Cloning,expression and characterization of laccase from Bacillus subtilis
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摘要: 为获得高效表达的产漆酶工程菌,将枯草芽孢杆菌（Bacillus subtilis）的漆酶（cotA）基因在大肠杆菌中表达。用PCR的方法从枯草芽孢杆菌（Bacillus subtilis）QM11基因组中扩增cotA基因,连接载体pET22b构建成表达载体pET22b-cotA,转化至E.coli BL21（DE3）进行诱导表达,最终通过SDS-PAGE检测蛋白表达情况。克隆得到的cotA基因含有1542个核苷酸,由513个氨基酸组成,预测相对分子量为58 ku,理论等电点为5.91,无信号肽,二级结构以β-折叠和无规卷曲为主,其中β-折叠占26.71%,无规卷曲占52.05%,与NCBI已公布的Bacillus subtilis漆酶cotA基因（Gen Bank:GQ184294.1）碱基序列有12个碱基的差异,其编码的氨基酸序列有4个发生了改变。通过SDS-PAGE检测,目标蛋白相对分子质量约为54 ku,与预测相对分子量基本相符。Abstract: To express laccase cotA gene from Bacillus subtilis in Escherichia coli expression system,cotA gene was cloned from Bacillus subtilis QM11 genome DNA by PCR. Plasmid p ET22 b containing cotA gene was constructed and transferred into competent Escherichia coli BL21(DE3) and the expression of recombinant protein was proved by SDS-PAGE.The gene cotA contained 1542 nucleotides,comprising one open reading frame encoding a polypeptide of 513 amino acids with 58 ku of relative molecular weight and predicted p I value of 5.91 and there was no signal peptide in cotA. The secondary structure of cotA was mainly β-sheet and random coil,β-sheet was 26.71% and random coil was 52.05%. There were twelve-site mutations between the nucleotide sequence from Bacillus subtilis QM11 and cotA(NCBI Gen Bank :GQ184294.1) from Bacillus subtilis,which could result in four amino acid substitution. The molecular weight of cotA gene protein was estimated to be 54 ku by SDS-PAGE.
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