PREPARATION OF BOVINE SERUM ALBUMIN MONOMER FOR CONJUGATION EXPERIMENTS BY USING DIFFERENT TYPES OF CHROMATOGRAPHY MEDIA

Abstract

The presence of some sugar contaminants and high molecular mass (HMW) derivatives detected in commercially available bovine serum albumin (BSA) preparations precluded the use of such reagents for basic molecular biology experiments. In this study the effectiveness of three separation methods of monomeric BSA from contaminants were examined: gel filtration on Sephadex G-200, HW-55S resin, and ion-exchange chromatography on DEAE-Sephadex A-50. Only chromatography on HW-55S allowed obtaining protein lacking HMW derivates and freeing of sugar contaminants, which was confirmed in SDS/PAGE and phenol/sulfuric acid analysis, respectively. Moreover, the determination of protein particle size by dynamic light scattering (DLS) at a level of 7 nm was in accordance with data for BSA monomeric molecules previously reported. Finally, the monomer BSA was subjected to modification with natural core oligosaccharide derived from Shigella lipopolysaccharide. The SDS/PAGE analysis of reaction products demonstrated different amounts of glycoconjugates compared to values obtained for commercial BSA. Only the pure protein modification permitted correct appraisal of obtained products. We propose gel filtration chromatography on HW-55S resin as simple and effective method of monomer BSA separation from commercial reagent before basic and applied research.

Keywords:

• bovine serum albumin
• DEAE-Sephadex A-50
• HW-55s
• protein modification
• Sephadex G-200
• size-exclusion chromatography

ACKNOWLEDGMENT

This study was supported by Grant No 3564/B/P01/2007/33 from the Ministry of Science and Higher Education of Poland and by research funded by the Wroclaw Research Center EIT + Ltd. in the task III.4: “Diagnosis and prevention of bacterial diseases.” This project was also implemented under the program: “The enterprising PhD student – Investment in Innovative Development of the Region” from Human Capital Operational Program, Priority VIII, Action 8.2 Knowledge transfer, 8.2.2 Sub-Regional Innovation Strategy. This project is co-financed by the European Union as part of the European Social Fund.