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Production and Characterization of Hirudin Variant-1 by SUMO Fusion Technology in E. coli

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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 His6-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.

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Acknowledgment

This study was supported by Jiangsu Province’s Outstanding Leader Program of Traditional Chinese Medicine, Jiangsu Province’s Nature Science Foundation (No. SBK20082754, No. SBK2009456).

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Authors and Affiliations

  1. Laboratory of Cellular and Molecular Biology, Jiangsu Province Institute of Traditional Chinese Medicine, 100#, Shizi Street, Hongshang Road, Nanjing, 210028, Jiangsu, China

    Wuguang Lu, Xueting Cai, Zhenghua Gu, Binbin Xia & Peng Cao

  2. Jiangsu Institute of Parasitic Diseases, Wuxi, 214064, Jiangsu, China

    Yuzheng Huang

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  1. Wuguang Lu
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  2. Xueting Cai
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  3. Zhenghua Gu
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Correspondence to Peng Cao.

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Lu, W., Cai, X., Gu, Z. et al. Production and Characterization of Hirudin Variant-1 by SUMO Fusion Technology in E. coli . Mol Biotechnol 53, 41–48 (2013). https://doi.org/10.1007/s12033-012-9511-1

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