批量离子交换层析和凝胶过滤层析在类人胶原蛋白I纯化中的应用

研究了利用批量离子交换层析与凝胶过滤层析相结合的方法纯化类人胶原蛋白I的最佳条件。分别考察了不同离子交换树脂、缓冲溶液pH、离子强度、进料蛋白浓度对批量离子交换的影响,以及不同凝胶过滤介质对凝胶过滤层析的影响。结果表明,采用阴离子交换树脂DEAE52吸附杂蛋白,缓冲液pH为7.0,NaCl浓度为0.2mol/L,进料蛋白浓度为40mg/mL,并采用凝胶过滤介质SephadexG-100进一步纯化后,类人胶原蛋白I的纯度可达到98.2%,总纯化倍数为3.1,总回收率为80.6%。SDS-PAGE分子量分析和N末端氨基酸序列分析均表明纯化后的类人胶原蛋白I与基因设计一致。

PURIFICATION OF HUMAN-LIKE COLLAGEN BIOPOLYMER I BY BATCH ION EXCHANGE CHROMATOGRAPHY COMBINED WITH GEL FILTRATION CHROMATOGRAPHY

The method of batch ion exchange chromatography combined with gel filtration chromatography to purify Human-like Collagen Biopolymer I was investigated. The different ion exchange resins, pH and ionic strength of buffer solution, the feed concentration of protein and different gel filtration resins were compared. The results showed that applying anion exchange resin DEAE52, pH of buffer solution was 7.0, the NaCl concentration was 0.2mol/L, the feed concentration of protein was 40mg/mL and applying gel filtration resin Sephadex G-100, were more effective. The purity of Human-like Collagen Biopolymer I could reach 98.2% and the purification fold multiple was 3.1. The total recovery ratio was 80.6%. The molecular weight and N terminus amino acids sequence analysis showed which consistent with that deduced from DNA sequence.