High level expression, purification and characterization of human kallikrein-1 in Pichia pastoris.
- Author:
Xiudong HUANG
1
;
Shusheng WANG
;
Peixin CHEN
;
Jun WANG
;
Yaoguo CHEN
;
Xuegong PAN
;
Zhifang CAO
Author Information
1. Department of Research & Development, Wangxing Biopharmaceuticals Co., Ltd, Shanghai 201026, China. hxd2002@hotmail.com
- Publication Type:Journal Article
- MeSH:
Amino Acid Sequence;
Base Sequence;
Chromatography, Ion Exchange;
methods;
Genetic Vectors;
genetics;
Humans;
Kidney;
metabolism;
Molecular Sequence Data;
Pichia;
genetics;
metabolism;
Recombinant Proteins;
biosynthesis;
genetics;
isolation & purification;
Tissue Kallikreins;
biosynthesis;
genetics
- From:
Chinese Journal of Biotechnology
2008;24(7):1186-1193
- CountryChina
- Language:Chinese
-
Abstract:
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.
