摘要
为了使碱性果胶酶基因能在毕赤酵母中获得非诱导表达,利用聚合酶链式反应(PCR)技术,以实验室保藏菌株毕赤酵母EIM-60基因组为模板,设计引物扩增出编码碱性果胶酯裂解酶的基因PGL,大小约为1 206 bp。酶切后连接到毕赤酵母组成型表达载体pGAPZαA的多克隆位点上,成功构建了重组载体pGAPZαA-PGL,将其电转化入毕赤酵母GS115进行异源表达。SDS-PAGE银染结果表明,该重组质粒在毕赤酵母中获得了异源表达,分子量为44.3 ku。通过考马斯亮蓝法蛋白浓度试剂盒测定其蛋白含量为0.430 g/L,通过BandScan软件分析目的蛋白占总蛋白的41.4%,即0.178 g/L。
In order to obtain a non-induction expression of alkaline pectinase gene in Pichia pastoris, an alkaline polygalacturaonate lyase （PGL） encoding gene as a primer was designed taking genome from Pichia EIM-60 collected in the lab as template and amplified using PCR with its length of I 206 bp. After enzyme-digestion the gene was linked to multiple cloning sites of P. pastoris component type expression vector pGAPZaA, and successfully established recom- binant vector pGAPZaA-PGL, and then was electro-transformed into P. pastoris GS115 to carry out heterogenie expression. SDS-PAGE silver staining results showed that the recombinant plasmid had gained the heterogenic expression in the P. pastoris, and the molecular mass was about 44.3 ku. Coomassie （Bradford） Protein Assay Kit showed that the content of protein was O. 430 g/L, and BandScan software analysis showed that the amount of goal protein accounted for 41.4% of total protein, i.e. 0. 178 g/L.
出处
《微生物学杂志》
CAS
CSCD
2012年第6期33-36,共4页
Journal of Microbiology
基金
福建省发改委产业化关键技术重点项目(闽发改投资(2009)958号)
