丝氨酸蛋白酶抑制剂可抑制昆虫细胞表达的蛋白质降解(英文)  

Serine Protease Inhibitor Inhibits Degradation of the Proteins Expressed in Insect Cells

作　　者：韩俊海[1] 吕海芹[1] 臧宇辉[1] 朱洁[1] 连玉官 陈涛[1] 秦浚川[1] 

机构地区：[1]南京大学生命科学院医药生物技术国家重点实验室,南京210093

出　　处：《南京大学学报（自然科学版）》2004年第5期566-575,共10页Journal of Nanjing University（Natural Science）

基　　金：National Natural Science Foundation of Cina(30270703)

摘　　要：干细胞因子是一种多功能细胞因子,能在多级造血水平与其他细胞因子协同作用促进造血干/祖细胞及各种血细胞的存活、增殖和分化。重组人二连体干细胞因子具有比干细胞因子单体更高的比活,可以避免副反应。重组人二连体干细胞因子在Sf9细胞和大肠杆菌中表达时,发现有特异性降解。片段缺 失实验证实切割位点位于重组人二连体干细胞因子的145位到165位氨基酸之间。丝氨酸蛋白酶抑制剂aprotinin和PMSF能抑制这种特异性降解。试验了不同浓度的aprotinin和PMSF对Sf9细胞存活和重组人二连体干细胞因子产量的影响,显示当aprotinin的浓度为 1.0μg/mL时,重组人二连体干细胞因子的产量是没有加aprotinin时产量的2倍,而且aprotinin可以完全抑制这种特异性降解。Stem cell factor (SCF) is a hematopoietic cytokine that promotes the survival, proliferation, and differentiation of hematopoietic cells. The recombinant dual human stem cell factor (rdhSCF) possesses higher specific activity than the monomer form of hSCF and might be preferred since it could be administered at low doses to avoid significant mast cell activation while stimulating hematopoietic recovery. In this report, degradation was found when rdhSCF was expressed in Sf9 cells and E. coli cells. The deletion analysis of rdhSCF confirmed that the cleavage site locates in the region from 145 to 165 amino acid of rdhSCF. Only serine protease inhibitor aprotinin and PMSF could inhibit the site-specific degradation. The influences of aprotinin and PMSF on Sf9 cell survivals and rdhSCF yield were measured at different concentrations. When aprotinin concentration was 1.0 μg/mL, the highest expression level of rdhSCF was achieved, which was about two times higher than without adding protease inhibitor. Aprotinin could completely inhibit degradation of rdhSCF expressed in insect cells.

关 键 词：重组人二连体干细胞因子 杆状病毒表达系统 SF9细胞 丝氨酸蛋白酶抑制剂 APROTININ PMSF 

分 类 号：Q78[生物学—分子生物学]