Coprinopsis cinerea漆酶基因的克隆及其在毕赤酵母中的表达

Cloning of laccase gene form Coprinopsis cinerea and study on its expression in Pichia pastoris

通过反转录反应从Coprinopsis cinerea okayama 7#130中克隆得到laccase 1,应用SignalP 3.0 Server软件分析其氨基酸序列后,设计不含信号肽序列的新引物扩增得到漆酶基因,构建重组酵母表达载体pPIC9K-lac1,电击转化毕赤酵母GS1115,并用甲醇诱导表达,之后研究重组酶的酶学性质。克隆得到的laccase 1碱基数为1 593 bp,编码530个氨基酸,其中信号肽包含18个氨基酸。SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)显示重组蛋白大小约为65 kDa。酶学性质研究表明,该酶最适反应温度为45℃,最适pH值为4.3。成功分泌表达的漆酶活力达到1.108 U/mL。

The laccase gene 1 was cloned by RT-PCR from Coprinopsis cinerea okayama 7#130,and through analyzing its amino-acid sequence using software SignalP 3.0 Server,a new primer without signal peptide sequence was designed and laccase 1 was obtained through its extension.Construction of Pichia pastoris was conducted for expressing plasmid pPIC9K-lac1,and then transformed into Pichia pastoris GS115 by electric shock and induction expression was effected using methanol.The properties of recombinant enzyme were studied.The full length was 1 593 bp of laccase 1,which coded 530 amino-acids,among them,18 amino acids of signal peptide sequence.SDS-PAGE showed that obvious protein band was 65 kDa.The optimal fermentative conditions of it： 45 ℃,and pH=4.3.The enzyme activity of secretion expression achieved 1.108 U/mL.