摘要
应用RT-PCR方法从人胚胎干细胞中扩增出Sox2基因,构建pET28b-Sox2表达载体.用IPTG诱导转化pET28b-Sox2表达载体的大肠杆菌BL21(DE3),并优化表达条件为37℃IPTG0.8 mmol/L诱导4 h.以Ni-NTA亲和层析法纯化Sox2重组蛋白,对变性蛋白进行柱上和透析复性,复性后蛋白得率为0.7 mg/g湿菌重.
To express Sox2 protein, the cDNA was cloned from human ES ceils by RT- PCR and constructed into a prokaryotic expression vector to make pET28b-Sox2, which was transformed into Escherichia coli （E. coli） BL21（DE3） ceils for expression. IPTG induction of the Sox2 expression was optimized to 0.8 mmol/L for 4 hours at 37~C. Re- combinant protein expression was identified by the standard SDS-PAGE and Western blot. The recombinant protein was purified by Ni-NTA affinity chromatography and refolded on column or by dialysis. The final concentration of purified active protein was 0.7 mg/g wet cell weight.
出处
《华中师范大学学报（自然科学版）》
CAS
CSCD
2008年第1期102-105,共4页
Journal of Central China Normal University：Natural Sciences
基金
国家863计划项目(2006AA02A102)
上海市科技攻关项目(06dj14001)
关键词
SOX2
原核表达
纯化
复件
Sox2 gene
prokaryotic expression
purification
refolding
