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Construction, Expression and Refolding of a Bifunctional Fusion Protein Consisting of C-Terminal 12-Residue of Hirudin-PA and Reteplase

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Abstract

To obtain a bifunctional protein simultaneously showing bioactivity of anticoagulant and fibrinolytic for use in the treatment of thrombotic diseases, we constructed a fusion protein (HV12p–rPA) containing C-terminal 12-residue of hirudin-PA (HV12p) and reteplase (rPA). The fusion protein, in which HV12p was linked to rPA via Gly–Gly–Gly, was successfully expressed in an inactive form of inclusion bodies in Escherichia coli. HV12p–rPA was identified by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The expression level of HV12p–rPA was optimized by an orthogonal method and finally enhanced from 12 % to approximate 30 %. We also deeply investigated the condition of renaturation of HV12p–rPA, and the inactive protein was partly renatured through various conditions. The refolding efficacy of HV12p–rPA estimated by the recovery of fibrinolytic activity varied from 0.03 % to 16.6 % and the anticoagulant activity fluctuated in the range from 41 to 2,297 ATU/mg. Bioassays indicated that the resulted fusion protein, as expected, exhibited both fibrinolytic and anticoagulant activities. These works laid a foundation for further characterization of HV12p–rPA.

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Abbreviations

DBA:

Diaminobenzidine

GSSG:

Oxidized glutathione

GSH:

Reduced glutathione

HRP:

Horse radish peroxidase

HV12p:

C-terminal 12-residue of hirudin-PA

rPA:

Reteplase

HV12p–rPA:

HV12p–Gly–Gly–Gly–rPA

IPTG:

Isopropyl thiogalactoside

PCR:

Polymerase chain reaction

PVDF:

Polyvinylidene fluoride

SDS-PAGE:

Sodium dodecylsulfate-polyacrylamide gel electrophoresis

SEC:

Size-exclusion chromatography

t-PA:

Tissue-type plasminogen activator

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Acknowledgments

This study was supported by the Open Foundation of the State Key Laboratory of Biochemical Engineering (China Grant Number: 2005KT2-23, No. KF2008-7) and the Ministry of Education Doctor Foundation (Grant Number: 20040316005).

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Authors and Affiliations

  1. Key Laboratory of Drug Targeting and Drug Delivery System, Ministry of Education, West China School of Pharmacy, Sichuan University, Chengdu, 610041, China

    Ling Gao, Chun Zhang, Lingling Li, Lan Liang, Xuan Deng & Rong Yu

  2. State Key Laboratory of Biochemical Engineering, Institute of Process Engineering, Chinese Academy of Sciences, Beijing, 100080, China

    Ling Gao & Zhiguo Su

  3. School of Life Science & Technology, China Pharmaceutical University, Nanjing, 210009, China

    Wutong Wu

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  1. Ling Gao
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  2. Chun Zhang
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  3. Lingling Li
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  4. Lan Liang
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  5. Xuan Deng
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  6. Wutong Wu
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  7. Zhiguo Su
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  8. Rong Yu
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Corresponding authors

Correspondence to Zhiguo Su or Rong Yu.

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Ling Gao and Chun Zhang contributed equally to this work and should be considered co-first authors.

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Gao, L., Zhang, C., Li, L. et al. Construction, Expression and Refolding of a Bifunctional Fusion Protein Consisting of C-Terminal 12-Residue of Hirudin-PA and Reteplase. Protein J 31, 328–336 (2012). https://doi.org/10.1007/s10930-012-9407-8

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  • DOI: https://doi.org/10.1007/s10930-012-9407-8

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