTAGCCTGGATCGTGATCCGGAATTTAAACGT TTTGTTAATCAGCATCTGTGTGGTAGCCATCTGGTTGAAGCACTGTATCTGGTTTGTGGTGAACGTGGTTTTTTTTATACCCCGAAAACCAAACGTGTATTGTTGAACAGTGTTGTACCAGCATTTGTAGCCTGTATCAGCTGGAAAATTATTGTAATTAA

SEQ ID NO 31:
Nucleotide sequence of TrpLE DDDDK Glargine:
ATGAAAGCCATCTTTGTGCTGAAAGGTAGCCTGGATCGTGATCCGGAATTTGATGATGATGATAAA TTTGTTAATCAGCATCTGTGTGGTAGCCATCTGGTTGAAGCACTGTATCTGGTTTGTGGTGAACGTGGTTTTTTTTATACCCCGAAAACCCGTCGTGGTATTGTTGAACAGTGTTGTACCAGCATTTGTAGCCTGTATCAGCTGGAAAATTATTGTGGTTAA

SEQ ID NO 32:
Nucleotide sequence of TrpLE KR Glargine:
ATGAAAGCAATTTTTGTTCTGAAAGGTAGCCTGGATCGTGATCCGGAATTTAAACGT TTTGTTAATCAGCATCTGTGTGGTAGCCATCTGGTTGAAGCACTGTATCTGGTTTGTGGTGAACGTGGTTTTTTTTATACCCCGAAAACCCGTCGTGGTATTGTTGAACAGTGTTGTACCAGCATTTGTAGCCTGTATCAGCTGGAAAATTATTGTGGTTAA

SEQ ID NO 33:
Nucleotide sequence of TrpLE DDDDK LISPRO:
ATGAAAGCCATCTTTGTGCTGAAAGGTAGCCTGGATCGTGATCCGGAATTTGATGATGATGATAAA TTTGTGAACCAGCATCTGTGTGGTAGCCATCTGGTTGAAGCACTGTATCTGGTTTGTGGTGAACGTGGTTTTTTCTATACCAAACCGACCAAACGTGGTATTGTTGAACAGTGTTGTACCAGCATTTGTAGCCTGTATCAGCTGGAAAACTATTGCAATTAA

SEQ ID NO 34:
Nucleotide sequence of TrpLE KCH LISPRO:
ATGAAAGCCATCTTTGTGCTGAAAGGTAGCCTGGATCGTGATCCGGAATTTGATGATGATGATAAA TTTGTGAACCAGCATCTGTGTGGTAGCCATCTGGTTGAAGCACTGTATCTGGTTTGTGGTGAACGTGGTTTTTTCTATACCAAACCGACCAAAGGTATTGTTGAACAGTGTTGTACCAGCATTTGTAGCCTGTATCAGCTGGAAAACTATTGCAATTAA

SEQ ID NO 35:
Nucleotide sequence of TrpLE DDDDK ASPART:
ATGAAAGCCATCTTTGTGCTGAAAGGTAGCCTGGATCGTGATCCGGAATTTGATGATGATGATAAA TTTGTTAATCAACATTTGTGTGGTTCTCATTTGGTTGAAGCTTTGTATTTGGTTTGTGGTGAAAGAGGTTTTTTTTATACTGATAAAACTAAAAGAGGTATTGTTGAACAATGTTGTACTTCTATTTGTTCTTTGTATCAATTGGAAAATTATTGTAATTAA

Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based upon description provided herein. The embodiments herein and the various features and advantageous details thereof are explained with reference to the non-limiting embodiments in the description. Descriptions of some well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein. The examples presented herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the present disclosure.

Further, the examples provided herein are for better understanding of the instant disclosure. However, a person skilled in the art would be aware that the illustrations provided herein are only prospective in nature and can be extrapolated under the purview of the instant disclosure.

EXAMPLES
The utility of the present invention is demonstrated by construction of expression plasmids wherein codon optimized gene sequence is inserted into expression vector under the control of T7/ PL/ PR or PhoA promoters but not limited to these and expressed in but not limited to BL21DE3 cells. Insulin and all its analogues are expressed in L-B-C-A, wherein L is a leader peptide varying from 5 to 124 amino acids, B is B chain of insulin comprising of 31 amino acids, A is A chain of insulin comprising of 21 amino acids and C is the connecting peptide which can be any one of these, but not limited to K/ R/ KR/ RR/ RK/ KK/ DDDDK.

Example 1: Expression of TrpLE DDDDK Insulin with KR or R or DDDDK as connecting peptide:

1. Isolation of NdeI and BamHI vector backbone of pET41b(+):
About 5.3µg of plasmid pET41b(+) was suspended in Cutsmart buffer (NEB), to this 4µl of NdeI and 4µl of BamHI restriction enzyme was added. These were incubated at 37°C for 2 hours. Reaction mixture was electrophoresed on 1% agarose gel at 120V. Vector backbone of 5081bp (from which ~851bp of GST signal sequence removed) was excised and gel eluted. This was used for ligation.

2. Synthesis of TrpLE(DDDDK) Insulin gene:
The coding region of TrpLE and Insulin gene (SEQ ID No:1 and SEQ ID No:3) was synthesized after codon optimization using codons commonly found in highly expressed E.coli genes and designed to include convenient restriction sites for cloning into pET41b(+) as shown in FIG. 2.

3. Isolation of TrpLE DDDDK Insulin:
3a. For TrpLE DDDDK Insulin with KR as connecting peptide:
Coding region of TrpLE DDDDK Insulin was amplified using TRPINSFP and INSRP primers (Table 1). The amplicon of ~ 240bp obtained was suspended in Cutsmart buffer (NEB), to this 5µl of NdeI and 5µl of BamHI restriction enzyme was added. These were incubated at 37°C for 2 hours. Reaction mixture was electrophoresed on 2% agarose gel at 120V. Insert of ~240bp was excised and gel eluted. This was used for ligation.

Table 1: Primer sequences
SL No Primer Name Primer Sequence
1 TRPINSFP GGGAATTCCATATGAAAGCCATCTTTGTGCT
2 INSRP AATAATGCGGGATCCTTAATTGCAATAG
3 TRPGLRFP1 GGATCGTGATCCGGAATTTAAACGTTTTGTTAATCAGCATCTGTG
4 TRPGLRFP2 TTTTGTTCTGAAAGGTAGCCTGGATCGTGATCCGGA
5 TRPNFP CGACCATATGAAAGCAATTTTTGTTCTGAAAGGTAGC
6 GLRRP ATACGGGATCCTTAACCACAATAATTTTCCAGC

3b. For TrpLE DDDDK Insulin with R as connecting peptide:
TrpLE DDDDK Insulin with R as connecting peptide was synthesized as ABB 55and coding region was cloned into pET41b (+) plasmid at NdeI and BamHI as explained in 3a.

3c. For TrpLE DDDDK Insulin with DDDDK as connecting peptide:
For introducing DDDDK as connecting peptide sequence, an overlap PCR was performed using TRPINSFP, BCAFP, BCARP and INSOMPRP (Refer Table 2). Once the full length TrpLE DDDDK Insulin with DDDDK as connecting peptide was obtained it was cloned into pET41b (+) at NdeI and BamHI as explained in 3a.

4. Ligation transformation and screening :
pET41b (+) digested with NdeI and BamHI restriction enzyme yielded 5081bp vector band. TrpLE DDDDK Insulin digested with NdeI and BamHI restriction enzyme gave 240 bp insert band. Based on the concentration of vector and insert, ligation reaction was set in the ratio 1:3 and 1:5. These two reaction mix was incubated overnight at 16ºC + 0.5°C. Next day, this ligation mixture was transformed into E.coli DH5a competent cells. Transformation mix was plated onto LB Kan (25µg/ml) plates. These plates were incubated at 37°C for overnight growth. Clones obtained were screened by Colony PCR and PCR positive clone was used for further analysis.

5. Small scale expression studies:
TrpLE DDDDK Insulin /pET41b (+) Clone #1 (FIG. 3), transformed into BL21 competent cells, showed several well isolated colonies. Subsequently, several colonies were inoculated into Super broth and simultaneously streaked onto LB kanamycin plates. These plates and tubes were incubated at 37°C + 1ºC. When the culture O.D600 reached ~ 0.7 – 0.9; an equivalent of 0.5 O.D cells was harvested. This was followed by overnight induction with 1mM IPTG at 37°C + 1ºC and 180 rpm. The next day an equivalent of 0.5 O.D cells were collected. Both uninduced and one vial of induced culture was spun at 8,000rpm and media was discarded. Cells were sonicated and sample was prepared by adding 20µl of loading buffer and 80µl of PBS. 25 µl of samples were loaded onto Tricine SDS – PAGE (FIG 9). Small scale expression studies were carried out similarly for TrpLE DDDDK Insulin with R as connecting peptide (FIG 10) and TrpLE DDDDK Insulin with DDDDK as connecting peptide (FIG 11).

Results: Tricine PAGE image (FIG 9, FIG 10 and FIG 11) clearly demonstrates that there was no expression seen in uninduced samples compared to induced samples; which shows a significant amount of TrpLE DDDDK insulin expressing after inducing with IPTG.

Example 2: Expression of TrpLE KR Insulin:

1. Isolation of NdeI and BamHI vector backbone of pET41b(+):
About 7µg of plasmid pET41b (+) was suspended in Cutsmart buffer (NEB), to this 3µl of NdeI and 3µl of BamHI restriction enzyme was added. These were incubated at 37°C for 2 hours. Reaction mixture was electrophoresed on 1% agarose gel at 120V. Vector backbone of 5081bp (from which ~851bp of GST signal sequence removed) was excised and gel eluted. This was used for ligation.
2. Synthesis of TrpLE KR Insulin gene:
The coding region of TrpLE and Insulin gene (SEQ ID No: 2 and SEQ ID No: 3) was synthesized after codon optimization using codons commonly found in highly expressed E.coli genes and designed to include convenient restriction sites for cloning into pET41b(+) as shown in FIG. 2.

3. Isolation of TrpLE KR Insulin cDNA from ABB 16:
About 8 µg of plasmid TrpLEKR Insulin/pMA (synthesized as ABB 16/pMA from Geneart) was suspended in Cutsmart buffer (NEB), to this 5µl of NdeI and 5µl of BamHI restriction enzyme was added. These were incubated at 37°C for 2 hours. Reaction mixture was electrophoresed on 1.8% agarose gel at 120V. Insert of ~222bp was excised and gel eluted. This was used for ligation.
4. Ligation transformation and screening:
pET41b (+) digested with NdeI and BamHI restriction enzyme yielded 5081bp vector band. TrpLE KR Insulin digested with NdeI and BamHI restriction enzyme gave ~ 222 bp insert band. Based on the concentration of vector and insert, ligation reaction was set in the ratio 1:5 and 1:8. These two reaction mix was incubated overnight at 16ºC + 0.5°C. Next day, this ligation mixture was transformed into E.coli DH5a competent cells. Transformation mix was plated onto LB Kan (25µg/ml) plates. These plates were incubated at 37°C for overnight growth. Eight clones were screened by colony PCR using TRPINSFP and INSRP and T7FP and T7RP primers. All eight colonies four were found to be positive. Hence clone 5 was used for further analysis.

5. Small scale expression studies:
TrpLE KR Insulin /pET41b (+) Clone #1 (FIG. 4), transformed into BL21 competent cells, showed several well isolated colonies. Subsequently, several colonies were inoculated into Super broth and simultaneously streaked onto LB kanamycin plates. These plates and tubes were incubated at 37°C + 1ºC. When the culture O.D600 reached ~ 0.7 – 0.9; an equivalent of 0.5 O.D cells was harvested. This was followed by overnight induction with 1mM IPTG at 37°C + 1ºC and 180 rpm. The next day an equivalent of 0.5 O.D cells were collected. Both uninduced and one vial of induced culture was spun at 8,000rpm and media was discarded. Sample was prepared by adding 20µl of loading buffer and 80µl of PBS. 25 µl of samples were loaded onto Tricine SDS – PAGE (FIG 12).

Results: Tricine PAGE image (FIG 12) clearly demonstrates that there was no expression seen in uninduced samples compared to induced samples; which shows a significant amount of TrpLE KR insulin expressing after inducing with IPTG.

Example 3: Expression of TrpLE KR Glargine:

1. Synthesis of TrpLE KR Glargine gene:
The coding region of TrpLE was added to Glargine gene (SEQ ID No: 2 and SEQ ID No: 6) by three consecutive PCR’s. Glargine gene was first amplified using TRPGLRFP1 and GLRRP (refer Table 1); amplicon obtained was used as template for next PCR. A second PCR reaction was set using TRPGLRF2 and GLRRP. Amplicon obtained was used as template for final PCR using TRPNFP and GLRRP (Refer Table 1). Final PCR product obtained was cloned into TA vector for sequence verification.

2. Isolation of TrpLE KR Glargine cDNA from TrpLE KR Glargine/TA:
2.7µg of TrpLE KR Glargine/TA was suspended in Cutsmart buffer (NEB), to this 2.5µl of NdeI and 2.5µl of BamHI restriction enzyme was added. These were incubated at 37°C for 3 hours. Reaction mixture was electrophoresed on 1.8% agarose gel at 120V. Insert of ~222bp was excised and gel eluted. This was used for ligation.

3. Isolation of NdeI and BamHI vector backbone of pET41b(+):
About 3.5µg of plasmid pET41b(+) was suspended in Cutsmart buffer (NEB), to this 2µl of NdeI and 2µl of BamHI restriction enzyme was added. These were incubated at 37°C for 3 hours. Reaction mixture was electrophoresed on 1% agarose gel at 120V. Vector backbone of 5081bp (from which ~851bp of GST signal sequence removed) was excised and gel eluted.This was used for ligation.

4. Ligation transformation and screening :
pET41b (+) digested with NdeI and BamHI restriction enzyme yielded 5081bp vector band. TrpLE KR Glargine digested with NdeI and BamHI restriction enzyme gave 222 bp insert band. Based on the concentration of vector and insert, ligation reaction was set in the ratio 1:3 and 1:5. These two reaction mix was incubated overnight at 16ºC + 0.5°C. Next day, this ligation mixture was transformed into E.coli DH5a competent cells. Transformation mix was plated onto LB Kan (25µg/ml) plates. These plates were incubated at 37°C for overnight growth. Twelve clones were screened by Colony PCR using TRPNFP and GLRRP and T7FP and T7RP primers. Of twelve colonies eleven were found to be positive. Hence clone 5 was used for further analysis.

5. Small scale expression studies:
TrpLE KR Glargine /pET41b (+) Clone #5 (FIG. 5), transformed into BL21 competent cells, showed several well isolated colonies. Subsequently, several colonies were inoculated into Super broth and simultaneously streaked onto LB kanamycin plates. These plates and tubes were incubated at 37°C + 1ºC. When the culture O.D600 reached ~ 0.7 – 0.9; an equivalent of 0.5 O.D cells was harvested. This was followed by overnight induction with 1mM IPTG at 37°C + 1ºC and 180 rpm. The next day an equivalent of 0.5 O.D cells were collected. Both uninduced and one vial of induced culture was spun at 8,000rpm and media was discarded. Sample was prepared by adding 20µl of loading buffer and 80µl of PBS. 25 µl of samples were loaded onto Tricine SDS – PAGE (FIG 14).

Results: Tricine PAGE image (FIG 14) clearly demonstrates that there was no expression seen in uninduced samples compared to induced samples; which shows a significant amount of TrpLE KR Glargine expressing after inducing with IPTG.

Example 4: Expression of TrpLE DDDDK Glargine:
1. Isolation of NdeI and BamHI vector backbone of pET41b(+):
About 10µg of plasmid pET41b(+) was suspended in Cutsmart buffer (NEB), to this 5µl of NdeI and 5µl of XhoI restriction enzyme was added. These were incubated at 37°C for 2 hours. Reaction mixture was electrophoresed on 1% agarose gel at 120V. Vector backbone of 5081bp (from which ~851bp of GST signal sequence removed) was excised and gel eluted.This was used for ligation.

2. Synthesis of TrpLE DDDDK Glargine gene:
The coding region of TrpLE DDDDK and Glargine gene (SEQ ID No: 1 and SEQ ID No: 6; was synthesized as ABB 33) after codon optimization using codons commonly found in highly expressed E.coli genes and designed to include convenient restriction sites for cloning into pET41b(+) as shown in FIG. 2.

3. Isolation of TrpLE DDDDK Glargine cDNA from ABB 33:
Approximately 5.5µg of plasmid TrpLE DDDDK Glargine/pMK (synthesized as ABB 33/pMK from Geneart) was suspended in Cutsmart buffer (NEB), to this 5µl of NdeI and 5µl of XhoI restriction enzyme was added. These were incubated at 37°C for 2 hours. Reaction mixture was electrophoresed on 1.8% agarose gel at 120V. Insert of ~231bp was excised and gel eluted. This was used for ligation.

4. Ligation transformation and screening:
pET41b (+) digested with NdeI and BamHI restriction enzyme yielded 5081bp vector band. TrpLE DDDDK Glargine digested with NdeI and XhoI restriction enzyme gave ~ 222 bp insert band. Based on the concentration of vector and insert, ligation reaction was set in the ratio 1:3. These two reaction mix was incubated overnight at 16ºC + 0.5°C. Next day, this ligation mixture was transformed into E.coli DH5a competent cells. Transformation mix was plated onto LB Kan (25µg/ml) plates. These plates were incubated at 37°C for overnight growth. Of eight clones obtained, three clones were screened by restriction digestion analysis. All three were found to be positive. Of which clone 6 was used for further analysis.

5. Small scale expression studies:
TrpLE DDDDK Glargine /pET41b (+) Clone #6 (FIG. 6), transformed into BL21 competent cells, showed several well isolated colonies. Subsequently, several colonies were inoculated into Super broth and simultaneously streaked onto LB kanamycin plates. These plates and tubes were incubated at 37°C + 1ºC. When the culture O.D600 reached ~ 0.7 – 0.9; an equivalent of 0.5 O.D cells was harvested. This was followed by overnight induction with 1mM IPTG at 37°C + 1ºC and 180 rpm. The next day an equivalent of 0.5 O.D cells were collected. Both uninduced and one vial of induced culture was spun at 8,000rpm and media was discarded. Sample was prepared by adding 20µl of loading buffer and 80µl of PBS. 25 µl of samples were loaded onto Tricine SDS – PAGE (FIG 12).
Results: Tricine PAGE image (FIG 14) clearly demonstrates that there was no expression seen in uninduced samples compared to induced samples; which shows a significant amount of TrpLE KR Glargine expressing after inducing with IPTG.

Example 5: Comparison of Met – Insulin (without fusion tag) expression with TrpE Insulin; and Met Glargine (without fusion tag) expression with TrpE Glargine:
Synthetic codon optimized Insulin and Glargine gene without any fusion tag was cloned into pET41b (+). Small scale expression studies were carried out to evaluate the expression levels (Fig No. 15).
Results: Tricine PAGE image (FIG 15) clearly demonstrates that there was less or no expression seen in clones without TrpLE tag compared to induced samples of TrpLE Insulin and TrpLE Glargine; which shows a significant amount of expression after inducing with IPTG.

Example 6: Expression of TrpLE DDDDK Lispro:
1. Isolation of NdeI and BamHI vector backbone of pET41b(+):
About 10µg of plasmid pET41b(+) was suspended in Cutsmart buffer (NEB), to this 5µl of NdeI and 5µl of XhoI restriction enzyme was added. These were incubated at 37°C for 2 hours. Reaction mixture was electrophoresed on 1% agarose gel at 120V. Vector backbone of 5081bp (from which ~851bp of GST signal sequence removed) was excised and gel eluted. This was used for ligation.

2. Synthesis of TrpLE DDDDK Lispro gene:
The coding region of TrpLE DDDDK and Lispro gene (SEQ ID No: 1 and SEQ ID No: 11); was synthesized as ABB 48) after codon optimization using codons commonly found in highly expressed E.coli genes and designed to include convenient restriction sites for cloning into pET41b(+) as shown in FIG. 2.

3. Isolation of TrpLE DDDDK Lispro cDNA from ABB 33:
Approximately 4.4µg of plasmid TrpLE DDDDK Lispro/pMA-T (synthesized as ABB 48/pMA-T from Geneart) was suspended in Cutsmart buffer (NEB), to this 4µl of NdeI and 4µl of XhoI restriction enzyme was added. These were incubated at 37°C for 2 hours. Reaction mixture was electrophoresed on 1.8% agarose gel at 120V. Insert of ~231bp was excised and gel eluted. This was used for ligation.

4. Ligation transformation and screening:
pET41b (+) digested with NdeI and BamHI restriction enzyme yielded 5081bp vector band. TrpLE DDDDK Lispro digested with NdeI and XhoI restriction enzyme gave ~ 231 bp insert band. Based on the concentration of vector and insert, ligation reaction was set in the ratio 1:3. These two reaction mix was incubated overnight at 16ºC + 0.5°C. Next day, this ligation mixture was transformed into E.coli DH5a competent cells. Transformation mix was plated onto LB Kan (25µg/ml) plates. These plates were incubated at 37°C for overnight growth. Eight clones were screened by colony PCR, all eight colonies were found to be positive. Of which clone 1 and clone 6 was used for further analysis.

5. Small scale expression studies:
TrpLE Lispro /pET41b (+) Clone #6 (FIG. 8), transformed into BL21 competent cells, showed several well isolated colonies. Subsequently, several colonies were inoculated into super broth and simultaneously streaked onto LB kanamycin plates. These plates and tubes were incubated at 37°C + 1ºC. When the culture O.D600 reached ~ 0.7 – 0.9; an equivalent of 0.5 O.D cells was harvested. This was followed by overnight induction with 1mM IPTG at 37°C + 1ºC and 180 rpm. The next day an equivalent of 0.5 O.D cells were collected. Both uninduced and one vial of induced culture was spun at 8,000rpm and media was discarded. Sample was prepared by adding 20µl of loading buffer and 80µl of PBS. 25 µl of samples were loaded onto Tricine SDS – PAGE (FIG 16).

Results: Tricine PAGE image (FIG 16) clearly demonstrates that there was no expression seen in uninduced samples compared to induced samples; which shows a significant amount of TrpLE DDDDK Lispro expressing after inducing with IPTG.

Example 7: Expression of TrpLE KCH DDDDK Lispro:

The utility of the present invention is demonstrated by construction of expression plasmids wherein codon optimized gene sequence (Seq ID 01 and Seq ID 11) was inserted in expression vector and expressed in BL21DE3 cells. In the present invention the connecting peptide between A chain and B chain is K.

1. Isolation of NdeI and BamHI vector backbone of pET41b(+):
About 10µg of plasmid pET41b(+) was suspended in Cutsmart buffer (NEB), to this 5µl of NdeI and 5µl of XhoI restriction enzyme was added. These were incubated at 37°C for 2 hours. Reaction mixture was electrophoresed on 1% agarose gel at 120V. Vector backbone of 5081bp (from which ~851bp of GST signal sequence removed) was excised and gel eluted. This was used for ligation.

2. Synthesis of TrpLE DDDDK KCH Lispro gene:

The coding region of TrpLE DDDDK and KCH Lispro gene (SEQ ID No: 1 and SEQ ID No: 11; was synthesized as ABB 52) after codon optimization using codons commonly found in highly expressed E.coli genes and designed to include convenient restriction sites for cloning into pET41b(+) as shown in FIG. 2.

3. Isolation of TrpLE DDDDK KCH Lispro cDNA from ABB 52:
Approximately 4.9µg of plasmid TrpLE DDDDK KCH Lispro/pMA-T (synthesized as ABB 52/pMA-T from Geneart) was suspended in Cutsmart buffer (NEB), to this 4µl of NdeI and 4µl of XhoI restriction enzyme was added. These were incubated at 37°C for 2 hours. Reaction mixture was electrophoresed on 1.8% agarose gel at 120V. Insert of ~228bp was excised and gel eluted. This was used for ligation.

4. Ligation transformation and screening:
pET41b (+) digested with NdeI and BamHI restriction enzyme yielded 5081bp vector band. TrpLE DDDDK KCH Lispro digested with NdeI and BamHI restriction enzyme gave ~ 228 bp insert band. Based on the concentration of vector and insert, ligation reaction was set in the ratio 1:3. These two reaction mix was incubated overnight at 16ºC + 0.5°C. Next day, this ligation mixture was transformed into E.coli DH5a competent cells. Transformation mix was plated onto LB Kan (25µg/ml) plates. These plates were incubated at 37°C for overnight growth. Three clones were screened by colony PCR, all three colonies were found to be positive. Of which clone 2 and clone 3 was used for further analysis.

5. Small scale expression studies:
TrpLE Lispro /pET41b (+) Clone # 3 (FIG. 8), transformed into BL21 competent cells, showed several well isolated colonies. Subsequently, several colonies were inoculated into super broth and simultaneously streaked onto LB kanamycin plates. These plates and tubes were incubated at 37°C + 1ºC. When the culture O.D600 reached ~ 0.7 – 0.9; an equivalent of 0.5 O.D cells was harvested. This was followed by overnight induction with 1mM IPTG at 37°C + 1ºC and 180 rpm. The next day an equivalent of 0.5 O.D cells were collected. Both uninduced and one vial of induced culture was spun at 8,000rpm and media was discarded. Sample was prepared by adding 20µl of loading buffer and 80µl of PBS. 25 µl of samples were loaded onto Tricine SDS – PAGE (FIG 17).

Results: Tricine PAGE image (FIG 17) clearly demonstrates that there was no expression seen in uninduced samples compared to induced samples; which shows a significant amount of TrpLE DDDDK KCH Lispro expressing after inducing with IPTG.

Example 8: Expression of Insulin with truncated Thioredoxin tags:

In this study we describe the usage of various sequences derived from other protein as a fusion partner or leader peptide for the expression of proteins or peptides. Regions varying from as long as 90 amino acids to as short as 15 amino acids were used as fusion partner that promotes the accumulation of expressed proteins as inclusion bodies in the cytoplasm.

The current invention involves the usage of short fragments derived from other well-known proteinswhich might serve in itself as a fusion tag such as Thioredoxin, Glucagon, Glucagon like peptides, GCSF, IFN alpha, IFN beta etc., and enhance the production of heterologous proteins in E.coli.

A full length Thioredoxin gene with His tag (SEQ ID NO: 14; FIG 19) was fused to N – terminus of Insulin (SEQ ID NO: 3) under the control of T7 promoter in pET41b (+) vector at NdeI and BamHI sites, and expressed in E.coli BL21DE3 cells. Since Insulin is of lesser molecular weight (~ 6.08 kDa) compared to that of Thioredoxin (~13.5kDa), the molar ratio of expressed protein (FIG 18) to that of tag will result in lesser yield of Insulin.

One of the drawback of full length Thioredoxin protein is that it causes oxidation of proteins resulting in lesser yield. Hence, by using truncated Thioredoxin, the protein of interest is not oxidized, thus giving rise to improved yield of protein of interest. This is particularly applicable to peptides and proteins that are amenable to oxidation.

Hence, truncated thioredoxin fragments are employed. Initially, thioredoxin tag was truncated into 6 fragments from both N and C terminus as shown in Figure 20 and 21. These fragments are fused to Insulin gene by PCR (refer Table 2 for Primer sequences) and expressed in BL21DE3 cells.

For creating N – terminus truncations each fragment as shown in FIG 20 (designated as ANTRXINS, BNTRXINS and CNTRXINS) was amplified separately and fused to insulin gene by overlapping PCR (refer Table 2 for primers and sequences). The coding region obtained was cloned into TA and sequence verified. Coding sequence was excised from TA and cloned into pET41b (+) vector at NdeI and BamHI site.

For creating C – terminus truncations each construct as shown in FIG 21 (designated as DCTRXINS, ECTRXINS and FCTRXINS) was amplified using primers (Table 2) and coding sequence was cloned into TA vector and sequence verified. Subsequently they were sub cloned into pET41b (+) at NdeI and BamHI site.

All the six pET41b(+) constructs: ANTRXINS, BNTRXINS, CNTRXINS, DCTRXINS, ECTRXINS and FCTRXINS (SEQ ID 16,17,18,19,20 and 21) was cloned into pET41b(+) were transformed into BL21DE3 cells and small scale expression studies were carried out to evaluate the expression levels. FIGs 22, 23, 24, 25, 26 and 27.

While all fragments showed increased expression (as depicted in table 4), two fragments of 30 amino acids (ANTRXINS) and 24 amino acids (FCTRXINS) showed a much increased expression of the Insulin protein (Fig 22 and 27). Based on the expression levels seen on Tricine SDS PAGE these two fragments (ANTRX and FCTRX) were further truncated into tags of 10 – 20 amino acids each (Seq IDs 22, 23, 24, 25 and 26) and fused with Insulin gene (SEQ ID 3) and cloned into pET41b(+) under the control of T7 promoter at NdeI and BamHI. These were transformed into E.coli BL21DE3 cells and small scale expression studies were carried out. Expression levels were checked on Tricine SDS PAGE (FIGs 28, 29 and 30).

Results: From the Tricine PAGE images (FIG 22, 23, 24, 25, 26 and 27) which clearly shows that amount of Proinsulin expressed with few truncated thioredoxin tags showed better / equivalent expression compared to that of full length thioredoxin fusion to insulin (FIG 19). Of all the truncations ANTRX and FCTRX showed higher level of expression, when compared with others. Hence ANTRX and FCTRX were further truncated into 5 more peptides (Seq IDs 22, 23, 24, 25 and 26) and used as fusion tags for insulin expression. There were variations in the expression level as seen in (FIG 28, 29 and 30). Wherein FCTRX (1 – 15) Insulin, FCTRX (10 – 24) Insulin and ANTRX (19 – 30) showed better expression of protein compared to FCTRX (10 – 24) tag, whereas in FCTRX (5 – 20) Insulin there was no expression seen.

Table 2: Primers used for creating truncations in Thioredoxin and fusing it with Insulin gene
SL No Construct Primer Name Primer Sequence
1 ANTRX INS TRXHISINSFP GGAGATTCCATATGAGCGATAAAATTATTCACCTGAC
ANOVPFP GACGACGATGACAAATTTGTTAATCAGCATCTGTGTGG
ANOVPRP AAATTTGTCATCGTCGTCTGCCCAGAAATCGACGAGG
INSOMPRP CCAGTATGGCGGATCCTTAATTGCAATAGTTTTCGAGC
2 BNTRX INS TRXHISINSFP GGAGATTCCATATGAGCGATAAAATTATTCACCTGAC
ANOVPFP GACGACGATGACAAATTTGTTAATCAGCATCTGTGTGG
BNOVPRP AAATTTGTCATCGTCGTCGTTCAGTTTTGCAACGGTC
INSOMPRP CCAGTATGGCGGATCCTTAATTGCAATAGTTTTCGAGC
3 CNTRX INS TRXHISINSFP GGAGATTCCATATGAGCGATAAAATTATTCACCTGAC
ANOVPFP GACGACGATGACAAATTTGTTAATCAGCATCTGTGTGG
CNOVPRP AAATTTGTCATCGTCGTCGGTTGCCGCCACTTCACC
INSOMPRP CCAGTATGGCGGATCCTTAATTGCAATAGTTTTCGAGC
4 DCTRX INS DCTRXFP TTCCATATGGAGTGGTGCGGTCCGTGC
INSOMPRP CCAGTATGGCGGATCCTTAATTGCAATAGTTTTCGAGC
5 ECTRX INS ECTRXFP TTCCATATGATCGATCAAAACCCTGGCA
INSOMPRP CCAGTATGGCGGATCCTTAATTGCAATAGTTTTCGAGC
6 FCTRX INS FCTRXFP TTCCATATGAAAGTGGGTGCACTGTCT
INSOMPRP CCAGTATGGCGGATCCTTAATTGCAATAGTTTTCGAGC

Table 3: Primers used for creating truncations in ANTRX and FCTRX sequence and fusing it with Insulin
SL No Construct Primer Name Primer Sequence
1 ANTRXINS (19 – 30) ANTR 19-30FP GACATATGAAAGCGGACGGGGCGATCC
INSOMPRP CCAGTATGGCGGATCCTTAATTGCAATAGTTTTCGAGC
2 FCTRXINS ( 1 – 15) FCTR 1-10FP GTCATATGAAAGTGGGTGCACTGTCTAAAGGTCAGGACGACGATGACAAATTTG
INSOMPRP CCAGTATGGCGGATCCTTAATTGCAATAGTTTTCGAGC
3 FCTRXINS (5 – 20) FCTR 1-15FP ACCATATGAAAGTGGGTGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGACGATGACAAATTTG
INSOMPRP CCAGTATGGCGGATCCTTAATTGCAATAGTTTTCGAGC
4 FCTRXINS (10 -24) FCTR 5-20FP GTCATATGGCACTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGACGACGATGACAAATTTG
INSOMPRP CCAGTATGGCGGATCCTTAATTGCAATAGTTTTCGAGC
5 FCTRXINS (1 – 10) FCTR 10-24FP ATCATATGCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCGGTTCTGGTTCTGACGACGATGACAAATTTG
INSOMPRP CCAGTATGGCGGATCCTTAATTGCAATAGTTTTCGAGC

Table 4: Insulin constructs with fusion tags
Sl no Tag Sequence Protein Expressed Expression level
1 TrpE Seq Id. 1 and 2 Insulin +++
2 ANTRXINS Seq Id. 20 Insulin +++
3 BNTRXINS Seq Id. 21 Insulin ++
4 CNTRXINS Seq Id. 22 Insulin +++
5 DCTRXINS Seq Id. 23 Insulin ++
6 ECTRXINS Seq Id. 24 Insulin ++
7 FCTRXINS Seq Id. 25 Insulin +++
8 ANTRXINS (19- 30) Seq Id. 26 Insulin +++
9 FCTRXINS (1 – 15) Seq Id. 27 Insulin +++
10 FCTRXINS (5 – 20) Seq Id. 28 Insulin -
11 FCTRXINS (10 – 24) Seq Id. 29 Insulin ++
12 FCTRXINS (1 – 10) Seq Id. 30 Insulin +

Note : +++ - Very good expression, ++ - Good expression, + - Moderate expression.

Example 9: Expression of TrpLEDDDDK Aspart in Saccharomyces cerevisiae:
The utility of the present invention is further demonstrated by construction of expression plasmids wherein codon optimized gene sequence (SEQ ID 01 and SEQ ID 12) was inserted in expression vector and expressed in Saccharomyces cerevisiae cells.

Expression of TrpLE Aspart in Saccharomyces cerevisiae:
The coding region for Aspart gene in L - B (1-30) – C – A(1-21) form (SEQ ID No: 1 and 12) was synthesized; wherein L is a leader peptide varying from 5 to 124 amino acids, B is B chain of insulin comprising of 31 amino acids, A is A chain of insulin comprising of 21 amino acids and C is the connecting peptide which can be any one of these, but not limited to K/ R/ KR/ RR/ RK/ KK/ DDDDK. Aspart coding gene was codon optimized for yeast using codons most commonly found in highly expressed yeast genes and designed to include convenient restriction sites like XhoI, EcoRI and NotI for cloning into pADB9 as shown in FIG. 31.

The obtained synthetic gene and pADB9 vector was subjected to restriction digestion using XhoI and EcoRI. Insert band of 234bp along with pADB9 vector backbone of ~ 5448bp was excised and gel eluted. Both the fragments were ligated in the ratio 1:3. The ligation mixture was transformed into E.coli DH5a cells and plated onto LBA.

The construction of Aspart/pADB9 is illustrated in Fig 32. Aspart/ pADB9 contains the DNA sequence GALp – MFa leader – (L-B-C-A) – CYC1t. This plasmid was linearized at AgeI site and transformed into Saccharomyces cerevisiae by Lithium acetate method. From the overnight grown culture, 0.2 O.D600 cells were transferred into fresh 100ml YPD media and allowed to grow at 30°C until it reaches an O.D600 of 0.6. 100ml of culture was harvested by centrifugation and washed with 25ml of sterile water. Cells were resuspended in 1ml of fresh 100mM LiAc in TE. Again the cells were harvested by centrifugation, followed by resuspension in 500µl of 100mM LiAc in TE.

In a fresh vial 0.1ml of cells, 0.1µg of linearized plasmid DNA, 100µg of salmon sperm DNA and 600µl of sterile PEG/LiAc (40% PEG 3350 + 100mM LiAc in TE) was added and incubated at 30°C and 200 rpm for 30 minutes. After incubation 70µl of DMSO was added and gently mixed. Heat shock was given for cells at 42°C for 15mins and then plated onto YPD plates with 100µg/ml G418.

Transformant colonies were picked after 3 days and subjected to higher antibiotic selections. Colonies which showed resistance to higher concentration of G418 were taken for induction with 2% galactose. After 2 days samples were electrophoresed on Tricine SDS PAGE and visualized after commassie staining (FIG 33).

Results: TrpLE Aspart expression as shown in FIG 33, was significant on Day 2, by Day 4 the expression levels reduced due to protease degradation. Hence a PEP4 protease knock out would help in retaining the product intact.

Example 10: Knocking out PEP4 in Saccharomyces cerevisiae (by insertional inactivation):
The utility of the present invention is further demonstrated by construction of expression plasmids wherein codon optimized gene sequence (SEQ ID 01 and SEQ ID 12) was inserted in expression vector carrying flanking regions of PEP4 gene on either side of the linear vector. This was used to knock out PEP4 protease and in turn express Aspart under the control of GALI promoter in Saccharomyces cerevisiae cells.

To minimize proteolytic cleavages, protease gene PEP4 (encoding proteinase A) was knocked out respectively by insertional inactivation. PEP4 or Vacuolar aspartyl protease (proteinase A); required for posttranslational precursor maturation of vacuolar proteinases; important for protein turnover after oxidative damage. Reduced degradation of Aspart was observed by disruption of PEP4 gene.

~ 356bp from the start of PEP4 gene and ~ 358bp from the end of PEP4 gene was used as flanking regions for targeting PEP4 gene in the genome. PEP4 gene was knocked out by insertional inactivation; wherein PEP4 gene was disrupted and in turn an Aspart cassette was inserted in the genome at the same site.

TrpLE Aspart PEP4 pADB11 (FIG 34) was constructed by cloning PEP4R and PEP4L genes into pADB11. This vector was linearized with pvuII and transformed into Saccharomyces cerevisiae by LiAc method (refer example 7 for protocol). Transformants obtained were screened for PEP4 knock out by APNE assay. Positives from APNE assay were induced with 2% galactose. After 2 days samples were analyzed by Tricine SDS PAGE and visualized after commassie staining (FIG 35).

Results: TrpLE Aspart expression as shown in FIG 35, was found to be intact after 4 days of induction, PEP4 protease knock out helped in retaining the product intact.

Example 11: Cell Lysis
Insulin, glargine and lispro are expressed in E. coli, as intracellular inclusion bodies. After harvesting, cell lysis was carried out using 10-200mM Tris 1-10mM EDTA pH-8.0 to pH-9.0 as buffer through high pressure homogenizer at 1000±50 bar pressure in three passes. Three passes were given to achieve efficient cell lysis.

Example 12: Inclusion body washing:
Washing of Insulin and insulin analog precursor (such as glargine and lispro) inclusion body with 10mM to 200mM Tris, 1mM to 10mM EDTA, 0.5 M to 3M Urea as wash buffer 1 and 10mM to 200mM Tris, 1mM to 10mM EDTA, 0.1% to 2% Triton X 100 as wash buffer 2 followed by 10mM to 200mM Tris, 1mM to 10mM EDTA wash which finally increases Insulins precursor inclusion bodies purity to more than 75% ( refer Fig: 37 to 39).

Example 13: Protein solubilization & refolding:
Insulin and insulin analog precursors expressed as inclusion bodies were solubilized in 10 to 200 mM sodium bicarbonate 4-8M urea pH 9 to 12.5 at a ratio of 1 gram to 2 gram IB in 20 ml to 100ml of buffer. Solubilized inclusion bodies were diluted 1 to 5 times into 10 mM to 200 mM sodium bicarbonate pH 9 to 12.5 at temperature about 5-25°C and then added cysteine & cystine at concentration of 0.5 mM to 5 mM & 1 mM to 5.0 mM respectively and kept continuous stirring for about 14-20 hrs (refer Fig: 40 to 42).

Example 14: Protein concentration:
Protein concentration was performed on refolded precursor insulin and its analogues using hollow fiber membrane (MMWC 5kDa) at 9-25 psi TMP to reduce volume to 10-20 folds.

Example 15: Enterokinase digestion:
After concentration precursor protein (insulin and its analogues such as glargine, lispro and aspart) was subjected to enzyme digestion with Enterokinase to remove the trpE tag connected to Insulin and its analogue single chain. The reaction was carried out at temperature about 15 to 30°C under stirring for 16-20hours in presence of 1mM -10mM CaCl2, 10mM to 400mM NaCl at pH of 5.5 -10.5 in buffered solution. The amount of enterokinase used for reaction was around 2-10 units per mg of insulin precursor (Figs 43 to 45).

Example 16: Mixed mode anionic exchanger chromatography:
After enterokinase digestion, to purify the insulin single chain mixed mode anionic exchanger with hydrophobic interaction was used. Protein was bound at high conductivity and pH, and eluted at low conductivity and pH. Column was equilibrated with 10mM to 200mM tris, 10mM to 250mM NaCl pH 7.5 to 11.0. Protein was loaded at 5 gram to 30 gram per liter of resin. And then to loosely bound molecules were removed by washing. Column washing was carried out with 10mM to 200mM tris, 10mM to 100mM NaCl pH 7.0 to 10.0. Proinsulins/ analogues were separated from precursors by a gradient elution with elution buffer having pH 2.5 to 5.0 with conductivity range of 0.5mS to 9.0 mS. (refer Fig: 46 to 48).

Example 17: Protein digestion:
After purifying pro insulin (intact single chain containing A, B and connecting C peptide) and its analogues such as glargine, lispro and aspart, pro peptide was enzymatically cleaved to convert into active molecule (insulin, glargine, lispro and aspart) . Before trypsinization, protein was citra conylated by using citra conic anhydride in a range of 0.1 to 5% at alkaline pH and then trypsin (2 to 20 units per 1 mg of insulin, glargine and aspart) and CPB (0.2 to 1.5 units per 1 mg of ( insulin, lispro and aspart) enzymes were used. The reaction was carried out at temperature 2-30°C for 5-20 hours. To arrest the reaction and decitra conylate the protein, sample pH was adjusted to 1.5 to 3.0 and kept at 15-30deg C for 12-20 hours under stirring (Figs 49 to 51).

Example 18: Protein Precipitation:
Protein was precipitated after decitra conylation. The precipitation was performed by adding 0.1% to 2.0% zinc chloride, phenol in a range 0.05% to 2.0% and adjusting the pH to 5.5 to 7.0 using alkali at temperature 2°C-30°C. After settling of precipitate for 1 to 5 hours, supernatant was decanted and centrifuged to collect the precipitate/pellet.

Example 19: Reverse Phase Chromatography-1:
Human Insulin and its analogues (glargine, lispro and aspart) pellet was taken forward further purification through reverse phase chromatography. Precipitate was dissolved in acetic acid (1 to 5 Normal) and acetonitrile and sodium acetate were added at a final concentration of 10%-20% and 20mM -100mM respectively. The RP column (C8,10µ, 100Å) was equilibrated with 90%-80% of buffer A (5mM to 200mM sodium acetate, 10mM to 100mM Sodium perchlorate in water in a pH range of 2-5.0/25mM -500mM acetic acid) and 10% to 20% of buffer B (Acetonitrile) for 5 to 10 column volumes. Protein (insulin and its analogues) was loaded onto column at 2 to 20 gram per liter. The protein was eluted with linear gradient from 15%-25% to 20-35% Acetonitrile in 10 to 30cv’s. Desired protein proteins were collected in multiple fractions. Purity obtained (using analytical HPLC) at this stage is more than 97% with step recovery of 50%-80% (refer Fig: 52 to 54).

Example 20: Reverse Phase Chromatography-2:
Further purification of insulin and its analogues were carried out using preparative RP HPLC to achieve more than 99.5% (by pharmacopeial RP method). Elution fractions of previous steps were pooled and reloaded onto reverse phase chromatography column (C8,10µ, 100Å) with different conditions.

The RP column (C8,10µ, 100Å) was equilibrated with 95%-90% of buffer A (10mM to 200mM Acetic acid/ 5mM -100mM sodium acetate, pH 3-7) and 5 to 10% of buffer B (IPA) for 5 to 10 column volumes. Protein (insulin and its analogues) was loaded onto column at 2 gram to 20 gram per liter. The protein was eluted with linear gradient from 10%-14% to 20-25% B in 10 to 30cv’s. Desired protein proteins were collected in multiple fractions and pH was adjusted to 5.5-8.5 using tris buffer. Purity obtained (using analytical HPLC) at this stage is more than 99.5% with step recovery of 50%-80%. (refer Fig: 55 to 60 and 65 to76).

Example 21: Protein Crystallization:
Crystallization on was performed by adding 0.1% to 2.0% zinc chloride and adjusting the pH to 5.5 to 7.0 using acetic acid at temperature range of 2°C -30°C. After settling of crystals for 1 to 5 hours, supernatant was decanted and centrifuged to collect the crystals.

Example 22: Lyophilization:
Protein crystals were freeze dried by changing temperatures from -45oC to 5oC under vacuum for 65 hours.

Example 23: Determination of intact mass:
Intact mass was determined using LC ESI MS for in-house insulin and its analogues. It is found that the intact mass of reference product matches with in-house product (Refer Fig: 61 and 62).

Example 24: SDS PAGE analysis:
Purity and impurity profile for insulins and its analogues were studied using a tris tricine silver stained SDS PAGE using reference standard. A single compact band was observed for in-house insulins and its analogues as well as the innovator product (Refer Fig: 63, 64 ,77 and 78).

Example 25: Expression of insulin and its analogs in Pichia pastoris:
For P. pastoris growth, a glycerol vial is used to inoculate MGY medium in shake flask and incubated at 30oC for 24 hours. A well grown seed is transferred to production fermentor having salt based defined media containing orthophosphoric acid, MgSO4, K2SO4, KOH and glycerol. The batch is induced with methanol after 24 hour of growth and a high cell density of 500 g/L wet cell weight is achieved after exponential feeding of methanol. The broth is harvested after cycle time of about 10 days. The cell free supernatant containing the precursor is recovered after subjecting the supernatant to centrifugation at 10000 g force for 20 minutes.

Some non-limiting advantages offered by the method of the present disclosure are as follows:
• Increased yield of recombinant proteins. About 5 – 20% of protein to cell mass ratio was obtained with fusion tags in the present invention compared to 3% of protein to cell mass ratio without fusion tag.
• Increased stability of recombinant proteins owing to formation of inclusion bodies with the proteins to prevent rapid intracellular degradation of the desired protein, said increased stability can also be correlated with the drastic increase in the yield of fusion protein;
• Protection of produced proteins from damage such as damage from oxidation, cleavage etc.;
• Applicability of the method to produce multiple proteins including insulin and its analogs;
• Applicability of the method to produce heterologous as well as homologous proteins;
• Flexibility with regard to choice of vector/host cell

Additional embodiments and features of the present disclosure will be apparent to one of ordinary skilled in art based upon the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein. Further, the disclosure herein provides for examples illustrating the above described embodiments, and in order to illustrate the embodiments of the present disclosure, certain aspects have been employed.

However, the examples used herein are intended merely to facilitate an understanding of ways in which the embodiments herein may be practiced and to further enable those of skill in the art to practice the embodiments herein. Accordingly, the following examples should not be construed as limiting the scope of the embodiments herein. It is an object line of the present disclosure to overcome at least one of the disadvantages of the prior art.

Unless the context clearly requires otherwise, the words “comprise”, “comprising”, and the like are to be construed in an inclusive sense throughout the description and the claims, as opposed to an exclusive or exhaustive sense. ,CLAIMS:We Claim:
1. A method for obtaining recombinant insulin or analogue thereof, said method comprising:
e) designing an expression construct comprising leader sequence - B – C – A, wherein the ‘leader sequence’ is selected from tryptophan operon leader (TrpLE) or fragment derived from thioredoxin protein; ‘B’ represents B chain of the insulin or analog thereof; ‘A’ represents A chain of the insulin or analog thereof; and ‘C’ represents connecting peptide; and wherein the leader sequence is fused to N-terminal of the B-chain;
f) cloning the expression construct into a vector;
g) transforming a host cell with the vector; and
h) culturing the host cell to obtain the insulin or analog thereof.
2. The method of claim 1, wherein the expression construct comprises promoter selected from inducible promotor or constitutive promoter; and wherein said promoter is not a tryptophan (Trp) promoter.
3. The method of claim 2, wherein the promoter is selected from a group comprising T5, T7, AOX, GAP, GAL, TPI, TEF, CUP1, PL, PR, TDH and ILV and PHOA, or any combination thereof.
4. The method of claim 1, wherein the insulin analog is selected from a group comprising glargine, aspart, lispro, glulisine and insulin detemir, or any combination thereof.
5. The method of claim 2, wherein the leader sequence comprises a cleavage site selected from a group comprising nucleotide sequence corresponding to DDDDK, KR, KK, RR, RK, K and R, or any combination thereof; and wherein the leader sequence is conjugated to N-terminal of the B-chain via said cleavage site.
6. The method of claim 5, wherein the leader sequence comprising cleavage site is selected from a group comprising SEQ ID No. 1, SEQ ID No. 2, SEQ ID No. 14 and combinations thereof.
7. The method of claim 6, wherein the connecting peptide comprises nucleotide sequence corresponding to any one of DDDDK, KR, KK, RR, RK, K and R, or any combination thereof.

8. The method of claim 1, wherein the leader - B – C – A sequence is selected from a group comprising SEQ ID No. 29 to 35.
9. The method of claim 1 wherein the expression construct is cloned into the vector in tandem repeats in copies ranging from about 2 to about 20, preferably about 12.
10. The method of claim 1, wherein the vector is selected from a group comprising pET series, TA vector and combinations thereof.
11. The method of claim 1, wherein the host cell is a prokaryote selected from a group comprising E.coli K12, E.coli BL21DE3, E.coli K12 DE3 and combinations thereof, or a eukaryote selected from a group comprising Pichia pastoris, Saccharomyces cerevisiae, Hansenula sp., Yarrowia sp., Kluveromyce sp. and combinations thereof.
12. The method of claim 1 wherein the transformation of vector into host cell is carried out by technique selected from a group comprising heat shock, electroporation, spheroplast and combinations thereof.
13. The method of claim 1 wherein the culturing of the host cell yields precursor of insulin or analog thereof; and said precursor is converted to functional insulin or analog thereof by:
a) cleavage of the leader peptide and the connecting peptide; and
b) cleavage of C-terminal extension of the B-chain.
14. The method of claim 1, wherein the expressed precursor of insulin or analog thereof accumulate as inclusion bodies in the cytoplasm of host cell.
15. The method of claim 13, wherein the leader peptide and the connecting peptide is cleaved by protease enzyme selected from a group comprising kexin, trypsin and a combination thereof; and the C-terminal extension of the B-chain is cleaved by a protease enzyme carboxypeptidase B.
16. The method of claim 15, wherein the protease enzymes are employed in modes selected from a group comprising batch, fed-batch, continuous, immobilized mode or any combination thereof.
17. The method of claim 1 or claim 13, wherein the functional insulin or analog thereof obtained is purified by a technique selected from a group comprising ion exchange chromatography, hydrophobic chromatography, multiple high pressure reverse phase chromatography or any combination thereof.
18. The method of claim 1 or 13, wherein purity of the obtained functional insulin or analog thereof ranges from about 95% to about 100%, preferably about 97% to about 99.8%.

Dated this 16th day of August, 2015

DURGESH MUKHARYA
IN/PA-1541
Of K&S Partners
Mob: +91 7349778249
Agent for the Applicant(s)
To:
The Controller of Patents,
The Patent Office, at: Chennai