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

摘要:

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120 h酶活力最高, 平均可达101.71 U/mL±3.33 U/mL; 其活力比未优化重组酵母株(31.83 U/mL±4.85 U/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58 kD。

Abstract:

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 GS115 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 58 kD.