Title of Invention	"AN IMPROVED PROCESS FOR THE SINGLE STEP ISOLATION OF ALBUMIN"
Abstract	An improved process for the single step isolation of albumin which comprises, adsorbing serum or plasma on the dye ligands as herein described, washing of the unadsorbed material and elution of adsorbed material using buffer as herein described at pH 8 and salt such as 2M NaCl to obtain albumin.

Full Text

The present invention relates to an improved process for the single step isolation of albumin. More particularly it relates to the process to obtain a single band from serum or plasma on SDS-PAGE (sodium dodecyl sulphate - polyacrylamide gel electrophoresis) using commercially available dye ligands. This finds application in pharmaceutical and biotech industries. Most of the blood proteins such as albumin have the therapeutic value, the methodologies for their isolation and purification are well established [Cohn, E.J. HUGHES, J.R.,weare, J.H. J. Am. Chem. Soc. 1947, 69, 1753-1757; Burnouf, T.J.Chromatography. 1995, 664, 3-15]. However, continuous efforts are going on to develop economical process to achieve homogeneous, highly purified and physiologically active blood proteins [Gebauer, K.H., Thommes. J., and Kula, M. -R. Biotechnology and Bioengineering. 1997, 54(2) 192-197]. The established processes comprise different unit operations such as, clarification, centrifugation, filtration, ultra filtration and chromatographic methods as adsorption, ion exchange, size exclusion and affinity (immuno). The multistep isolation process poses significant economical problems. To minimize the number of isolation and purification steps and enhancement of purity is a major thrust area. Although the different chromatographic methods are available, the dye ligand chromatography is at fore front due to its high specificity and low cost [Scopes. R.K.Anal. Biochemistry. 1987, 165, 235-246]. It was reported that the albumin could be selectively removed from plasma using blue dye ligand however, isolated albumin do not show single band on electrophoresis [Travis, J., Bowen, J., Tewksbury, D., Johnson, D., and Pannell, R. Biochem. J. 1976, 157. 301-306]. So far, no process is reported which have isolated albumin in a single step operation that will give single band on SDS-PAGE. We have claimed a process for the single step isolation of albumin to obtain a single band on SDS-PAGE using commercially available dye ligands. The object of the present invention is to provide a process for the single step isolation of albumin to obtain a single band on SDS-PAGE (sodium dodecyl sulphate-polyacrylamide gel electrophoresis) from serum or plasma. Accordingly the present invention provides an improved process for the single step isolation of albumin which comprises, adsorbing serum or plasma on the dye ligands as herein described, washing of the unadsorbed material and elution of adsorbed material using buffer as herein described at pH 8 and salt such as 2M NaCl to obtain albumin. In one of the embodiment of the present invention, the serum or plasma, which may be, obtained from animal source preferably horse and human source. In another embodiment of the present invention, the dye ligands may be any commercially available dye ligands, preferably blue, green and red. In still another embodiment of the present invention, the buffer for equilibration, adsorption, washing, and elution may be acetate, borate, tris etc, preferably phosphate. In a feature of the present invention, the serum or plasma without any pretreatment was adsorbed on the different dye ligands. The elution was carried out with5 ml sodium phosphate buffer pH8, and 2M NaCl. The protein content of the elution was estimated with the folin phenol reagent [Lowry, O.K., Rosebrough, N.N., Farr, A.C. and Randall, R.J. J.Biol. Chem. 1951, 193, 265-275]. In another feature, the identity and purity of the eluted protein was carried out by SDS-PAGE [Weber, K., Osborn, M, J. Biol. Chem. 1969, 244, 4406], The 20µ1 samples were loaded on the 5%, 7.5%, 10% slab gel. Prior to the application, all the samples were diluted (1:1) with incubation mixture (1% SDS, 1%2-Mercaptoethanol, 0.02%Bromophenol Blue and 36%Urea prepared in O.IM phosphate buffer pH7) and kept in a walerbath at 37°C for 1h. The electrophoresis was run at constant current (62 milliamps) for 5h at 22±2°C. The gels were stained with coomassie brilliant blue R250. The experiments were repeated with the application of 40µ1 samples. The process of the present invention is described by following examples, which are illustrative only and should not be construed to limit the scope of the present invention in any manner. Example 1: The horse serum (17 mg protein) was loaded on the different dye ligands packed in a column (20mm x 8mm). The elution obtained by phosphate buffer pH 8, and 2 M NaC1 subjected to protein estimation and SDS-PAGE. The single band was observed from blue, green and red dye ligands on SDS-PAGE. On the basis of electrophoretic mobility, the bands were identified as albumin. Example 2: The horse serum (17 mg protein) was loaded on blue dye ligand. After washing the unadsorbed material, the adsorbed material was eluted with phosphate butler pH 8. 3.34 mg protein was obtained in elution, which showed single band on SDS-PAGE and identified as albumin on the basis of electrophoretic mobility. Example 3: The horse serum (17 mg protein) was loaded on blue dye ligand. After washing the unadsorbed material, the adsorbed material was eluted with 2 M NaCl. 3.0 mg protein was obtained in elution, which showed single band on SDS-PAGE and identified as albumin on the basis of electrophoretic mobility. Example 4: The horse serum (17 mg protein) was loaded on green dye ligand. After washing the unadsorbed material, the adsorbed material was eluted with phosphate buffer pH 8. 4.0 mg protein was obtained in elulion, which showed single band on SDS-PAGE and identified as albumin on the basis of electrophoretic mobility. Example 5: The horse serum (17 mg protein) was loaded on green dye ligand. After washing the unadsorbed material, the adsorbed material was eluted with 2 M NaCl. 0.9 mg protein was obtained in elulion, which showed single band on SDS-PAGE and identified as albumin on the basis of electrophoretic mobility Example 6: The horse serum (17 mg protein) was loaded on red dye ligand. After washing the unadsorbed material, the adsorbed material was eluted with phosphate bufler pH 8. 1.0 mg protein was obtained in elution, which showed single band on SDS-PAGE and identified as albumin on the basis of electrophoretic mobility. Example 7: The horse plasma ( 12 mg protein) was loaded on the different dye ligands packed in a column (20mm x 8mm). The elution obtained by phosphate buffer pH 8, and 2 M NaCl subjected to protein estimation and SDS-PAGE. The single band was observed from blue, green and red dye ligands on SDS-PAGE. On the basis of electrophoretic mobility, the bands were identified as albumin. Example 8: The human serum (36.5 mg protein) was loaded on the different dye ligands packed in a column (20mm x 8mm). The elution obtained by phosphate buffer pH 8, and 2 M NaCl subjected to protein estimation and SDS-PAGE. The single band was observed from blue, green and red dye ligands on SDS-PAGE. On the basis of electrophoretic mobility, the bands were identified as albumin. We Claim: 1. An improved process for the single step isolation of albumin which comprises, adsorbing serum or plasma on the dye ligands as herein described, washing of the unadsorbed material and elution of adsorbed material using buffer as herein described at pH 8 and salt such as 2M NaCl to obtain albumin. 2. A process as claimed in claim 1 wherein the serum or plasma, used is animal serum 3. A process as claimed in claim 1 to 2 wherein the dye ligands used is triazine blue, triazine green & triazine red. 4. A process as claimed in claim 1 to 3 wherein the buffer for equilibration, adsorption, washing, and elution used in acetate, borate, tris, preferably phosphate, 5. A process for the single step isolation of albumin as substantially described herein before with reference to examples.

Documents:

188-del-2000-abstract.pdf

188-del-2000-claims.pdf

188-del-2000-correspondence-others.pdf

188-del-2000-correspondence-po.pdf

188-del-2000-description (complete).pdf

188-del-2000-form-1.pdf

188-del-2000-form-2.pdf

188-del-2000-form-4.pdf