重组人kallistatin蛋白在毕赤酵母中的高效表达及生物活性分析

High level expression of recombinant human kallistatin in Pichia pastoris and its bioactivity

摘要:

为了研究kallistatin (Kal) 的生物活性，本实验构建了可分泌表达Kal的毕赤酵母菌株。首先通过PCR方法从pAAV-Kal中扩增出Kal cDNA，并克隆至酵母表达载体pPIC9，得到甲醇酵母分泌型表达载体pPIC9-Kal，然后将载体线性化并电击转化毕赤酵母GS115 (his4)，通过MD平板筛选出阳性表达菌株。阳性表达菌株在BMMY培养基 (pH 7.0) 中29℃培养，经2％甲醇诱导表达96 h，摇瓶表达量可达14 mg/L。表达上清经Phenyl Superose、Heparin Sepharose FF分离纯化，目的蛋白纯度达到98％，分子量为58 kDa。生物活性实验显示，所得到的Kal蛋白具有较好的抗氧化活性，过氧化物酶活性达到 (163±4) U/(mg?min)，可有效降低H2O2对LX-2细胞的氧化损伤。另外，重组产生的Kal还能抑制HUVEC细胞的增殖。本研究首次成功地利用毕赤酵母表达系统分泌表达了有生物活性的Kal，为继续开展其抗肿瘤活性奠定了基础。

Abstract:

In order to research the bioactivity of kallistatin (Kal), we obtained the recombinant Kal using Pichia pastoris expression system. Kal cDNA was amplified from pAAV-Kal and inserted into pPIC9 vector to generate a recombinant vector of pPIC9-Kal. Then, pPIC9-Kal was linearized and transformed into Pichia pastoris strain GS115 (His4) by electroporation. The positive transformants were selected by MD plate and confirmed by PCR. High level of Kal was obtained in BMMY medium (pH 7.0) after 96 hours induction of 29?C and 2% methanol, with the highest yield of 14 mg/L in shake flask culture. Kal protein was purified from the supernatant with Phenyl Superose and Heparin Sepharose FF chromatograph. The recombinant Kal had a molecular weight of 58 kDa with 98% purity, showing by SDS-PAGE. Moreover, it had a high peroxidase activity (163±4) U/(mg?min), which could protect LX-2 cell against oxidation of H2O2. Recombinant Kal also effectively inhibited HUVEC proliferation. In this report, we successfully established the expression system using Pichia pastoris and obtained the bioactive recombinant human Kal. It lays a foundation for its further anti-cancer therapy.