人激肽释放酶-1在甲醇酵母中高水平表达、纯化与鉴定

High Level Expression, Purification and Characterization of Human Kallikrein-1 in Pichia pastoris

将自肾脏cDNA中获得的人激肽释放酶-1（Human kallikrein1，hK1）基因插入到pPICZαA载体中，构建甲醇酵母分泌型表达载体pPICZα-hK1，该载体转化毕赤酵母（Pichia pastoris）宿主菌X33，通过提高YPD平板和培养液中抗生素Zeocin的浓度（500～700μg／mL），筛选能高水平表达重组人激肽释放酶-1（Recombinant human kallikrein1，rhK1）的Ppastoris工程菌株。在30L发酵罐中发酵的表达条件为30℃、pH6．0，诱导64h时，发酵上清中rhK1产量达到6500u／L（约1．25g／L）。表达的rhK1有N-型糖基化程度不同的两种类型，分别是分子量偏犬的rhK1-H和分子量略小的rhK1-L。通过苯基疏水作用、Cu2＋螯合以及阴离子交换层析纯化，从每升发酵上清中可获得0．28g的rhK1-H和0．62g的rhK1-L，纯化的总得率约为72％，纯度不低于96％。到目前为止，该研究获得的rhK1产量高于其他研究者的结果。

Human kallikrein-1 （hK1） gene was cloned from kidney tissues cDNA, it was inserted into the plasmid pPICZαA, then the yeast expression vector pPICZα-hK1 was constructed. After transformed into Pichia pastoris host X33, high-level expression transformants were screened by escalating the concentration of Zeocin （from 500 to 700 μg/mL） of YPD plate and medium. When temperature was 30℃, pH 6.0 with induction duration of 64 hours in the 30 L fermenter, the highest yield can reach about 6500 u/L （1.25 g/L）. The variation of glycosylation resulted in two kinds of molecules, i.e. rhK1 -H with a heavy molecular weight and rhK1-L with a light one. rhK1 was purified from the supernatant through Phenyl hydrophobic interaction, Cu2＋-charged Chelating and Anion-exchange chromatography. 0.28 g rhK1-H and 0.62 g rhK1-L can be purified from one liter supernatant. The yield recovery was 72% with a purity of 〉96%. So far our yield of rhK1 is superior than known recombinant expression method reported by other researchers.