改构的野生革耳漆酶基因在毕赤酵母中的表达和活性鉴定  

Expression and Purfication of the Modified Gene of Laccase from Panus rudis in Pichia pastoris

作　　者：杨建强[1] 刘刚[1] 汤国营[1] 戴红梅[1] 朱厚础[1] 

机构地区：[1]军事医学科学院生物工程研究所,北京100071

出　　处：《生物技术通讯》2005年第3期255-258,共4页Letters in Biotechnology

摘　　要：在野生革耳漆酶的cDNA序列3'端添加15个核苷酸后得到突变基因lccT。将其克隆入表达载体pPICZαA,重组质粒pPICZαA/lccT经PmeⅠ线性化,电击转化毕赤酵母X-33细胞。经过菌落PCR鉴定的转化子pPICZαA/lccT-X-33在甲醇诱导后分泌表达活性重组漆酶LCCT,比未添加该段序列的野生革耳漆酶基因lcc在毕赤酵母中表达的活性提高2倍多。其培养液上清经凝胶过滤层析和离子交换层析后,得到了电泳纯的漆酶蛋白,以ABTS为底物在pH4.5时测得比活为3.68U/mg。根据SDS-PAGE测定的重组漆酶LCCT的表观相对分子质量为62600。A modified gene lccT obtained by adding 15 nucleotides to 3′ terminal of the cDNA encoding the laccase from Panus rudia, and then cloned into the expression vector pPICZαA. The recombinant plasmid pPICZαA/lccT linearized with PmeⅠ was transferred into Pichia pastoris X-33 by electroporation method. After direct PCR screening, the transformant pPICZαA/lccT-X-33 were cultured in liquid medium and secreted active LCCT after methanol induction. The activity of LCCT which encoded gene had been modified was two times higher than that which encoded gene had unmodified. The recombinant LCCT was purified to electrophoretic homogeneity by sequential steps of gel filtration and ion-exchange charomatography. The special activity of the purified protein at pH4.5 was 3.68 U/mg, the apparent molecular mass was approximately 62.6 kDa determined by SDS-PAGE.

关 键 词：毕赤酵母 酶基因 活性鉴定 野生 SDS-PAGE cDNA序列 离子交换层析 凝胶过滤层析 相对分子质量 PCR鉴定 突变基因 表达载体 重组质粒 X-33 电击转化 活性重组 分泌表达 ABTS 核苷酸 漆酶 线性化 PME 转化子 lcc 培养液 酶蛋白 

分 类 号：Q786[生物学—分子生物学] TQ925[轻工技术与工程—发酵工程]