摘要
用化学法合成克鲁维酵母的菊粉酶基因的信号肽序列 (INU) ,将其嵌入酵母附加型表达质粒pYES2 ,得到一套新型的分泌表达载体pYES2 I,pYES2 Ⅱ ,pYES2 Ⅲ。然后用PCR方法分别扩增大肠杆菌的天冬酰胺酶基因 (ASN)和短芽孢杆菌α 乙酰乳酸脱羧酶 (ALDC)基因 ,连接到INU下游 ,得到重组质粒pASN和pALDC。分别将这两个重组质粒转化酿酒酵母菌株IN VScⅠ中表达 ,胞内和胞外的酶活分析表明ASN和ALDC基因都能在酿酒酵母中分泌表达 ,表明菊粉酶信号肽序列能很好地将酿酒酵母中的重组蛋白分泌到胞外。稳定性分析表明 ,重组酵母菌株在没有选择压力的条件下连续接种培养 1 0 0h 。
The DNA fragment ecoding the Signal peptide of inulinase of Kluyveromyces smarxianu was synthesized chemically.This fragment was cloned in\|frame in the expression vector pYES\-2 of Saccharomyces cerevisiae , resulting in a set of new secreting expression vectors pYES\-2Ⅰ,pYES\-2Ⅱ,pYES\-2Ⅲ.The L\|Asparaginase gene ( ASN ) of E.coli and α acetylactate decarboxylase gene ( AL DC) of B.brevis which were amplified by PCR and cloned into the new vectors respectively were transformed into Saccharomyces cerevisia ,and most of enzyme activities were secreted into the medium.The new secreting expression vectors still have excellent segregational stability even after growth for 100h in the absence of selective pressure.
出处
《微生物学报》
CAS
CSCD
北大核心
2002年第4期431-435,共5页
Acta Microbiologica Sinica
基金
国家"86 3"资助项目 ( 1 0 1 0 6 0 7 0 1 )~~
关键词
酿酒酵母
附加型
分泌表达载体
构建
信号肽
Saccharomyces cerevisiae, Signal peptide, Secreting expression vector
