拟康氏木霉eg1基因的克隆及在酿酒酵母中的分泌表达

从拟康氏木霉3.3002基因组中克隆了内切葡聚糖酶EGI基因,该基因全长1566 bp,由3个外显子2个内含子组成,编码461个氨基酸.编码蛋白EGI的N端为22aa组成的信号肽,其后依次为催化结构域、连接肽和结合结构域.采用重叠PCR法获得无内含子的内切葡聚糖酶基因eg1,并将其成熟肽编码序列插入酿酒酵母分泌型表达载...

Cloning of egl Gene from Trichoderma pseudokoningii and Its Secretory Expression in Saccharomyces cerevisiae

A gene（eg1） for an endoglucanaseⅡ（EGI） was cloned from Trichoderma pseudokoningii strain 3. 3002.The gene was composed of 1566 bp,interrupted by 2 introns,and coding for 461 amino acid residues.The deduced amino acid sequence of EGI revealed a multi-domain structure composed of a 22aa signal peptide,a catalytic domain, a linker,and a cellulose-binding domain from the N-terminus.The eg1 gene with no introns was obtained by overlap PCR,and the sequence encoding the mature peptide was inserted into the Saccharomyces cerevisiae secretion vector pYEa.The recombinant expression plasmid pYEα-Pegl was constructed and then transformed into S.cerevisiae,along with the yeasts transformed with vector pYEa as controls.After induction of galactose,the resulting S.cerevisiae transformant secreted a recombinant ECI that had enzymatic properties detected by congo red assay and enzyme activity assay. The expression was confirmed by a protein band in the SDS-PAGE,which is a little larger than predicted ECI.