CALB脂肪酶在毕赤酵母中的组成型表达及纯化

The Constitutive Expression of CALB in Pcihia pastoris and Purification of Product

根据南极假丝酵母脂肪酶calb的成熟多肽序列,人工合成了由毕赤酵母(Pichia pastoris)偏爱密码子组成的calb基因序列(仅成熟肽)。将该基因克隆至穿梭质粒pGAPZαΑ中,获得的重组质粒pGAPZαA-calb经线性化后转入毕赤氏酵母GS115菌株,构建成组成型表达CALB的酵母工程菌株。酵母转化子在YPD培养基上传代10次后,其外源基因未丢失。经摇瓶发酵4天后,CALB水解活力为14.5U/ml。经脱盐和阴离子交换两步纯化后,其纯度达到了95%以上,目的蛋白的含量达到了220mg/L。

The artificially synthetic gene calb（only mature peptide sequence） was inserted into a yeast constitutive vector,pGAPZαA.Under the control of the promoter GAP（glyceraldehydes 3-phosphate dehydrogenase）CALB was expressed constitutively.The yeast transformants still had the foreign gene after ten times of passage in YPD medium.The activity of CALB in shake flask cultured 4 days achieved 14.5U/ml.And after two steps of purification（the desalinization and DEAE column）,purity of the final protein achieved 95% and protein concentration has achieved 220mg/L.