重组类人胶原蛋白的分离纯化

目的 建立重组类人胶原蛋白分离及纯化工艺。方法 将工程菌株E.coli BL21 3.1在L1523型12.8L自控发酵罐中培养14h,发酵菌体经超声破碎、硫酸铵沉淀后,依次经DEAE-52和Sephadex G-100柱层析分离纯化。结果 发酵菌体干重达到71g/L,表达量为菌体蛋白量的29.4％,最终产物纯度达98％,回收率为80.5％,纯化倍数为3.4。结论 纯化的类人胶原蛋白达电泳纯,相对分子质量为90 000,N端序列为NH_2-H-D-P-V-V-L-Q-R-R-D-W-E-N-P-G,均与其因序列设计一致。

Separation and Purification of Recombinant Human Collagen-like Protein

Objective To develop a method for the purification of recombinant human collagen-like protein. Methods The recombinant E. coli BL21 3.1 strain with human collagen-like protein gene after being incubated in 12.8L Bioengineering L1523 fermentor for 14h was subject to ultrasonication and precipitation with ammonium sulfate,then purified by DEAE-52 and Sephadex G-100 column chromatography.Results The dry weight of thallus in zymotic fluid reached 71g/L, and the expressed product contained 29.4% of somatic protein. The purity, recovery rate and purification fold of final product were 98 %, 80.5 % and 3.4 respectively . Conclusion The obtained recombinant human collagen-like protein, with a relative molecular weight of 90 000, was electrophoretically pure.The amino acid sequence at its N-terminal,NH2-H-D-P-V-V-L-Q-R-R-D-W-E-N-P-G,was identical to that designed.