关键词: MUC1-MBP融合蛋白, 离子交换层析, 疏水层析, 超滤技术, 内毒素, 显色基质法鲎试剂

Abstract:

bstract:Objective
 To evaluate the effectiveness of ultrafiltration technology in endotoxin removal from purified recombinant MUC1-MBP fusion protein (MUC1-MBP) and to demonstrate the effect of  ultrafiltration on  endotoxin removal.Methods  CM Sepharose FF weak cation exchange （CM) （CM　group）,CM combined with Phenyl Sepharose 6 FF exchange (C6) (CM+C6  group),CM combined with  ultrafiltration (CM+ultrafiltration  group),and CM combined with C6 and  ultrafiltration (CM+C6+ultrafiltration group) were used to purify the MUC1-MBP from E.coli.and remove endotoxin;the expression level of endotoxin  was detected by Chromogenic End-point Tachypleus Amebocyte Lysate.Results There was a single band at the expected molecular weight of 62 000 by SDS-PAGE analysis.and the purity>96% by Quantity One analysis.The endotoxin levels in CM group and CM +C6 group were quite high and there was no significant difference  between two groups (P>0.05);the endotoxin level in CM+ultrafiltration group was significantly lower than that in CM group,and there was significant difference (P<0.01)；the endotoxin level in  CM+C6+ultrafiltration group was significantly decreased compared with CM+C6 group (P<0.01);there was no significant differences of endotoxin level between CM+ultrafiltration group and CM +C6+ultrafiltration group (P>0.05).Conclusion The effects of CM or CM combined with C6 on endotoxin removal are quite poor,especially C6;CM combined with ultrafiltration are quite effective on endotoxin removal,and   ultrafiltration plays an important role in endotoxin removal.

Key words: MUC1-MBP fusion protein, ion exchange chromatography, thin layer chromatography, ultrafiltration technology, endotoxin, chromogenic end-point tachypleus amebocyte lysate