麦芽寡糖基海藻糖合酶基因的克隆及在大肠杆菌中的表达

Cloning and Expression of Maltooligosyltrehalose Synthase from Sulfolobus acidocaldarius in Escherichia coli

应用PCR方法,从芝田硫化叶菌B12中扩增了麦芽寡糖基海藻糖合酶基因(TreY),扩增产物大小为2.2kb,将扩增片段与pUCm-T连接,得到重组质粒pUCm-T-TreY,CaCl2法转化DH5α,并筛选阳性克隆。用BamHI/NdeI分别双酶切pUCm-T-TreY和表达载体pET21a,连接后得重组pET21a-TreY,转化大肠杆菌DE3并进行了表达。表达产物经SDS-PAGE分析,得到一条分子量约为74kDa的特异条带,同核苷酸序列所推导的值相符。

The gene of mahooligosyhrehalose synthase from Sulfolobus acidocaldarius B12 was amplified by the polymerase chain rection （PCR）, ligated the fragment with pUCm-T vector by T4 DNA ligase and transformed the competent cells Ecoli DH5α. Identifying the fragment by enzyme digest and sequencing. The recombinant plasmid was called pUCm-T-TreY. Restriction endonucleases BamHI and NdeI were used to cleave both pUCm-T-TreY and expression vector PET21a, cloned the object gene fragment to expression vector by T4 DNA ligase, we named the recombinant plasmid pET21a-TreY, transformed recombinant plasmid pET21 a-TreY to Ecoli DE3 by heat shock and expressed TreY protein. The molecular weight of expressed TreY detected by SDS-PAGE was about 74KD, which is conformed with that deduced from nucleotide sequence.