角毛壳菌几丁质酶基因chi58多点突变及在毕赤酵母中的高效表达

Chitinase Gene Overexpression of Chaetomium cupreum in Pichia pastoris by Multi-site Mutations

应用重叠延伸PCR技术(gene splicing by overlap extension PCR,gene SOEing),简称SOE-PCR对角毛壳菌(Chaetomium cupreum)的几丁质酶基因chi58进行多点突变。依据毕赤酵母密码子偏爱性,将毕赤酵母中编码Arg使用频率几乎为0的密码子CGC突变为使用频率高的AGA,构建了含有正确突变的酵母表达载体pPIC9K-chi58A,电转化毕赤酵母GS115,获得的重组酵母株在诱导120h酶活力最高,平均可达101.71U/mL±3.33U/mL;其活力比未优化重组酵母株(31.83U/mL±4.85U/mL)提高了约3倍,且经10代传代培养后遗传稳定性良好。表达产物的SDS-PAGE分析表明,酶蛋白分子大小为58kD。

By using gene SOEing PCR, Arg （CGC） in the expression fragment of chitinase gene （chi58） from Chaetomium cupreum was mutated synonymously to Arg （AGA）, which is a bias code of Pichia pastoris yeast. The expression plasmid pPIC9K-chi58A was constructed and transformed into GSll5 strain through electroporation. The chitinase activity reached 101.71 U/mL±3.33 U/mL after induced with 120 h, which was the 3 fold of the original strain （31.83 U/mL±4.85 U/mL）. Additionally, recombinant yeast showed good genetic stability after 10 cycle. SDS-PAGE analysis suggested that the weight of protein was 58kD.