高温中性蛋白酶基因的克隆及毕赤酵母表达研究

Cloning of Thermophilic Neutral Protease Gene and Expression of Pichia pastoris

对地衣芽孢杆菌XJT9503高温中性蛋白酶进行了克隆,并在毕赤酵母中进行了表达。以高温中性蛋白酶产生菌地衣芽孢杆菌XJT9503基因组DNA为模板,通过PCR扩增,获得高温中性蛋白酶(Tnp)基因的特异性片段,大小约为1 kb。通过EcoRI、NotI双酶切后,将该基因连入毕赤表达载体pPIC9K,并整合到毕赤酵母SMD1168基因组中,构建的基因工程菌,进行甲醇诱导表达。结果表明,基因工程菌发酵72 h,有最大酶活,为19 U/mL;酶的最适作用温度为65℃,与原始酶的最适作用温度相符。证明研究成功克隆了高温中性蛋白酶基因,并在毕赤酵母中正确表达。

XIT9503 Thermophilic neutral protease （Tnp） gene is cloned and expressed in Pichia pastoris in this research. The tnp gene was amplified through PCR by using XJT9503 genomic DNA as template, the gene digested with EcorI, NotI and ligased to the express vecter pPIC9K and then transformed into Pichia pastoris SMD1168, the recombinant strain can produce thermophilic neutral protease induced by methanol. The largest yield of Tnp activity was measured 19 U/mL the genetic engineering strains were fermented for 72 h. The optimal temperature of the expressed Tnp was 65℃ as same as original Tnp＇s. The result suggested the Tnp gene was doned and expressed in Pichia pastoris successfully.