FIELD OF THE INVENTION The present invention relates to a recombinant growth factor vascular endothelial growth factor with fully functioning active peptide, expressible in expression system(s), and a process of preparation thereof.

BACKGROUND AND PRIOR ARTS OF THE PRESENT INVENTION

Growth factors are endogenous signaling polypeptides between cells which acts by binding to specific receptors on the surface of their target cells. These help in stimulating cell proliferation, growth, differentiation and healing.

Growth factors have a good role in recruiting many attractive agents which enhance the rate of healing around the wound area. They will help in regulating the cellular responses required for wound healing process such as migration, proliferation and differentiation.

Vascular endothelial growth factor (VEGF) is one of the best studied angiogenic factor which stimulates angiogenesis, by up regulating the angiogenic factors such as Fibroblast Growth Factor (FGF), PDGF (Platelet Derived Growth Factor) and epidermal growth factor. VEGF is a 45 kDa heterodimeric heparin-binding signal polypeptide expressed in endothelial cells, comes under the sub family of growth factors, is also known as vascular permeability factor. VEGF is primarily involved in vasculogenesis (de novo formation of embryonic circulatory system) and angiogenesis (the growth of blood vessels from pre-existing vasculature). Under hypoxic conditions, when the blood supply is inadequate VEGF plays an important role in restoring oxygen to tissues. Though the VEGF and its receptors primarily expressed in endothelial cells, it is expressed well in non-endothelial cells like tumor cells, macrophages, platelets, keratinocytes, and renal mesangial cells. So, the up regulation of VEGF and VEGF receptors can initiate tumor.

In mammals, VEGF family comprises five (5) groups members namely VEGF-A (placenta growth factor), VEGF-B, VEGF-C and VEGF-D. VEGF proteins encoded in viruses (VEGF-E) and snake venom (VEGF-F) have been identified. Among them both VEGF-A and VEGF-D found to enhance wound healing as well as angiogenesis.

VEGF has a prominent role in several facets of the wound repair process by initiating angiogenesis, it is a strong inducer of vascular permeability in the skin. As it increases angiogenesis, it results in the formation of more blood vessels around the wound region and thereby improving more supply of oxygen and blood around the wound region.

In sum, wound healing is a chronic process, which involves the recruitment of several signals which helps in the proliferation, differentiation of the wound area. So, protein molecules which enhance the rate of wound healing will help in faster healing of wounds. The growth factors can perform a number of events which ultimately results in the wound healing. In our invention we are developing such a growth factor which can do some positive actions in the wound area

In this invention the inventors have achieved the development of mature VEGF using prokaryotic expression system, with a minimal production cost and capable of delivering to a suitable scaffold vehicle to the wound site which heal the chronic wounds by recruiting more blood vessels to the wound site, thus, initiates a better healing.

Recombinant human VEGF have been found critical in treating surgical wounds as well as in the therapy of diabetic foot ulcers.

US 11541848 discloses purifying of a peptide, polypeptide or protein from a biological sample said method comprising subjecting the biological sample to affinity chromatography to purify human origin VEGF-B. US 08915795 isolates VEGF that can stimulate at least one endothelial cell biological activity.

WO 1999037671 A1 isolated nucleic acid molecules that encode novel molecules related to the VEGF family to stimulate angiogenesis, by administration of a therapeutically effective amount of a VEGF-R antagonist.

US 10792480 discloses VEGF-C protein and gene, mutants thereof. And uses thereof by purifying and isolating a polypeptide which is capable of binding to the Flt4 receptor tyrosine kinase and the extracellular domain of Flt4 receptor tyrosine kinase and stimulates Flt4 autophosphorylation. US 6180107 B1 discloses VEGF II, a heterodimer protein which, stimulates mitogenesis of mammalian vascular endothelial cells, and proven to be useful for the promotion of vascular development and repair.

US 20030166547 A1 disclosed a method of inducing angiogenesis in a tissue in need of angiogenesis. US 20030215921 A1 discloses a biologically active VEGF 2 polypeptide, a nucleic acid vector construct, which can be administrated in chronic limb ischemic patients.

US 20030215921 A1 worked on VEGF-2. US5194596 A discloses the production of VEGF. US 5194596 A isolated VEGF selected from the group consisting of bovine vascular endothelial cell growth factor of 120 amino acids, and human VEGF of 121 amino acids.

OBJECTS OF THE PRESENT INVENTION: It is therefore an object of this invention to develop a growth factor which helps in bringing more blood vessels to the wound area. Another objective of the study is to create active peptide which enhances wound healing in a very short time period.

Yet another objective of this invention is to combine the developed growth factor with another growth factors which helps in wound healing

Still another objective of the invention is to deliver the growth factors to a suitable scaffold which suites the skin.

SUMMARY OF THE INVENTION In one embodiment, the present invention discloses a recombinant growth factor vascular endothelial growth factor with fully functioning active peptide, expressed in prokaryotes, with a Glutathione S - Transferase (GST) tag, purified to 80-90 %, a fully functioning active peptide, so it doesn’t need any further post translational modification for its action. The prokaryotic expression system makes the recombinant growth factor VEGF-D cost effective.

The fully functioning active peptide is synthesized by specially selecting the active sequence which targets angiogenesis. In another embodiment, using the low-cost procedure of protein isolation protocol and bulk protein purification protocol by eluting out all the bacterial impurities using affinity columns which can be reused.

In another embodiment, primers for reverse transcriptase-polymerase chain reaction (RT-PCR) for cloning the c-DNA obtained from VEGF, comprising a reverse primer and a forward primer under SEQUENCE ID NO.: 1 and 2 respectively, has been disclosed. In yet another embodiment, an active peptide, a 352 base pair (bp) fragment from the whole VEGF-D gene) which specifically regulates the cellular functions down regulates wound healing, is prepared. The forward primer is 28 bp with the GC percentage is 44.8, and a melting temperature of 70.9° C, said sequence is deliberated under SEQUENCE ID NO.: 2, and the reverse primer is 32 bp with the GC percentage is 43.8, and a melting temperature of 72.5° C, said sequence is detailed under SEQUENCE ID NO.: 1.

In yet another embodiment, the peptide is having a purity of 80-90%, which does not make allergic reactions in humans, a functionally active peptide of vascular endothelial growth factor (VEGF) of SEQUENCE ID NO.: 3 for enhancing angiogenesis especially in wounds by binding to the receptors in the wound cells without undergoing any post-translational modifications.

In still another embodiment, the present invention discloses primers for reverse transcriptase-polymerase chain reaction (RT-PCR) for cloning the c-DNA obtained from VEGF, comprising a forward primer and a reverse primer.

In still another embodiment, the present invention discloses a process of preparation of VEGF for enhancing angiogenesis especially in wounds by binding to the receptors in the wound cells without undergoing any post-translational modifications, said process comprising the steps of isolating the RNA of VEGF (SEQUENCE ID NO.: 3) performed through washing with water, grounding using motor and pestle in liquid nitrogen, adding RNA lysis solution and homogenizing for short pulse using tissue homogenizer, treating with DNase I for 15 min and purifying by phenol chloroform and precipitating with ammonium acetate and isopropanol; RT-PCR of VEGF using primers of SEQUENCE ID NO.: 1 and 2, and collecting the purified c DNA performed by mixing 7 pi of isolated RNA with 1 mΐ of VEGF, and incubating for 5 minutes at 70° C, followed by cooling down using ice, adding with 4 mΐ of 5X RT-PCR buffer, 10 mM dNTP, 0.5 mΐ of RNAse inhibitor of 30 U / mΐ, and finally mixing with 5 mΐ of nuclease free deionized water followed by incubation at 37° C for 5 minutes, adding 0.5 mΐ of 200 U / mΐ M-MuLV reverse transcriptase, again incubating at 42° C for 60 minutes for the reaction to occur, stopping said reaction by heating at 70° C for 10 minutes, followed by purifying of amplified VEGF; characterized in that the reaction mixture of PCR comprises 10.5 pi of nuclease free deionized water, 1 pi each of VEGF forward and reverse primers, 1 mΐ of 10 mM dNTP, 2 mΐ of 25 mM magnesium chloride (MgCh), 2.5 mΐ of 10 X PCR buffer, 2 mΐ of 1 U / mΐ Taq polymerase, and 5 mΐ of cDNA obtained, making a resultant volume of 25 mΐ; ligating the clones as obtained with the vector pTZ57R/T comprising essentially of adding 1 mΐ of pTZ57R/T to 10 mΐ of VEGF; along with 1.5 mΐ 10 X cohesive end ligase buffer followed by adding 1 mΐ of PEG 4000 and BSA each and finally adding 0.5 mΐ of T4 DNA ligase, expressing in XL1 Blue by electroporation and screening using blue white screening method; isolating the plasmid and VEGF forward clone confirmed with 364 base pair and 2884 base pair using the restriction enzyme Bam HI, ligating to the vector pGEX4Tl having a size of 4929 base pair by incubating a reaction mixture at overnight, characterized in that said reaction mixture constitutes, 8 mΐ each of pTZ / VEGF, 10X Bam H I Buffer, 10 U / mΐ Bam HI restriction enzyme, along with 63.5 mΐ of nuclease free deionized water., wherein the amplified product is screened and selected by transforming XL1 blue cells; preparing the competent cells comprising essentially of adding XL1 Blue strain is added into 5 ml of LB broth at 37°C rotating at 200 rpm overnight; inoculating 2 ml of this initial day’s culture to 100 ml of LB broth, and incubating at 37°C rotating at 200 rpm till the time the OD reaches 0.4; transferring to 50 ml sterile polypropene tubes duly sealed thereafter with paraffin; followed by inoculation on ice, and then keeping for rotation at 4,500 rpm for 10 minutes at 4°C, decanting of the supernatant, suspending the pellets in 5 ml ice cold CaCh of 200 mM; keeping in ice for 20 minutes, resuspending the pellets in 15 ml of 80 mM CaCh, keeping on ice for 20 minutes, and storing in fridge for further use; transformation is performed through incubating 100 mΐ of competent cells and 1 mΐ of plasmid in a sterile tube for 30 minutes on ice, applying heat shock at 42° C for 90 seconds with immediate storing in ice, followed by adding 500 mΐ of LB broth media, keeping at 37° C for 45 minutes at 350 rpm; confirming the insert using EcoRv and EcoRI double digestion of pGEX4Tl - VEGF, confirming VEGF single insert in reverse orientation; and expressing VEGF proteins induced using IPTG as this triggers the lac operon gene in the pGEX4Tl vector; isolating the protein comprising essentially of inoculating 1 ml culture of VEGF into 100 mL of LB ampR media and incubating at 37°C at 200 rpm, adding 0.1 mM IPTG when the OD reaches 0.6, incubating for 3 hours at 200 rpm at 37°C, transferring to centrifuge tubes wherein the centrifugation is performed at 500 g for 15 minutes at 4°C, keeping the obtained pellets aside and resuspending using 1 ml of IX PBS, followed by adding lysozyme at a concentration of 1 mg/ ml, vortex ting and keeping in ice for 30 minutes, adding 10 ml of 0.2% Triton to each tube, and passing of solution through 5 ml syringes, keeping the tubes are then kept at 4°C for 30 minutes, centrifuging at 3000g for 30 min at 4°C, adding 1 mM DTT to the supernatant, and separating by Glutathione S transferase (GST) column chromatography with a glutathione Sepharose 4B column; wherein the protein is further purified through pre-equilibrating using the column of IX PBS in 10 bed volume, clarifying the crude protein sample with 0.22 mm filter syringe and passing through the column, cleaning column with 2 ml of IX PBS between each sample application, eluting the column with 6 ml of reduced glutathione buffer, collecting the elution fractions, following re-calibration of the column with 20 ml of IX PBS.

In a further embodiment, a pharmaceutical composition comprising essentially of SEQUENCE ID NO.: 3 along with other additives, carriers, excipients and other filler materials, is disclosed.

In a still further embodiment, a method of healing wounds through enhanced angiogenesis by the application of SEQUENCE ID NO.: 3 or through the application of pharmaceutical composition has been disclosed.

BRIEF DESCRIPTION OF ACCOMPANYING DRAWINGS The illustrated embodiments of the subject matter will be best understood by reference to the drawings, wherein like parts are designated by like numerals throughout. The following description is intended only by way of example, and simply illustrates certain selected embodiments of devices, systems, and processes that are consistent with the subject matter as claimed herein, wherein:

Figure 1: a. 352 bp-long VEGF-D gene fragment used for expressing mature peptide; and b. Over expression of VEGF on IPTG induction.

DETAILED DESCRIPTION OF THE INVENTION

At the very outset of the detailed description, it may be understood that the ensuing description only illustrates a form of this invention. However, such a form is only exemplary embodiment, and without intending to imply any limitation on the scope of this invention. Accordingly, the description is to be understood as an exemplary embodiment and teaching of invention and not intended to be taken restrictively.

Throughout the description and claims of this specification, the phrases “comprise” and“contain” and variations of them mean“including but not limited to”, and are not intended to exclude other moieties, additives, components, integers or steps. Thus, the singular encompasses the plural unless the context otherwise requires. Wherever there is an indefinite article used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

Thus, the terms“comprises”,“comprising”, or any other variations thereof used in the disclosure, are intended to cover a non-exclusive inclusion, such that a device, system, assembly that comprises a list of components does not include- only those components but may include other components not expressly listed or inherent to such system, or assembly, or device.

In other words, one or more elements in a system or device proceeded by “comprises... a” does not, without more constraints, preclude the existence of other elements or additional elements in the system, apparatus or device.

Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with an aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith. All the features disclosed in this specification (including any accompanying claims, abstract and drawings), and / or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not restricted to the details of any foregoing embodiments. The invention extends to any novel one, or any novel combination, of the features disclosed in this specification including any accompanying claims, abstract and drawings or any parts thereof, or to any novel one, or any novel combination, of the steps of any method or process so disclosed. The reader's attention is directed to all papers and documents which are filed concurrently with or before this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference. Post filing patents, original peer reviewed research paper shall be published.

The following descriptions of embodiments and examples are offered by way of illustration and not by way of limitation.

Unless contraindicated or noted otherwise, throughout this specification, the terms “a” and“an” mean one or more, and the term“or” means and/or. As used in the description herein and throughout the claims that follow, the meaning of “a.” “an,” and“the” includes plural reference unless the context clearly dictates otherwise. Also, as used in the description herein, the meaning of“in” includes “in” and“on” unless the context clearly dictates otherwise. The principle aim of the invention is to develop a functionally active peptide of VEGF, specifically of VEGF-D, for that the peptide of VEGF-D is designated, then it is coned into a resistant vector supplemented with a particular antibiotic resistance. Throughout the specification, instead of VEGF-D, VEGF has been used, and it may be understood that wherever the reference of VEGF with respect to this present invention has been mentioned, said VEGF is VEGF-D, sequence of which is appended as SEQUENCE ID NO.: 4.

The vector may be any vector selected from plasmids, phasmids, viral vectors, cosmids, and artificial chromosomes or the likes. The vector is a DNA/ RNA molecule used as a vehicle to artificially carry foreign genetic material into another cell, where it can be replicated and / or expressed, therefore, the vector must be free-floating, and able to carry a desired information.

Virtually all vectors that are used to deliver a gene segment, contain genes for antibiotic resistance. Once bacteria have been treated with a plasmid, scientists grow them in the presence of antibiotic. Only those cells that contain the plasmid will survive, grow and reproduce. The presence of antibiotic resistance gene helps as a selection marker. The cells which have taken up the plasmid will acquire resistance to the antibiotic and will be able to grow on the antibiotic media. The cells who have not taken the plasmid won't be able to survive on the antibiotic media. Antibiotic resistance is the ability of a microorganism to withstand the effects of an antibiotic. It is a specific type of drug resistance, which can be a useful way of implanting artificial genes into the microorganism. Antibiotic resistance is a consequence of evolution via natural selection. To ensure the retention of plasmid DNA in bacterial populations, an antibiotic resistance gene is included in the plasmid.

Any antibiotic resistant gene will have a specific segment ensuring the resistance against a particular antibiotic, including but not limited to ampicillin, streptomycin, amoxicillin, penicillin or the likes. In this present invention, an ampicillin resistant gene may be selected for the recombinant vector. However, in order to identify the sequence in vivo, there is required a proper tagging, said tagging may be chemical or using bioluminescence. For this invention, a glutathione S transferase (GST) tag has been used specifically to purify our protein of interest from the bacterial protein, said system is expressed in a prokaryotic host system.

The VEGF thus synthesized will specifically target the wound healing pathway. As the mature peptide has been synthesized, it can directly bind to the receptors in the wound cells without undergoing any further post translational modifications. Since the gene is cloned and expressed in a prokaryotic system, it makes the invention cost effective. The GST tag used to purify our protein of interest from the bacterial protein does its task to 80-90%.

The VEGF mature peptide with 352 bp, deliberated under SEQUENCE ID NO.: 3 can be isolated from a donor tissue where angiogenesis is high, said tissue may be any skeletal or involuntary or cardiac muscles.

Here for example it may be isolated from the human heart. Total RNA of human heart tissue was isolated and VEGF cDNA was prepared. The cDNA, thus obtained was then subject to polymerase chain reaction (PCR) using forward and reverse primers.

The total RNA was isolated from discarded heart tissue bits. These tissues are discarded, and although derived from the biological world, however, doesn’t constitute a part of the biological world, and may be considered like hairs and nails, the collection of which doesn’t take any pleasant or unpleasant effect on the donor. The tissue may be collected from anywhere, however, the collection point is Sree Chitra Tirunal Institute for Medical Sciences.

The tissue is washed with water, ground using motor and pestle in liquid nitrogen. To be finely powdered tissue, RNA lysis solution was added and homogenized for short pulse using tissue homogenizer. Isolated RNA was treated with DNase I for 15 min and purified by phenol chloroform and precipitated with ammonium acetate and isopropanol. The RNA pellet was dissolved in 15ul sterile water.

The present formulation is further clarified by giving the following exhibits. It must, however, be understood that these exhibits are only illustrative in nature and should not be taken as limitations to the capacity of the invention. Several amendments and improvements to the disclosed segments will be obvious to those skilled in the art. Thus, these amendments and improvements may be made without deviating from the scope of the invention.

Example 1:

RNA Isolation:

The total RNA is isolated from discarded heart tissue bits. The tissue is washed with water, ground using motor and pestle in liquid nitrogen. To be finely powdered tissue, RNA lysis solution is added and homogenized for short pulse using tissue homogenizer. Isolated RNA is treated with DNase I for 15 min and purified by phenol chloroform and precipitated with ammonium acetate and isopropanol. The RNA pellet was dissolved in 15 pi sterile water. Example 2:

Primer designing for the Reverse Transcription of VEGF:

Primer designing is the primordial part of this step, namely forward and reverse primers, which are designed based on the RNA obtained from the atrial appendage, and corresponding cDNA. The length of the reverse primer is 32 bp with the GC percentage is 43.8, and a melting temperature of 72.5° C, said sequence is detailed under SEQUENCE ID NO.: 1. Similarly, the length of the forward primer is 28 bp with the GC percentage is 44.8, and a melting temperature of 70.9° C, said sequence is deliberated under SEQUENCE ID NO.: 2. Example 3: Polymerase Chain Reaction (PCR) for the Reverse Transcription of VEGF:

7 mΐ RNA thus isolated is mixed with 1 mΐ of VEGF, and incubated for 5 minutes at 70° C, followed by cooling down using ice. This was then added with 4 mΐ of 5X RT-PCR buffer, 10 mM dNTP, 0.5 mΐ of RNAse inhibitor of 30 U/ mΐ, and finally mixed with 5 mΐ of nuclease free deionized water followed by incubation at 37° C for 5 minutes. Following this incubation, 0.5 mΐ of 200 U / mΐ M-MuFV reverse transcriptase is added. This mixture is again incubated at 42° C for 60 minutes for the reaction to occur, said reaction is then stopped by heating at 70° C for 10 minutes. The below Table showing the cycles of said PCR:

The reaction mixture of PCR comprises 10.5 mΐ of nuclease free deionized water, 1 mΐ each of VEGF forward and reverse primers, 1 mΐ of 10 mM dNTP, 2 mΐ of 25 mM magnesium chloride (MgCk), 2.5 mΐ of 10 X PCR buffer, 2 mΐ of 1 U / mΐ Taq polymerase, and 5 mΐ of cDNA obtained, thus making a resultant volume of 25 mΐ. Example 4:

Purification of amplified VEGF and its expression:

Complementary DNA (cDNA) of VEGF was made from mRNA isolated from discarded human heart tissue. VEGF having a size of 352 bp is amplified from the cDNA. and then the amplified VEGF was eluted from the gel using QIAquick PCR purification kit. The purified product was further confirmed by agarose gel electrophoresis with 360 base pair length.

In order to introduce TA overhangs to the amplified VEGF, it was then cloned to the vector pTZ57R/T (Fermentas Inc).

Example 5:

Ligation Reaction:

For the ligation, 1 mΐ of pTZ57R/T is added to 10 mΐ of VEGF; and in the mixture, 1.5 mΐ 10 X cohesive end ligase buffer is added. This is then followed by addition of 1 mΐ of PEG 4000 and BSA (bovine serum albumin) each, followed by final addition of 0.5 mΐ of T4 DNA ligase.

The TA cloned VEGF gene is expressed in XL1 Blue by electroporation and it is screened using blue white screening method. Later plasmid is isolated and VEGF forward clone is confirmed with 364 base pair and 2884 base pair using the restriction enzyme Bam HI, the forward primer for VEGF PCR constitutes Bam HI site.

Then VEGF/pTZ5 clones are ligated to the vector pGEX4Tl having a size of 4929 base pair, by incubating a reaction mixture at overnight, said reaction mixture constitutes 8 mΐ each of pTZ/ VEGF, 10X Bam H I Buffer, 10 U / mΐ Bam HI restriction enzyme, along with 63.5 mΐ of nuclease free deionized water. The amplified product is screened and selected by transforming XL1 blue cells.

Example 6:

Preparation of competent cells: On the initial day, XL1 Blue strain is added into 5 ml of LB media aka luria broth at 37°C rotating at 200 rpm overnight. 2 ml of this initial day’s culture is inoculated to 100 ml of LB broth, and incubated at 37°C rotating at 200 rpm till the time the OD (Optical Density) reaches 0.4. Then the culture was transferred to 50 ml sterile polypropene tubes duly sealed thereafter with paraffin. This is followed by inoculation on ice, and then keeping for rotation at 4,500 rpm for 10 minutes at 4°C. Then the supernatant is decanted, and the pellets are resuspended in 5 ml ice cold CaCL of 200 mM. This is then kept in ice for 20 minutes, resuspended the pellets in 15 ml of 80 mM CaCL, kept on ice for 20 minutes, and stored in fridge for further use.

Example 7:

T ransf ormation : Incubation is done with 100 pi of competent cells and 1 pi of plasmid in a sterile tube for 30 minutes on ice, then the heat shock is applied at 42° C for 90 seconds with immediate storing in ice, followed by adding 500 mΐ of LB broth media, keeping at 37° C for 45 minutes at 350 rpm.

Cloning is confirmed by blue white selection. Plasmid isolated insert was confirmed using restriction endonuclease digestion. EcoRv and EcoRI double digestion of pGEX4Tl - VEGF gave three bands at base pair 249 bp, 1905 bp and 3179 bp, confirmed VEGF single insert in reverse orientation. Expression of VEGF proteins are induced using IPTG as this triggers the lac operon gene in the pGEX4Tl vector. Example 8:

Protein isolation using IPTG induction Protocol:

1 ml culture of VEGF is inoculated into 100 mL of LB ampR media and incubated at 37°C at 200 rpm. When the OD reaches 0.6, 0.1 mM IPTG is added, then incubated for 3 hours at 200 rpm at 37°C. The cultures are then transferred to centrifuge tubes wherein the centrifugation is performed at 500 g for 15 minutes at 4°C. The pellets are then kept aside and resuspended using 1 ml of IX PBS (Phosphate Buffer Saline), followed by addition of lysozyme at a concentration of 1 mg/ ml, vortexed and kept in ice for 30 minutes. Then 10 ml of 0.2% Triton is added to each tube, and the solution is passed through 5 ml syringes. The tubes are then kept at 4°C for 30 minutes, centrifuged at 3000g for 30 min at 4°C, and to the supernatant 1 mM DTT is added.

The isolated crude protein which includes the bacterial as well as the protein of interest having 41 KDa was separated by Glutathione S transferase (GST) column chromatography .

An affinity chromatography procedure is done with a glutathione Sepharose 4B column. This column matrix helps in binding GST Fusion proteins, which upon on contact with glutathione reduced elution buffer decrease its affinity towards Sepharose 4B and bind off from the stationary phase. The purified proteins are qualitatively analyzed using SDS PAGE. The purity of the protein is ~ 80 - 90%.

Example 9:

Protein bulk purification protocol:

The column is pre-equilibrated using the column of IX PBS in 10 bed volume, and the crude protein sample is clarified with 0.22 mm filter syringe and passed through the column. The column is cleaned with 2 ml of IX PBS between each sample application. The column is then eluted with 6 ml of reduced glutathione buffer, and elution fractions are collected and stored. The column is then re equilibrated with 20 ml of IX PBS.

Example 10:

Pharmaceutical Composition: A pharmaceutical composition comprising essentially of SEQUENCE ID NO.: 3 along with other additives, carriers, excipients and other filler materials.

The pharmaceutical composition may be in the form of tablet or capsule or sublingual administration tablets or the likes, or liquid or suspension or injectable or patch or cream or ointment or gel like topical formulation for topical administration or the likes. Generally, the preferred route is oral or injectable.

A pharmaceutical composition for the preferred oral or sublingual route of administration can be obtained by combining SEQUENCE NO.: 3 with conventional pharmaceutical additives and excipients used in the art for sublingual preparations. Appropriate formulation methods are well known to the person skilled in the art; see, for instance, Pharmaceutical Dosage Forms: Tablets. Volume 1, 2nd Edition, Lieberman H A et ah; Eds.; Marcel Dekker, New York and Basel 1989, p. 354-356, and literature cited therein. Suitable additives comprise additional carrier agents, preservatives, lubricants, gliding agents, disintegrants, flavorings, and dyestuffs as state of the art knowledge is concerned. A disintegrating agent may be incorporated in the composition of the invention, wherein such an agent will accelerate the dispersion of the carrier particles. Examples of disintegrating agents according to the invention include cross-linked polyvinylpyrrolidone, carboxymethyl starch, natural starch, microcrystalline cellulose, cellulose gum and mixtures of in 1% to 10% of the composition. The modus operandi of disintegrating agent and the bio/ mucoadhesion promoting agent overlap somewhat, and it may be preferred that both functions are served by the same substance. However, it is important to note that these two categories of excipients are not equivalent, and there are efficiently functioning disintegrants which do not possess bio/mucoadhesive properties, and vice versa.

The use of solid, non-porous spheres allows to have a precise knowledge of the mass- surface relationship of particles and therefore, by virtue of a selection of the size of the spheres, that is to say of the radius or of a distribution of radii, to have a precise control of the rate of release of the active ingredient or active ingredients administered, thus, by avoiding overdosages or the need to compensate for underdosages, makes it possible to reduce the total administration of the biologically active substance SEQUENCE ID NO.: 3 to the minimum quantity required in order to obtain the desired therapeutic effect and thereby decrease the risk of producing undesirable secondary effects in the patient.

Microspheres may be prepared of decreasing the volume of solid material which has to be injected into a living organism, having the advantage of not using a low- melting excipient, those particles could agglutinate and cause handling problems upon injection along with the advantage of not introducing unnecessary solid excipient, more or less degradable, into the organism.

A number of substances may be combined with adjuvants not directly active on the receiving organism, said combination may comprise various pharmaceutically acceptable additive means for increasing the stability or chemical integrity of the biologically active substances, it being understood that they are not vector type excipients. In particular, it may become useful to decrease the melting point or to inhibit a decomposition reaction during the microsphere manufacturing process through melting-freezing or the likes. The microspheres may be classified and separated more finely and more reliably as a function of their size than irregularly shaped particles.

The formulation according to the present invention may also be provided in the form of microsphere powder in vials-ampoules ready for making into a suspension, or in the form of a suspension ready prepared in injectable ampoules ready for administering in human or veterinary medicine differing only in dosage. The suspension medium may be sterile water, a sterile saline solution, an oil containing the buffers, surfactants or preservatives conventionally employed in injectable suspensions by pharmacists, or any other substance or combination, which does not threaten the physical and chemical integrity of the substances in suspension and which is suitable for the organism which will receive it. If it is desired to avoid a sudden initial elevation of the level of active ingredient in the internal medium of the receiving organism, the use will be preferred, in the case of ready-for-use suspensions, of liquid vectors in which the said active ingredients are practically insoluble. In the case of active substances partially soluble in the lukewarm liquid vector but insoluble at cold temperature, it is preferable, from the pharmacological point of view, to avoid the formation of precipitates i.e. to avoid any undesired caking effect by preparing formulations in the form of separate microsphere powder and liquid vector which will be mixed only at the time of injection.

In veterinary applications where the duration of desired effect may be very long like for the case of lactation in adult female animals, diameters of some hundreds of microns may be used. If it is desired to limit the diameter of needles for injection syringes for the comfort of the patient, it is good to limit the diameter of the microspheres to 300 microns and more preferably to 100 microns. In contrast, for very short durations of desired effect, such as circadian, the diameter of the microsphere may be reduced to 1 micron.

For most applications in human medicine, it may be preferable to use microspheres whose diameter is between 0.1 to 100 microns.

An essential condition for achieving the dosage form according to the present invention is to have batches of calibrated microspheres, that is to say homogeneous in diameter. Microspheres may also be separated according to their diameter during the manufacture using cyclonic separators, by sieving using air suction or by sieving in a liquid medium. If more than 70% of the microspheres have diameters of between 70% and 130% of a specified diameter, the condition may be referred sufficient. Mixing of batches with different suitable diameters may be done following the ideal dissolution curve as determined for the present invention.

Means like mechanical abrasion may be used so is suspension of the product in the melted state in the form of microdrops, with stirring, in a liquid vector with which the said product is non-miscible, followed by solidification of the said product. Other method of obtaining porous microspheres like spraying, freezing and freeze-drying in a cold gas substances dissolved in a suitable solvent, may be employed.

Furthermore, the particles which are not in compliance with the specifications may be recycled. An additive may also be used in order to eliminate a phase transition, from a solid phase to another solid phase, which is likely to weaken the structure of the sphere. The process is also suited to mixtures of active substances in solid solution one inside the other. Now, the crux of the invention is claimed implicitly and explicitly through the following claims.

Each of the appended claims defines a separate invention, which for infringement purposes is recognized as including equivalents to the various elements or limitations specified in the claims. Depending on the context, all references below to the“invention” may in some cases refer to certain specific embodiments only. In other cases, it will be recognized that references to the“invention” will refer to subject matter recited in one or more, but not necessarily all, of the claims.

Groupings of alternative elements or embodiments of the invention disclosed herein are not to be construed as limitations. Each group member can be referred to a claimed individually or in any combination with other members of the group or other elements found herein. One or more members of a group can be included in or deleted from, a group for reasons of convenience and / or patentability. When any such inclusion or deletion occurs, the specification is herein deemed to contain the group as modified thus fulfilling the written description of all groups used in the appended claims.