Title of Invention

A POLYNUCLEOTIDE SEQUENCE, A VECTOR AND A METHOD TO EXPRESSING AND PURIFYING AN EPIDERMAL GROWTH FACTOR.

Abstract

The invention describes a polynucleotide sequence encoding a novel epidermal growth factor (EGF), a vector comprising a first nucleotide sequence encoding an epidermal growth factor; a second nucleotide sequence encoding a fusion tag; and, a third nucleotide sequence encoding a chemically cleavable site, and a promoter sequence to express the epidermal growth factor, wherein the third nucleotide sequence is between the first nucleotide sequence and the second nucleotide sequence. The invention further discloses a method for obtaining a transformed host cell with the said vector and procuring purified EGF.

Full Text

The present invention relates to a polynucleotide sequence, a vector and a method to expressing and purifying an epidermal growth factor. FIELD OF INVENTION The present invention particularly relates to a polynucleotide sequence encoding epidermal growth factor and a vector to express and purify an epidermal growth factor. More particularly, it relates to epidermal growth factor, which is a recombinant protein. The recmbinant epidermal growth factor (rEGF) according to this invention is over expressed by implanting a vector in a host before harvesting and purifying. Furthermore, the present invention relates to a method of purifying the epidermal growth factor (EGF). BACKGROUND Recombinant technology has been used to clone express and purify several protein including epidermal growth factor. Epidermal growth factor (EGF) is known to have therapeutic properties; for example, it is known to have excellent wound-healing properties. The epidermal growth factor gene has been chemically synthesized and cloned in bacteria and yeast. Epidermal growth factor has also been cloned and purified using streamline chromatography. Epidermal growth factor has also been produced using a cationic exchanger and subsequent cleavage by Carboxypeptidease B. However, most of these processes and the vectors used therein, suffer from poor recovery and purity of epidermal growth factor. Furthermore, some of these processes also suffer from having several steps. Alternatively EGF is produced isolating protein from natural sources. However, in addition to the multi-steps and prolonged time requirement for isolation of protein the continuous availability of natural source poses problems for commercial production. The prior art known to the inventor includes CA 1269055 corresponding to US 4743679. The patent describes recombinant DNA methods to produce fusion protein by introducing glutamyl residue at the point of attachment of the leader sequence and the first amino acid of EGF. It also provides biochemical methods for generating EGF from the fusion protein through specific enzymatic cleavage of the glutamyl residue preceding amino acid sequence of EGF. Korean Patent No. KR 9301386 relates to producing human EGF comprising construction of a vector containing a DNA sequence that encodes formation of a fused polypeptide of EGF gene attached through CP protein gene specific to Staphylococcus protein A, transferring to microorganism, growing the said transformed microorganism and isolating EGF from fusion protein by cleaving. WO2005005476 provides EGF having an N-terminal cap covalently bonded to amino terminus of EGF to delay inactivation of rEGF. According to the disclosure of the said patent stability of EGF is increased by attaching a small fragment of peptide, referred to as N-cap and having at least three amino acids, to the N- terminal of EGF. The preferred group of amino acids may be Ala-Arg-lle.Further, the rEGF is expressed in E. coli. US 5,652,120 describes a process for expression and purification of recombinant human EGF from E.coli. Another Us Patent No. 5,096,825 relates to a process for expression of recombinant human EGF in yeast cells. Both these patents differ from naturally occurring EGF in that they contain extra N-terminal methionine residue. Thus, it is apparent that the currently available methods are not satisfactory and the stability of the enzyme remains a problem as long as it is stored in aqueous form and above 0°C. Therefore, there is a need to obtain the epidermal growth factor in a more efficient manner. SUMMARY OF INVENTION The present invention relates to a polynucleotide sequence having EGF encoded therein, a vector and method to expressing and purifying an epidermal growth factor. The polynucleotide sequence comprises a first nucleotide sequence encoding the epidermal growth factor; a second nucleotide sequence encoding a fusion tag; and a third nucleotide sequence encoding a chemically cleavable site, wherein the third nucleotide sequence is between the first nucleotide sequence and the second nucleotide sequence. The vector comprises a first nucleotide sequence encoding the epidermal growth factor, a second nucleotide sequence encoding a fusion tag; and a third nucleotide sequence encoding a chemically cleavable site, and a promoter sequence to express the epidermal growth factor, wherein the third nucleotide sequence is between the first nucleotide sequence and the second nucleotide sequence. The fusion tag functions as a purification facilitator, for example, allowing the fusion protein to be purified using affinity purification. The cleavage site allows the EGF to be separated from the fusion tag, for example, through enzymatic cleavage. According to an embodiment the chemically cleavable site can be, but is not limited to, an cnterokinase cleavable site or a trypsin cleavable site. The fusion tag can be, but is not limited to, a poly-Histidine tag, preferably a six-Histidine tag (SEQ ID NO.: 3), or a poly-Arginine tag, preferable a six-Arginine tag (SEQ ID NO.: 4). Other cleavable sites and tags known in the art are also appropriate for the present invention. The preferred combinations are a poly-Arginine fusion tag and the a trypsin cleavable site, or a poly-Histidine fusion tag and a enterokinase cleavable site. A fusion protein containing a poly-Ilistindine tag can be affinity purified by binding to metal ions, such as cobalt or nickel. A fusion protein containing poly-Arginine tag can be affinity purified using a ion exchange mechanism, such as with a cationic resin, e.g. S-sepharose. According to another embodiment, a method for obtaining an epidermal growth factor comprises transforming a host cell with a vector, wherein the vector can comprise a first nucleotide sequence encoding the epidermal growth factor; a second nucleotide sequence encoding a fusion tag; and a third nucleotide sequence encoding a chemically cleavable site, wherein the third nucleotide sequence is between the first nucleotide sequence and the second nucleotide sequence and a promoter sequence to express the epidermal growth factor; harvesting the transformed host cell; and, obtaining the epidermal growth factor. According to a further embodiment, the obtaining step can further comprise separating the host cell; lysing the host cell; and, treating the host cell with a chaotropic agent to give an inclusion body. The chaotropic agent can comprise one of guanidine chloride and urea. The host cell may comprise E. coli cells, mammalian cells or yeast cells. The obtaining step can further comprise using a first chromatography to obtain a partially-purified epidermal growth factor. The first chromatography can comprise one of a convention chromatography, a cationic chromatography or a streamline chromatography. The partially-purified epidermal growth factor can be treated with an enzyme such as enterokinase or trypsin, to cleave the epidermal growth factor from the fusion tag. Thereafter, a second chromatography can be used to obtain the epidermal growth factor. The second chromatography can comprise one of a cationic chromatography and anionic chromatography. The epidermal growth factor can then be filtered for final use. The host cell appropriate for the present invention can be, but is not limited to, E. coli cell, a mammalian cell, plant cells, insect cells or a yeast cell. BRIEF INSCRIPTION OF FIGURES FIG. 1 illustrates a vector map in accordance with one embodiment of the present invention. FIG. 2 illustrates a vector map in accordance with another embodiment of the present invention. FIG. 3 illustrates a polynucleotide sequence (SEQ ID NO.: 1) in accordance with an embodiment of the present invention. FIG. 4 illustrates a sequence of a fusion protein (SEQ ID NO.: 2) in accordance with an embodiment of the present invention. FIG. 5 illustrates a flow chart of a process of purifying an epidermal growth factor in accordance with an embodiment of the present invention. FIG. 6 illustrates a flow chart of a process of purifying the epidermal growth factor in accordance with a further embodiment. DE I AILED DESCRIPTION As required, detailed embodiments of the present invention are disclosed herein; however, it is to be understood that the disclosed embodiments are merely exemplary of the invention, which can be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limiting, but merely as a basis for the claims and as a representative basis for teaching one skilled in the art to variously employ the present invention in virtually any appropriately detailed structure. Further, the terms and phrases used herein are not intended to be limiting but rather to provide an understandable description of the invention. The terms "a'" or "an", as used herein, are defined as one or more than one. The term "plurality'*, as used herein, is defined as two or more than two. The term "another', as used herein, is defined as at least a second or more. The terms "including" and/or "having", as used herein, are defined as comprising (i.e., open language). The present invention relates to a nucleotide sequence and a vector to express and purify epidermal growth factor. Furthermore, the present invention relates to a method of purifying epidermal growth factor as a fusion protein. According to an embodiment, a polynucleotide sequence can comprise a first nucleotide sequence encoding an epidermal growth factor; a second nucleotide sequence encoding a fusion tag; and a third nucleotide sequence encoding a chemically cleavable site, wherein the third nucleotide sequence is between the first nucleotide sequence and the second nucleotide sequence. As used throughout the specification and claims, "chemically cleavable site" can be one that can be treated with a chemical agent to break the peptide bonds at the site, thereby effectively separating the epidermal growth factor from the fusion tag. According to another embodiment, a vector can comprise a first nucleotide sequence encoding an epidermal growth factor; a second nucleotide sequence encoding a fusion tag; a third nucleotide sequence encoding a chemically clcavable site; and, a promoter sequence to express the epidermal growth factor, wherein the third nucleotide sequence is between the first nucleotide sequence and the second nucleotide sequence. According to an embodiment, the chemically cleavable site can be an enterokinase clcavable site and the fusion tag can be a poly-Histidine tag. FIG. 1 shows a map of a vector according to an embodiment, wherein the fusion tag is a six-Histidine tag and the chemically cleavable site is an enterokinase cleavable site. The expression of epidermal growth factor is under the influence of a heat inducible promoter, XPRPL. The cntcrokinase site has a nucleotide sequence coding for 5 Aspartic acid and lysine amino acid. The epidermal growth factor sequence was cloned in a pucl8 vector along with the enterokinase site and the histidine tag. Initially, pucl8 was digested with Smal and Pstl (obtained from New England Biolabs #141S and #140S, MA, USA). The insert was amplified from the pucl8 vector using gene specific primers. A heat inducible vector pGSlOO was digested with BamHI and Ecori. The insert was amplified; and the amplified insert was ligated into the vector of this invention designated as BBILlOOto give a plasmid. The plasmid was transformed in E.coli. Positive clones were selected on ampicillin plates and the clones were checked for expression of epidermal growth factor. As shown in FIG.l, the recombinant epidermal growth factor is under the control of APRPL promoter and the pMB 1 receptor and the ampicillin resistance gene. According to another embodiment, the chemically cleavable site is a trypsin cleavable site and the fusion tag encodes a poly-Arginine tag. FIG. 2 shows a map of a vector that can express recombinant epidermal growth factor in accordance with the embodiment. The sequence pertaining to the epidermal growth factor, the trypsin cleavable site at the N-tcrminal of the epidermal growth factor sequence and the six-Arginine amino acids at the C-terminal region of the epidermal growth factor sequence, were chemically synthesized. The synthetic gene was cloned in a PUC18 vector. Initially, PUC18 was digested with Smal and Pstl (New England Biolabs, #141S and #140S, MA, USA). The vector was transformed into a DH5a E. coli strain. Ampicillin positive colonies were selected and the clones were grown in Luria broth media. The plasmid was purified as described in Sambrook J, E.F. Fritsch and T. Maniatis,1989, Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. The inserted gene was amplified using gene specific primers. A heat inducible pGSIOO vector was digested with BamHI and EcorL The plasmid was transformed in E. coli and positive clones for ampicillin were selected and tested for expression of epidermal growth factor. In this case, the expression of the epidermal growth factor is under a Lac promoter. FIG. 3 discloses a polynucleotide sequence, SEQ ID NO.: 1, in accordance with an embodiment of the present invention. According to the embodiment, the chemically cleavable site is a Trypsin cleavable site and the fusion tag is a poly-Arginine tag. According to another embodiment, a fusion protein can comprise an epidermal growth factor, a fusion tag; and a chemically cleavable site between the epidermal growth factor and the fusion tag. FIG. 4 illustrates a sequence of the epidermal growth factor fusion protein, SEQ ID NO.: 2, in accordance with an embodiment of the invention. According to another embodiment, as shown in FIG. 5 a process for obtaining an epidermal growth factor comprises: transforming a host cell with a vector as per step 505, wherein the vector can comprise a first nucleotide sequence encoding the epidermal growth factor; a second nucleotide sequence encoding a fusion tag; and a third nucleotide sequence encoding a chemically cleavable site, wherein the third nucleotide sequence is between the first nucleotide sequence and the second nucleotide sequence; harvesting the host cell as per step 510; and, obtaining the epidermal growth factor as per step 515. According to a further embodiment shown in FIG. 6, the obtaining step further comprises as per step 605: separating the host cell. For example, after fermentation, the host cell needs to be separated from the supernatant. The obtaining step further comprises lysing the host cell as per step 605. After lysing the host cell, the obtaining step further comprises, as per step 605, treating the host cell with a chaotropic agent. For example, the chaotropic agent can comprise one of urea or guanidinc chloride, according to one embodiment. The chaotropic agent can also be another agent which can perform a similar function in a sodium acetate buffer. Those of skill in the art will appreciate that the chaotropic agent is not limited to the examples stated, and the invention shall have full scope of the claims. Treating the lysed cell with the chaotropic agent gives an inclusion body. The obtaining step further comprises subjecting the inclusion body to a first chromatography to give a partially-purified epidermal growth factor protein, as per step 610. The first chromatography can include the use of conventional chromatography, cationic chromatography or streamline chromatography, as is known to those skilled in the art. The partially purified epidermal growth factor protein is treated with an enzyme, as per step 615. For example, the enzyme can be enterokinase or trypsin, depending on the type of chemically cleavable site used in transforming the cell. The enzyme helps in cleaving at certain sites to separate the epidermal growth factor from the fusion tag. The obtaining step further comprises performing a second chromatography to give the epidermal growth factor as per step 620. The second chromatography can include the use of cationic or anionic chromatography to obtain the epidermal growth factor. The obtaining step further comprises filtering the epidermal growth factor as per step 625. According to one embodiment, master cell banks and working cell banks were prepared from selected clones, wherein the selected clones included sequences encoding trypsin and six-Arginine amino acid residues. The vials were stored at - 70°C and characterized for various parameters known to those skilled in the art of recombinant technology guidelines. A single cryovial of cells was used to develop the inoculum. Uric fly, in the first stage, 0.5 ml of the cells were inoculated in 5 ml Luria Broth with 50 ng/ml final concentration of ampicillin, grown at 37°C for 12 hours. In the second stage, 4 ml of inoculum was transferred to 50 ml Luria Broth with 50 ng/ml ampicillin. The culture was grown at similar conditions for the same period of time. In the third stage, 12.5 ml of inoculum was added to 250 ml of high cell density medium (HCDM) at the same time and conditions. 2000 ml of inoculum was transferred to 100 L HCDM, in 150 L fermentor and fermentation was carried out at 37°C, at an initial RPM of 3000, an initial volume of air/volume of media (VVM) of about 0.3 and a pH of 6.9. The batch mode was carried for about 12 to 13 hours by increasing the VVM to about 1.8 as well as the RPM to 800. The fed batch was carried out for 10 to 12 hours with 90% glucose. Subsequently the culture was induced by a combination of inducers and glucose. After 4 to 5 hours, the cells were harvested. Subsequent to fermentation, the cells were lysed and treated with urea, which provided a solubilized inclusion body. The inclusion body was passed through conventional Cationic Chromatography or Streamline Chromatography with SP matrix, to obtain a partially purified protein. The partially purified protein was treated with trypsin at appropriate concentration ranging from 1:250 to 1:1000 of trypsin to protein, to digest the arginine tag. Thereafter, a second round of cationic chromatography was carried out to provide the epidermal growth factor, which was filtered and ready for use. According to another embodiment, master cell banks and working cell banks were prepared from selected clones, wherein the selected clones included sequences encoding enterokinase and six-Histidine residues. The vials were stored at - 70°C and characterized for various parameters known to those skilled in the art of recombinant technology guidelines. A single cryovial of cells was used to develop the inoculum. Briefly, in the first stage, 0.5 ml of the cells were inoculated in 5 ml Luria Broth with 50 jig/ml final concentration of ampicillin, grown at 37°C for 12 hours. In the second stage, 4 ml of inoculum was transferred to 50 ml Luria Broth with 50 \ig/m\ ampicillin. The culture was grown at similar conditions for the same period of time. In the third stage, 12.5 ml of inoculum was added to 250 ml of high cell density medium (HCDM) at the same time and conditions. 2000 ml of inoculum was transferred to 100 L HCDM, in a 150 L fermentor and fermentation was carried out at 37°C, at an initial RPM of 3000, an initial volume of air/volume of media (VVM) of about 0.3 and a pH of 6.9. The batch mode was carried for about 12 to 13 hours by increasing the VVM to about 1.8 as well as the RPM to 800. The fed batch was carried out for 10 to 12 hours with 90% glucose. Subsequently the culture was induced by a combination of inducers and glucose. After 4 to 5 hours, the cells were harvested. Subsequent to fermentation, the cells were lysed and treated with urea, which provided a solubilized inclusion body. The inclusion body was purified with Ni NTA Affinity column in a conventional chromatography to obtain a partially purified protein. The partially purified protein was treated with enterokinase to digest the Histidine specific sequences. Anionic chromatography was used to separate the epidermal growth factor, which was then filtered and ready to use. In the foregoing specification, specific embodiments of the present invention have been described. However, one of ordinary skill in the art appreciates that various modifications and changes can be made without departing from the scope of the present invention as set forth in the claims below. Accordingly, the specification and figures are to be regarded in an illustrative rather than a restrictive sense, and all such modifications are intended to be included within the scope of present invention. The benefits, advantages, solutions to problems, and any element(s) that may cause any benefit, advantage, or solution to occur or become more pronounced are not to be construed as a critical, required, or essential features or elements of any or all the claims. The invention is defined solely by the appended claims including any amendments made during the dependency of this application and all equivalents of those claims as issued. WE CLAIM: 1. A polynucleotide sequence comprising: a first nucleotide sequence encoding an epidermal growth factor; a second nucleotide sequence encoding a fusion tag; and, a third nucleotide sequence encoding a chemically cleavable site, wherein the third nucleotide sequence is between the first nucleotide sequence and the second nucleotide sequence. 2. The polynucleotide sequence as claimed in claim 1, wherein the fusion tag comprises a six-Arginine fusion tag and the chemically cleavable site comprises a trypsin cleavable site. 3. The polynucleotide sequence as claimed in claim 1, wherein the fusion tag comprises a six-Histidine fusion tag and the chemically cleavable site comprises an enterokinase cleavable site. 4. The polynucleoptide sequence as claimed in claim 1, having the nucleotide sequence ofSEQ ID NO.: 1. 5. A vector comprising: a first nucleotide sequence encoding an epidermal growth factor; a second nucleotide sequence encoding a fusion tag; a third nucleotide sequence encoding a chemically cleavable site; and, a promoter sequence to express the epidermal growth factor, wherein the third nucleotide sequence is between the first nucleotide sequence and the second nucleotide sequence. 6. The vector as claimed in claim 5, wherein the fusion tag comprises a six-Arginine fusion tag and the chemically cleavable site comprises a trypsin cleavable site. 7. The vector as claimed in claim 5, wherein the fusion tag comprises a six-I listidine fusion tag and the chemically cleavable site comprises an enterokinase cleavable site. 8. A host cell transformed by implanting the vector of claim 5. 9. The host cell as claimed in claim 8, wherein the host cell is an E. coli cell, a mammalian cell, plant cells, insect cells or a yeast cell. 10. A fusion protein comprising: an epidermal growth factor; a fusion tag; and, a chemically cleavable site between the epidermal growth factor and the fusion tag. 11. The fusion protein as claimed in claim 10, wherein the fusion tag comprises a poly-Histidine tag or a poly-Arginine tag. 12. The fusion protein as claimed in claim 10, wherein the chemically cleavable site comprises a enterokinase cleavable site or a trypsin cleavable site. 13. The fusion protein as claimed in claim 10, having the amino acid sequence of SHQIDNO.:2. 14. A method of obtaining an epidermal growth factor, the method comprising steps of: transforming a host cell with a vector, wherein the vector comprises a first nucleotide sequence encoding the epidermal growth factor, a second nucleotide sequence encoding a fusion tag; a third nucleotide sequence encoding a chemically cleavable site, and a promoter sequence to express the epidermal growth factor; the third nucleotide sequence is between the first nucleotide sequence and the second nucleotide sequence; harvesting the host cell; and. obtaining the epidermal growth factor. 15. The method as claimed in claim 14, wherein the obtaining step further comprises: separating the host cell; lysing the host cell; and, treating the host cell with a chaotropic agent resulting in formation of an inclusion body, partially purifying the said inclusion body/fusion protein, cleaving and further purifying to obtain EGF. 16. The method as claimed in claim 15, wherein the chaotropic agent comprises one of guanidine chloride and urea. 17. The method as claimed in claim 15, wherein the partial purification in obtaining step further comprises: subjecting inclusion body to conventional chromatography to obtain a partially purified epidermal growth factor wherein the chromatography is a cationic chromatography and/or a streamline chromatography. 18. The method as claimed in claim 15, wherein the cleaving is effected by treating the partially purified epidermal growth factor with an enzyme, wherein the enzyme comprises one of enterokinase and trypsin. 19. The method as claimed in claim 15, wherein the further purification comprises subjecting the said cleaved EGF to chromatography comprising one of a cationic chromatography and anionic chromatography followed by separating the purified EGF by conventional methods such as filtration. 20. A polynucleotide sequence, a vector and a method to expressing and purifying an epidermal growth factor substantially as herein described with reference to drawings. Dated this 17th day of May 2006

Documents:

0642-che-2005-abstract.pdf

0642-che-2005-claims.pdf

0642-che-2005-correspondnece-others.pdf

0642-che-2005-description(complete).pdf

0642-che-2005-description(provisional).pdf

0642-che-2005-drawings.pdf

0642-che-2005-form 1.pdf

0642-che-2005-form 26.pdf

0642-che-2005-form 3.pdf

0642-che-2005-form 5.pdf

642-CHE-2005 AMANDED PAGES OF SPECIFICATION 19-03-2010.pdf

642-CHE-2005 CORRESPONDENCE OTHERS 16-04-2012.pdf

642-CHE-2005 FORM-13 16-04-2012.pdf

642-CHE-2005 POWER OF ATTORNEY 16-04-2012.pdf

642-che-2005 abstract 07-09-2009.pdf

642-CHE-2005 AMANDED CLAIMS 19-03-2010.pdf

642-che-2005 claims 07-09-2009.pdf

642-CHE-2005 CORRESPONDENCE OTHERS 07-09-2009.pdf