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

摘要:

将自肾脏cDNA中获得的人激肽释放酶-1(Human kallikrein 1, 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(500~700 μg/mL), 筛选能高水平表达重组人激肽释放酶-1(Recombinant human kallikrein 1, rhK1)的P. pastoris工程菌株。在30 L发酵罐中发酵的表达条件为30oC、pH 6.0, 诱导64 h时, 发酵上清中rhK1产量达到6500 u/L(约1.25 g/L)。表达的rhK1有N-型糖基化程度不同的两种类型, 分别是分子量偏大的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0.28 g的rhK1-H和0.62 g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。

Abstract:

Human kallikrein-1 (hK1) gene was cloned from kidney tissues cDNA, it was inserted into the plasmid pPICZaA, then the yeast expression vector pPICZa-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 mg/mL) of YPD plate and medium. When temperature was 30oC, 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.