Production and Characterization of Hirudin Variant-1 by SUMO Fusion Technology in E. coli
作者： Wuguang LuXueting CaiZhenghua Gu , Yuzheng HuangBinbin , XiaPeng Cao
DOI:
关键词: Affinity chromatography 、 Escherichia coli 、 Hirudin 、 Chemistry 、 Recombinant DNA 、 Fusion gene 、 Thrombin 、 Protease 、 Biochemistry 、 Fusion protein
摘要: Hirudin is the most potent non-covalent inhib- itor of thrombin. Several expression systems have been used to produce recombinant hirudin for pharmaceutical purposes. However, high active in Escherichia coli cytoplasm has not successful owing fact that heterogenetic small peptide easily degraded cell. To solve this problem, we constructed a form variant-1 (HV1) as fusion protein with ubiquitin-related modifier gene (SUMO) by use over-lap PCR. The His6-SUMO-HV1 was highly expressed E. BL21 (DE3) which SUMO-HV1 accounts over 30% soluble fraction. purified Ni-NTA affinity chromatography and cleaved SUMO- specific protease Ulp1 release HV1 natural N-terminal. (rHV1) further then Q anion-exchange chromatography. N-terminal sequencing result demonstrated rHV1 had same sequence native hirudin. MALDI-TOF/ MS analysis indicated molecular weight 6939.161 Da, similar theoretical 6,944 Da. Chromozym TH assay showed anti-thrombin activity 8,800 ATU/mg compa- rable
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