[High level expression, purification and characterization of human kallikrein-1 in Pichia pastoris].
Human kallikrein-1 (hK1) gene was cloned from kidney tissues cDNA, it was inserted into the plasmid pPICZalphaA, then the yeast expression vector pPICZalpha-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 microg/mL) of YPD plate and medium. When temperature was 30 degrees C, 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, Cu(2+)-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.
Department of Research & Development, Wangxing Biopharmaceuticals Co., Ltd, Shanghai 201026, China. hxd2002@hotmail.com
Xiudong
X
Huang
Shusheng
S
Wang
Peixin
P
Chen
Jun
J
Wang
Yaoguo
Y
Chen
Xuegong
X
Pan
Zhifang
Z
Cao
D000595:Amino Acid Sequence; D001483:Base Sequence; D002852:Chromatography, Ion Exchange / Q000379:methods; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D007668:Kidney / Q000378:metabolism; D008969:Molecular Sequence Data; D010843:Pichia / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification*; D020840:Tissue Kallikreins / Q000096:biosynthesis* / Q000235:genetics
D011994:Recombinant Proteins; D020840:Tissue Kallikreins