Abstract:Abstract: To prepare human proprotein convertase subtilisin/kexin type 9 (PCSK9), the plasmids named PCSK9-pPICZαA and PCSK9-pcDNA4.0 were constructed with cordon optimized, synthesized human PCSK9 genes. Then they were introduced into GS115 yeast cells by electroporation and suspension cultured Chinese hamster ovary (CHO) cells by transient transfection respectively to express his-tag PCSK9 protein. Human PCSK9 recombinant protein was successfully expressed in both Pichia pastoris and CHO cells. With the SDS-PAGE and Western-blot methods, it is easy to find that the size of the recombinant protein expressed in GS115 was larger than expected and the protein was also found degraded, while the protein expressed in CHO cells can be purified easily with a purity up to 90%, and the size is nearly the same as human native PCSK9 protein. After purification with a His/Ni2+ affinity column, the high specific anti-PCSK9 serum was successfully prepared by immunizing mice with the purified PCSK9. A rapid method of producing human PCSK9 recombinant protein has been developed with a yield of 5.6 mg/L, which laid the foundation for specific PCSK9 inhibitors development and further study of molecular structure and function as well as the relationship between them.
引用本文:
贺梦影, 张长江, 魏 红, 段小波, 谭 文. 在真核细胞表达制备重组人前蛋白转化酶枯草溶菌素9[J]. 生命科学研究, 2015, 19(6): 471-478.
HE Meng-Ying, ZHANG Chang-Jiang, WEI Hong, DUAN Xiao-Bo, TAN Wen. Preparation of Human PCSK9 in Eukaryotic Cells. Life Science Research, 2015, 19(6): 471-478.