重组人组织激肽释放酶结合蛋白的高密度毕赤酵母表达、纯化和生物学活性  

Expression of recombinant human kallistatin in Pichia pastoris by high density cell culture, and its purification and characterization

作　　者：张倩[1] 邢永梅[1] 刘嘉[1] 刁勇[1] 

机构地区：[1]华侨大学生物医学学院,华侨大学分子药物研究院,福建泉州362021

出　　处：《药学学报》2013年第7期1107-1112,共6页Acta Pharmaceutica Sinica

基　　金：国家自然科学基金资助项目(30973591,81271691);国际科技合作项目(2011DFG33320);华侨大学校级基金资助项目(11J0084*)

摘　　要：研究重组人组织激肽释放酶结合蛋白(rHKal)的高密度毕赤酵母表达、纯化以及生物学活性。以质粒pPIC9-Kal/GS115(His4)转化毕赤酵母细胞,在7.5 L培养罐内高密度细胞发酵,以1%～2%的甲醇诱导表达rHKal。发酵上清液经疏水层析、亲和层析和凝胶层析分离rHKal。通过MTT及小管形成实验评价rHKal对HUVEC的生物学活性。收获发酵上清液内rHKal的表达水平为50 mg.L 1,纯化后rHKal原液的纯度达98%以上。rHKal对HUVEC的增殖抑制活性呈剂量依赖性,但对其小管形成抑制作用的量效关系呈U型曲线。rHKal具有新生血管形成抑制活性,本研究为rHKal的生产提供了有效方法。Kallistatin （Kal） is a negative acute phase endogenous protein which can inhibit tumor angiogenesis, growth and metastasis effectively. To express and purify recombinant human kallistatin （rHKal）, and characterize its biological activity, P pastoris was transformed with pPIC9-Kal/GS115 （His4） to express rHKal. The fermentation was carried out in a 7.5 L bioreactor with high density cell culture. 1%-2% methanol was added to the medium to induce the expression of rHKal. The secretion was purified with phenyl sepharose, G-25 sepharose, heparin sepharose and Sephacryl S-100 chromatography. The biological activity of purified bulk rHKal on HUVEC was evaluated with MTT and tube formation assays. The final expression of rHKal in the supernatant reached 50 mg.L-1, the purity of bulk rHKal after purification was above 98%. A dose-dependent inhibition of rHKal on HUVEC proliferation was observed, however, a U-shaped dose-response curve of rHKal on capillary formation of HUVEC was revealed. The described protocol provides an effective means for lareoarinz rHKal that could be used for anti-angiogenesis therapy in the future.

关 键 词：组织激肽释放酶结合蛋白 高密度发酵 纯化 生物学活性 

分 类 号：R963[医药卫生—微生物与生化药学]