重组葡激酶的高效表达与纯化  

High-Level Expression and Purification of Recombinant Staphylokinase from Engineering Bacterium

作　　者：卢锦[1] 霍蕾[1] 田天丽[1] 徐非一[1] 刘玉应[1] 孙启玲[1] 

机构地区：[1]四川大学生命科学学院生物资源与生态环境教育部重点实验室,成都610064

出　　处：《四川大学学报（自然科学版）》2006年第6期1369-1373,共5页Journal of Sichuan University(Natural Science Edition)

摘　　要：通过发酵和纯化条件的研究,建立适合重组葡激酶工程菌的发酵和纯化工艺.对重组葡激酶工程菌发酵过程中的pH、饱和氧浓度、补料以及诱导时间进行考察,获得该工程菌的高效表达条件.在此条件下,重组葡激酶表达水平在50%以上,并以活性可溶性状态存在于细胞内;经渗滤,超滤浓缩、透析,DEAE-Sepharose FF层析和SP-Sepharose FF层析后,经SDS-PAGE和HPLC分析,纯度达到99%.在还原条件下进行SDS-PAGE分析,测得该酶相对分子质量为15.5kD,等电聚焦显示其等电点为8.4.The high-level expression of Staphylokinasegene in E. coli was obtained, at the same time , an effective, comfortable fermentation and purification procedure of recombinant staphylokinase （rSak） was established in this paper. During the fermentation process, the conditions of some expression factors, such as pH, DO saturation,supplement and induced time, et al, were regulated in order to obtain high-level rSak expression. Under given conditions, the rSak was expressed in a soluble , active form and covered over 50% of total soluble protein. Ultrafiltration was used to concentrate and desalt solutes retained by the membrane or to collect material passing through the membrane. After DEAE-Sepharose FF and SP-Sepharose FF chromatographies, the purity of rSak was more than 99 % by SDS-PAGE and HPLC analysis. Reduced SDS-PAGE analysis indicated that relative molecular weight of rSak was about 15.5kD,and polyacrylamide gel isoelectric focusing identified that the pI of rSak was 8.4.

关 键 词：重组葡激酶 工艺优化 高效表迭 

分 类 号：Q55[生物学—生物化学] Q93