金属螯合亲和层析在纯化重组类人胶原蛋白中的应用

Purification of Recombinant Human-like Collagen by Using Metal Chelated Affinity Chromatography

研究了应用金属螯合亲和层析法纯化重组类人胶原蛋白的条件。分别考察了不同金属离子、平衡缓冲液pH值及两种洗脱方法对纯化结果的影响。结果表明,用锌离子螯合介质,pH7.5磷酸盐缓冲液平衡,上样后用100mmol·L-1氯化铵洗脱纯化效果最佳。纯化后重组类人胶原蛋白的纯度达98%,回收率为85.4%,纯化倍数为3.4。SDS-聚丙烯酰胺凝胶电泳与N端序列分析结果均证明所得纯品为重组类人胶原蛋白。

Recombinant human-like collagen with a hexahistidine tail attacked to the carboxyl end could be easily purified by Metal Chelated Affinity Chromatography (MCAC). The affinity capability of MCAC with different immobilized metal ions (Zn2+, Cu2+ and Ca2+), the pH values of equilibrate buffer and different elution methods were compared. The results show that the effect of MCAC immobilized with Zn2+ is more effective than that with Cu2+ and Ca2+, the best equilibrate buffer pH is 7.5, and elution with 100 mmol&middotL-1 ammonium chloride is better than that with pH gradient. The purity of recombinant human-like collagen could be 98%, recovery is 85.4%, purification fold is 3.4. The protein has the collagen features and has only one stripe on its SDS-PAGE electrophotogram. The molecular weight is 90 ku and N terminus amino acids sequence is NH2-H-D-P-V-V-L-Q-R-R-D-W-E-N-P-G, which is consistent with that deduced from DNA sequence.