%0 Journal Article
%T High Level Expression, Purification and Characterization of Human Kallikrein-1 in Pichia pastoris<br>人激肽释放酶-1在甲醇酵母中高水平表达、纯化与鉴定
%A Xiudong Huang
%A Shusheng Wang
%A Peixin Chen
%A Jun Wang
%A Yaoguo Chen
%A Xuegong Pan
%A Zhifang Cao
%A <br>黄秀东
%A 王书生
%A 陈佩新
%A 王俊
%A 陈耀国
%A 潘学工
%A 曹之舫
%J 生物工程学报
%D 2008
%I 
%X 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.
%K kallikrein
%K Pichia pastoris
%K recombinant protein
%K S-2266 substrate<br>激肽释放酶
%K 毕赤酵母
%K 重组蛋白
%K S-2266生色底物
%U http://www.alljournals.cn/get_abstract_url.aspx?pcid=90BA3D13E7F3BC869AC96FB3DA594E3FE34FBF7B8BC0E591&jid=A66E90C274451689E69F6F0291467824&aid=3B6E164BDF040FFD1BA3FCE8719231E1&yid=67289AFF6305E306&vid=B91E8C6D6FE990DB&iid=DF92D298D3FF1E6E&sid=2E45C5B3364EA9F3&eid=8339CA1DD1F3C6AB&journal_id=1000-3061&journal_name=生物工程学报&referenced_num=0&reference_num=18