聚乙二醇共价修饰β-乳球蛋白的合成和分离纯化方法研究

Study on preparation and purification of PEGylated β-lactoglobutin

目的以β-乳球蛋白(β-lactoglobulin B,LG)为模型蛋白,探讨PEG化蛋白质的最佳合成条件和分离纯化方法。方法用不同摩尔比的NHS-PEG-MAL修饰LG,不同反应时间下,进行SDS-PAGE测定。采用ImageMaster图象分析系统定量凝胶中游离蛋白质,评价蛋白质的PEG化程度:采用灌注色谱技术,通过阳离子交换柱分离纯化反应混合物。结果SDS-PAGE法可使游离LG与PEG-LG达到完全分离。LG的PEG化程度随PEG量和反应时间的增加而提高。在最佳合成条件下,PEG化LG的程度接近95%。被纯化的PEG-LG的产率为84%,LG和PEG的摩尔比为1:7.3。结论对PEG化LG的合成、分析和分离纯化进行了较完整的研究,结果较满意。检定方法可用于其他蛋白质的PEG化研究。

OBJECTIVE Using β-lactoglobulin B （LG） as model protein, to optimize the reaction conditions of polyethylene glycolylated LG （PEG-LG） and study a novel strategy for separation and purification of PEGylated protein,METHODS LG was modified with different molar ratio of N-hydroxylsuccimide-PEG-maleimide （NHS-PEG-MAL,3 400） and sampled at different reaction time. SDS-PAGE was applied to determine the resulted PEGylated protein. The free LG band in SDS-gel was quantified by using the ImageMaster VDS-CL bio-imaging system, and then the degree of PEGylation was assessed, The resulted mixture after PEGylation were separated and purified by cation-exchange perfusion chromatography.RESULTS The free LG and PEG-LG were completely separated by SDS-PAGE. The degree of PEGylated LG increased with increasing mass of PEG and reaction time. In optimizing reaction conditions, the percentage of PEGylation approached to 95 %.The recovery of purified PEG- LG was 84 %, and the molar ratio of LG and PEG was 1：7.3. CONCLUSION PEG-modlfied LG is systemic investigated, the results are relatively satisfied. The assay methods can be applied to the study of other PEGylated proteins.