Chromatography for rapid buffer exchange and refolding of secretory leukocyte protease inhibitor

被引:37
作者
Hamaker, KH
Liu, JY
Seely, RJ
Ladisch, CM
Ladisch, MR
机构
[1] PURDUE UNIV, RENEWABLE RESOURCES ENGN LAB, POTTER CTR 1295, W LAFAYETTE, IN 47907 USA
[2] AMGEN BOULDER INC, PROC DEV DEPT, BOULDER, CO 80301 USA
关键词
D O I
10.1021/bp950071f
中图分类号
Q81 [生物工程学(生物技术)]; Q93 [微生物学];
学科分类号
071005 ; 0836 ; 090102 ; 100705 ;
摘要
A DEAE-cellulose stationary phase in a rolled configuration was used to separate recombinant secretory leukocyte protease inhibitor (rSLPI) from denaturants and reducing agents (3 M guanidine-HCl and 5 mM DTT) in less than 5 min to promote refolding of the protein to an active form. The mobile phase consisted of buffer and 500 mM NaCl, where NaCl suppressed binding of protein to this stationary phase. Separation of an initial concentration of 2 mg/mL protein from the other constituents resulted in 96% recovery of the rSLPI at an average concentration of 1.28 mg/mL. When incubated for 4 h at 20 degrees C, the fractionated rSLPI gave a 46% yield of properly refolded protein. The protein concentration was 6.4 times higher than that reported in a previously published method, where refolding was carried out by diluting the mixture of protein, denaturants, and reducing agents by a factor of 10. The results show that a combination of rapid chromatographic separation over a cellulosic stationary phase followed by protein refolding will significantly enhance process throughput by minimizing tankage, water requirements, and process time.
引用
收藏
页码:184 / 189
页数:6
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