Claims:We Claim,
1. A method of purification of glycoprotein using affinity column chromatography, the said method comprising:
- cross linking an insoluble guar gum with epichlorohydrin emulsion in 25mL 3N Sodium Hydroxide;
- stirring the mixture vigorously until it gets solidified and then keeping it at 40ºC in a water bath for 24 hours with occasional stirring followed by heating in an oven at 70 ºC;
- homogenizing the mixture in a mortar pestle to a particle size of about 30mm size in the presence of Sodium Phosphate buffer (pH 7.4);
- packing the homogenized guar gum into a column and equilibrating it with Phosphate buffer;
- washing the column with Phosphate buffer;
- applying the crude protein extract to the column;
- washing the unbound proteins using sodium phosphate buffer;
- eluting the bound protein using 10 mM D-galactose solution.
2. The method of purification of glycoprotein using affinity column chromatography as claimed in Claim 1, wherein the crude protein is extracted from defatted deskinned seeds of Jatropha curcas L using phosphate buffer with saline and precipitated using ammonium sulphate.
3. The method of purification of glycoprotein using affinity column chromatography as claimed in Claim 1, wherein the phosphate buffer is selected from the group of Sodium phosphate buffer, Potassium phosphate buffer having pH in the range of 7.2 - 7.6.
4. The method of purification of glycoprotein using affinity column chromatography as claimed in Claim 1, wherein the guar gum affinity column is washed with phosphate buffer until the absorbance reading of elutes at 280 nm reaches 0.02 ensuring removal of contaminant protein in the column.
5. The method of purification of glycoprotein using affinity column chromatography as claimed in Claim 1, wherein the unbound proteins on guar gum affinity column are washed with phosphate buffer until the absorbance reading of elutes at 280nm is 0.05.
6. The method of purification of glycoprotein using affinity column chromatography as claimed in Claim 1, wherein the purified sample is analyzed using 12% SDS-PAGE gel.
7. The method of purification of glycoprotein using affinity column chromatography as claimed in Claim 1, wherein the purified protein sample is analyzed by HR-MS analysis for determination of amino acid residues and conformation of curcin protein. , Description:DESCRIPTION OF THE INVENTION:
FIELD OF THE INVENTION:
The present invention relates to the field of biochemistry;
More particularly, the present invention relates to purification of glycoproteins.

BACKGROUND OF THE INVENTION:
Jatropha (Jatropha curcas L.) is a drought resistant, a multipurpose perennial plant belonging to Euphorbiaceae family. The plant exhibits antibacterial, antifungal and anticancer properties. Majorly all parts of Jatropha plant including leaf, fruits, latex, and bark can be used in different aliments as traditional medicine.
Nowadays, Jatropha is gaining importance for its oil which is regarded as potential fuel substitute. In addition to oil, it has a high protein content for which it is a supplement in animal feed and manure. Along with all these potential applications of Jatropha in biopharmaceuticals and bio-energy production, the seeds of Jatropha curcas are found to be toxic to humans and animals.
Jatropha oil is used to supplement the fuel requirements for lamps, cooking, and small diesel engines. Jatropha is gaining importance as biodiesel plants. Jatropha is considered as a second-generation biofuel plant that may provide a part of the non-conventional fuel supply. Biodiesel production using this seed oil produces large amount of solid waste called as seed cake. After removal of oil seed cake contains toxic protein Curcin and diterpene esters are called as phobolesters. As the protein content and other minerals are present in very high amount in this seed cake its detoxification will help to use it as manure and animal feed. It adds economical value to it and also reduces environmental damage caused due to non-proper disposal of seed cake.
Lectins are carbohydrate-binding proteins or glycoproteins of non- immune origin that are very specific for sugar moieties. Because of this specificity it agglutinates cells or precipitate polysaccharides. This unique property of lectin Curcin is employed in its purification by affinity chromatography. The lectin Curcin is found to be specific for galactose binding. Curcin a lectin is Ribosome inhibition protein (RIP). RIP’s exhibits different enzyme activities like RNA N- glycosidase, polynucleotide adenosine glycosidase, DNases and RNases. Curcin is also a potent antitumor protein. It specifically kills cancerous cells because of rich glycosylation on cell surface.
After removal of oil from Jatropha seeds there are limitations in using the seed cake as animal feed and manure due to the presence of toxic protein Curcin. The prior art reveals that there have been attempts to remove the Curcin protein by various methods viz. roasting of seeds which degrades various proteins from seeds, removal of protein using gel filtration chromatography.
There are problems with the present approach. Firstly, the gel filtration chromatography involves use of costly material like sepharose, sepharyl; and secondly, the high costs associated with purification essentially limit the scale up of the purification method. Therefore, in light of foregoing discussion, there exists a need to overcome the drawbacks associated with existing prior arts.
The present invention describe A method of purification of glycoprotein using affinity column chromatography which aims to overcome or mitigate the aforementioned problems with the prior art.

OBJECTS OF INVENTION
The primary object of the invention is to provide a method of purification of glycoprotein using affinity column chromatography that overcomes shortcomings of the prior arts;
Another object of the invention is to provide a single step purification method using affinity column chromatography;
Further object of the invention is to provide Jatropha seed cake, free from toxic protein to facilitate its use as manure, animal feed as well as in targeted drug delivery system.

SUMMARY OF THE INVENTION
Embodiments of the present disclosure present technological improvements as solution to one or more of the above-mentioned technical problems recognized by the inventors in conventional practices and existing state of the art.
The present disclosure seeks to provide a method of purification of glycoprotein using affinity column chromatography.
According to an aspect of the present invention, total protein is extracted from defatted skinned seeds of Jatropha curcas L. The extracted protein is precipitated by Ammonium Sulphate precipitation method. The toxic protein present in Jatrophacurcas L. is lectin and therefore haemagglutination assay is used as standard test during isolation of Curcin protein. In the study, 60% crude protein fraction showed positive agglutination for B(Rh+) blood group which shows presence of galactose specific lectin.
According to further aspect of the present invention, affinity chromatography is used to purify proteins which bind non-covalently and reversibly to specific molecules known as ligands.
According to an aspect of the present invention, the affinity chromatography column comprises guar gum beads having free galactose sugar (ligand) on its surface whichbinds the target protein Lectin Curcin completing the protein purification in single step.
The objects and the advantages of the invention are achieved by the process elaborated in the present disclosure.

BRIEF DESCRIPTION OF DRAWINGS
The foregoing Summary, as well as the following detailed description of preferred embodiments of the invention, will be better understood when read in conjunction with the appended drawings. The accompanying drawings constitute a part of this specification and illustrate one or more embodiments of the invention. Preferred embodiments of the invention are described in the following with reference to the drawings, which are for the purpose of illustrating the present preferred embodiments of the invention and not for the purpose of limiting the same. In the drawings:
Fig. 1 is a graph showing affinity chromatography of Curcin protein wherein absorbance of the elutes at 280nm is plotted against the collected fractions of saturations of protein.
Fig. 2 is an image of the SDS-PAGE of proteins after purification by affinity chromatography.
Fig. 3 is an image of BLAST (Basic Local Alignment Search Tool) result page for the Curcin protein purified in accordance with the embodiments of the present invention.
Fig. 4 is an image of HR-MS analysis of Curcin protein. The peaks represent the total ion current chromatogram of purified protein.
Fig. 5 is MASCOT PMF analysis of Curcin protein.
Fig. 6 is Peptide comparison using BLAST search and similarity of curcin with Ricin A chain.

DETAILED DESCRIPTION
The following detailed description illustrates embodiments of the present disclosure and ways in which the disclosed embodiments can be implemented. Although some modes of carrying out the present disclosure have been disclosed, those skilled in the art would recognize that other embodiments for carrying out or practicing the present disclosure are also possible.
The present invention provides a method of purification of glycoprotein using affinity column chromatography.
The present invention is particularly well-suited to purification of D-Galactose binding Lectin from seeds of Jatropha Curcas L. It will be understood that the invention may be applied to other glycoproteins.
Affinity chromatography is a technique used to purify proteins which bind non-covalently and reversibly to specific molecules known as ligands.
According to an embodiment of the present invention, guar gum beads having free galactose sugar (ligand)on its surface is used. The target protein being galactose-specific, retention of lectin curcin is completed in single step purification. This is called as carbohydrate affinity chromatography of lectins.
The guar gum is commonly available in market and is purchased from the supplier - Sigma Aldrich.
According to an embodiment of the present invention, insoluble guar gum is cross-linked with epichlorohydrin emulsion in 25 mL 3N sodium hydroxide. The mixture is stirred vigorously until itgets solidified and then kept at 400C in a water bath for24 hours with occasional stirring followed by heating in an oven at 700C. It is then homogenized in a mortar pestle to a particle size of about 30 mm size in the presence of Sodium Phosphate buffer (pH 7.4). The homogenized guar gum is then packed into a column and equilibrated with Sodium Phosphate buffer (pH7.4).
According to an embodiment of the present invention, Sodium Phosphate buffer washes are given to the affinity chromatography column containing gaur gum and the absorbance of elutes is taken at 280 nm until the reading was 0.02 to ensure that there is no contaminant protein present in the column.
According to a preferred embodiment of the present invention, 100µl of 60% crude protein extract is applied to the column. The unbound proteins are washed out using sodium phosphate buffer. The 1 ml fractions are collected and the absorbance of elutes is taken at280 nm until it became less than 0.05, to ensure all unbound protein to elute out.
According to a preferred embodiment of the present invention, the bound protein is then eluted using 10 mM D-galactose solution, and the absorbance is measured at 280 nm.

Working Example
Preparation of Seed Meal:
The fruits of Jatropha curcas L. were collected from locally grown plants. Selected mature fruits were washed, sun dried for 3-4 days and divided manually into three parts (Pericarps, seed coats, embryos). The mature embryos of seeds were ground finely with a mortar and pestle defatted with two volumes of petroleum ether, at 60-70°C for 24 hours. The resulting powder was left to dry at room temperature for 24 hours.
Extraction of Curcin:
Extraction of Curcin from Jatropha curcas L. was carried out according to the standard procedures 10 as follows: 100 g of mature embryos defatted powder was extracted with 1L of 5 mM-sodium phosphate buffer (SPB), pH 7.2, containing 0.2 M NaCl. The mixture was stirred on a magnetic stirrer for 2-3 h and left overnight at 4°C. The clear supernatant was collected by centrifugation at 3000 g for 20 min. The protein was precipitated with 20%, 40%, 60%, 80% and 100% ammonium sulphate, (NH4)2SO4 saturation. 12.8 gm, 3.29 gm, 3.56 gm, 3.84 gm and 3.45 gm of solid ammonium sulphate was gradually added and the precipitate was collected for the respective saturation of proteins with constant stirring for 30- 60 min. The protein fractions were desalted by dialysis for 24-48 h at 4 ºC against a flow of Sodium Phosphate buffer with 0.2 M 3, 10.
Haemagglutination Assay of Crude Protein Preparations:
The sugar specificity and concentration of lectin can be assayed by haemagglutination assay. All protein fractions were screened for haemagglutination activity against A, B, and O blood group samples of human origin to find out the presence of lectin. Serial twofold dilutions of 20%, 40%, 60%, 80% and 100% saturation protein samples of 0.1 ml aliquot were incubated with 0.1 ml of 4% erythrocyte suspension on slides. The slides were observed format or button formation. The positive hemagglutination showed mat formation.
Protein Estimation in Crude Extracts of Proteins:
Protein concentration was estimated by Lowry’s and Bradford’s methods for 20%, 40%, 60%, 80% and 100% ammonium sulphate saturation fractions.
FTIR (Fourier-Transform Infrared Spectroscopy) analysiswas carried out of crude protein to find out components of aqueous extract.
The various saturation fractions of proteins viz. 20%, 40%, 60%, 80%, and 100% were run on 12% SDS PAGE and native PAGE gel to find out some proteins present in crude extracts before its purification by affinity chromatography.
Desalting of protein samples is carried out by dialysis against phosphate buffer and is referred to as ‘crude protein.’ The 60% saturation gave a huge amount of precipitated protein. Protein was estimated in all ranges of percent saturation from 20% to 100% by Lowry’s and Bradford’s method. The resultant yield ranged from 0.80 mg/ml to 71.44 mg/ml with a maximum yield of 71.44 mg/ml obtained in 60% saturation. As a huge amount of total protein was precipitated in 60% fraction FTIR analysis was carried out to confirm the presence of total proteins and to ensure no other contamination was present in the given fraction. Crude protein was subjected to FTIR analysis which demonstrated peaks for N-H stretching, C=0 stretching, C=O bending, N=N bending of only amino acids representing the presence of only proteins. The proteins of all five fractions were analyzed using 12% SDS-PAGE before purification by affinity chromatography. It showed 4-6 protein bands with the intense band in 60% fraction. They are referring to the high concentration of protein in 60% saturation. The crude protein samples were also analyzed using 12% native gel. The number of bands observed in the native gel was 2-3 for all saturation. The crude protein samples were assessed for haemagglutination activity where protein obtained in 60% saturation showed the positive haemagglutination indicating the presence of lectin in the sample derived from 60% saturation. The haemagglutination was carried out on A, B and AB human blood group samples a positive haemagglutination was seen with B+ve blood group sample indicating its specificity towards galactose binding. In the present invention, affinity chromatography using guar gum for lectin was used for curcin purification.
The insoluble guar gum is cross-linked with epichlorohydrin emulsion in 25mL 3N Sodium Hydroxide. The mixture is stirred vigorously until it gets solidified and kept at 40ºC in a water bath for 24 hours with occasional stirring followed by heating in an oven at 70 ºC. Then it is homogenized in a mortar pestle to a particle size of about 30mm size in the presence of Sodium Phosphate buffer (pH 7.4). The homogenized guar gum is then packed into a column and equilibrated with Sodium Phosphate buffer (pH 7.4).
Then several Sodium Phosphate buffer washes were given to the column, and the absorbance of elutes was taken at 280 nm until the reading reaches 0.02 which ensures removal of contaminant protein in the column. Then 100 µl of 60% crude protein extract was applied to the column as it indicated the presence of lectin in it. The unbound proteins are washed out using sodium phosphate buffer. The 1ml fractions were collected. The absorbance of elutes was taken at 280 nm until it became less than 0.05, to ensure all unbound protein to elute out. The bound protein was then eluted using 10 mM D-galactose solution, and the absorbance was taken at 280 nm. The purified sample was analyzed using 12% SDS-PAGE gel.
Fig. 1 describes a graph showing affinity chromatography of Curcin protein wherein absorbance of the elutes at 280nm is plotted against the collected fractions of saturations of protein. The absorbance of the eluted sample was 0.205 and concentration was 1.884 mg/ml.
Fig. 2 is an image of the SDS-PAGE of proteins after purification by affinity chromatography and molecular weight determination with the standard molecular marker. A single band was observed in Lane 2 on the SDS-PAGE. The molecular weight of purified protein band on the gel was 28 kDa similar to the reported molecular weight of Curcin.
Fig. 3 is an image of BLAST (Basic Local Alignment Search Tool) result page for the Curcin protein purified in accordance with the embodiments of the present invention. The BLAST showed good query coverage and 80 to 100% similarity with very less E value. The ricin A Four proteins had more than 100% query coverage. The max score for the given query was 636.The RIP domain was only present in the BLAST result. Though curcin lectin showed haemagglutination and sugar inhibition assay specific for galactose no conserved domain for carbohydrate binding domain was observed in BLAST results.
The purified protein sample was analyzed by HR-MS analysis for determination of amino acid residues and conformation of curcin protein. The m/z values of samples and MASCOT PMF (Peptide Mass Fingerprinting) analysis resulted into 20 peptides. These peptides by pairwise sequence alignment using NCBI BLASTp showed similarity with Ricin chain A. As Ricin chain A is similar in sequence with Curcin protein it was confirmed in the sample isolated from seeds.