title: Production and characterization of hirudin variant-1 by SUMO fusion technology in E. coli.
abstract: Hirudin is the most potent non-covalent inhibitor of thrombin. Several expression systems have been used to produce recombinant hirudin for pharmaceutical purposes. However, high expression of active hirudin in Escherichia coli cytoplasm has not been successful owing to the fact that heterogenetic small peptide is easily degraded in the cell. To solve this problem, we constructed a recombinant form of the hirudin variant-1 (HV1) as a fusion protein with the small ubiquitin-related modifier gene (SUMO) by use of over-lap PCR. The fusion gene His(6)-SUMO-HV1 was highly expressed in E. coli BL21 (DE3) in which the SUMO-HV1 accounts for over 30% of the soluble fraction. The fusion protein was purified by Ni-NTA affinity chromatography and cleaved by a SUMO-specific protease Ulp1 to release the HV1 with natural N-terminal. The recombinant HV1 (rHV1) was further purified by Ni-NTA affinity chromatography and then by Q anion-exchange chromatography. N-terminal sequencing result demonstrated the purified rHV1 had the same N-terminal sequence as the native hirudin. MALDI-TOF/MS analysis indicated that the molecular weight of the purified rHV1 protein was 6939.161 Da, which was similar to the theoretical molecular weight of rHV1 6,944 Da. The Chromozym TH assay result showed that the anti-thrombin activity of purified rHV1 was 8,800 ATU/mg and comparable to the specific activity of native hirudin.
authors.affiliation: Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, 100#, Shizi Street, Hongshang Road, Nanjing, 210028 Jiangsu, China.
authors.forename: Wuguang
authors.initials: W
authors.lastname: Lu
authors.forename: Xueting
authors.initials: X
authors.lastname: Cai
authors.forename: Zhenghua
authors.initials: Z
authors.lastname: Gu
authors.forename: Yuzheng
authors.initials: Y
authors.lastname: Huang
authors.forename: Binbin
authors.initials: B
authors.lastname: Xia
authors.forename: Peng
authors.initials: P
authors.lastname: Cao
mesh_terms: D000925:Anticoagulants / Q000494:pharmacology; D002846:Chromatography, Affinity / Q000379:methods; D003546:Cysteine Endopeptidases / Q000378:metabolism; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000254:growth & development / Q000378:metabolism*; D015870:Gene Expression; D006629:Hirudins / Q000096:biosynthesis* / Q000235:genetics* / Q000494:pharmacology; D008970:Molecular Weight; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics; D025841:Small Ubiquitin-Related Modifier Proteins / Q000235:genetics* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000925:Anticoagulants; D006629:Hirudins; D011993:Recombinant Fusion Proteins; D025841:Small Ubiquitin-Related Modifier Proteins; D003546:Cysteine Endopeptidases; C118083:Ulp1 protease