Synthesis and High Expression of Chitin Deacetylase from Colletotrichum lindemuthianum in Pichia pastoris GS115
Synthesis and High Expression of Chitin Deacetylase from Colletotrichum lindemuthianum in Pichia pastoris GS115
复制标题
炭疽菌几丁质脱乙酰酶的合成及其在毕赤酵母 GS115 中的高表达
DOI：
10.4014/jmb.1112.12026
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发表时间：
2012-09-01
影响因子：
2.8
通讯作者：
Ma, Lixin
中科院分区：
文献类型：
--
作者：
Kang Lixin;Chen, Xiaomei;Ma, Lixin
根据毕赤酵母（Pichia pastoris）的密码子使用偏好性，优化了编码林德木硫炭疽菌（Colletotrichum lindemuthianum）几丁质脱乙酰酶（Chitin deacetylase，CICDA）的基因，并利用重叠延伸PCR技术体外合成了该基因。利用组成型表达载体pHMB 905 A在毕赤酵母GS115中进行分泌表达。甲醇诱导72 h后，在110 mg/l培养上清中表达量最高，几丁质脱乙酰酶活性为77.27 U/mg。SDS-PAGE、质谱和去糖基化实验表明，重组蛋白部分糖基化，表观分子量为33 kDa。重组蛋白的氨基酸序列通过质谱法确认。
A gene, CICDA, encoding chitin deacetylase from Colletotrichum lindemuthianum, was optimized according to the codon usage bias of Pichia pastoris and synthesized in vitro by overlap extension PCR. It was secretorily expressed in P. pastoris GS115 using the constitutive expression vector pHMB905A. The expression level reached the highest with 110 mg/l culture supernatant after 72 h of methanol induction, which comprised 77.27 U/mg chitin deacetylase activity. SDS-PAGE, mass spectrometry, and deglycosylation assays demonstrated that partial recombinant protein was glycosylated with an apparent molecular mass of 33 kDa. The amino acid sequences of recombinant proteins were confirmed by mass spectrometry.