摘要
用 PCR的方法 ,在大鼠 Gs alpha亚基的 C端引入了 6个外源组氨酸 (即 6×His- Tag)并以此为纯化标记 .构成的表达载体 p QE60 /rat Gsα( L)在大肠杆菌 BL2 1 ( DE3)中获得了稳定的表达 .经 DEAE- Sephacel离子交换柱和 Ni- NTA Agarose亲和层析获得纯化的具有较高 [35S]- GTPγS结合活力的重组大鼠 Gs
In an attempt to facilitate purification,six histidines were added by PCR as purification tag to the C terminal of rat Gs alpha subunit.Both the constructed expression plasmid (pQE60/rat Gsα(L)) and helper plamid (pREP4) were transformed into E.coli BL21(DE3).At low temperature (30℃) and low IPTG (30 μmol/L), the recombinant rat Gsα was stably expressed.The target protein was purified by DEAE Sephacel ion exchange column and Ni NTA Agarose affinity column.The purified recombinant protein showed an apparent molecular weight of 52 kD examined by SDS PAGE with silver staining and had higher [ 35 S] GTP γS binding activity.
出处
《中国生物化学与分子生物学报》
CAS
CSCD
2000年第1期28-31,共4页
Chinese Journal of Biochemistry and Molecular Biology
基金
中科院重大项目!( KJ95 1-B1-6 0 9)
国家自然科学基金委重点项目!( 3 973 0 13 0 )资助
关键词
大鼠Gsalpha亚基
原核表达
纯化
G蛋白
Rat Gs alpha subunit,6×His Tag,Expression,[ 35 S] GTP γS binding activity
