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Refolding of a Staphylokinase Variant Y1-Sak by Reverse Dilution

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Abstract

To develop more potent thrombolytic agents with fibrinolytic and antiplatelet aggregation activity, staphylokinase (Sak) variant Y1-Sak, a recombinant mutant of the Staphylococcus aureus protein Sak, was constructed. Y1-Sak formed an insoluble inclusion body when overexpressed in Escherichia coli strain JF1125. To obtain an optimized refolding process, dilution refolding was used to optimize refolding conditions. The results revealed that additive l-arginine and refolding temperature played critical roles in the refolding of Y1-Sak. Subsequently, two refolding methods, gel filtration and reverse dilution, were investigated to refold Y1-Sak. The results indicated that the fibrinolytic activity and recovery of Y1-Sak from gel filtration were lower than those from reverse dilution. Reverse dilution refolding successfully reduced the side reaction of refolding with the help of l-arginine, and the fibrinolytic activity and recovery of Y1-Sak were significantly improved. Functional analysis revealed that refolded Y1-Sak by reverse dilution possessed fibrinolytic and antiplatelet aggregation activities. Moreover, the immunogenicity of Y1-Sak was significantly reduced.

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Abbreviations

Sak:

staphylokinase

Y1-Sak:

a staphylokinase variant in which N-terminal 15 amino acid residues were deleted and Ser16, Lys74, Glu75, Arg77, Lys109, Phe 111 was substituted with Lys, Ala, Ala, Ala, Arg, and Asp

rt-PA:

recombinant tissue-type plasminogen activator

CV:

column volumes

IBs:

inclusion bodies

Gu-HCl:

guanidine–HCl

RGD:

Arg-Gly-Asp

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Authors and Affiliations

  1. College of Life Science, Hebei Normal University, 113 Yuhua East Road, Shijiazhuang City, Hebei Province, 050016, People’s Republic of China

    Jintian He, Ruiguang Xu & Jinlin Feng

  2. College of Environmental Science and Engineering, Hebei University of Science and Technology, Shijiazhuang City, Hebei Province, 050018, People’s Republic of China

    Gaizhen Wang & Jinlong Wang

  3. Key Laboratory of Molecular Medicine, Ministry of Education, Fudan University, 130 Dong-an Road, Shanghai, 200032, People’s Republic of China

    Huabo Su & Houyan Song

Authors
  1. Jintian He
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  2. Gaizhen Wang
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  3. Ruiguang Xu
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  4. Jinlin Feng
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  5. Jinlong Wang
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  6. Huabo Su
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  7. Houyan Song
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Correspondence to Jintian He.

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He, J., Wang, G., Xu, R. et al. Refolding of a Staphylokinase Variant Y1-Sak by Reverse Dilution. Appl Biochem Biotechnol 151, 29–41 (2008). https://doi.org/10.1007/s12010-008-8150-2

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  • DOI: https://doi.org/10.1007/s12010-008-8150-2

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