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Chinese Journal of Biotechnology ; (12): 1186-1193, 2008.
Artigo em Chinês | WPRIM | ID: wpr-275405

RESUMO

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.


Assuntos
Humanos , Sequência de Aminoácidos , Sequência de Bases , Cromatografia por Troca Iônica , Métodos , Vetores Genéticos , Genética , Rim , Metabolismo , Dados de Sequência Molecular , Pichia , Genética , Metabolismo , Proteínas Recombinantes , Genética , Calicreínas Teciduais , Genética
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