Research Article Open Access

Purification of A Recombinant Thrombin-like Enzyme, Gloshedobin by Egg Yolk Antibody-Coupled Adsorbents

Qing Yang1, Xuyu Lei1, Jianqiang Xu1 and Lijia An1
  • 1 Dalian University of Technology, China

Abstract

The gloshedobin, a snake venom thrombin-like enzyme was biosynthesized in the soluble form and purified by egg yolk antibody-coupled adsorbents from E. coli. As His-tag is not favored from the point of view of high-level and soluble expression, we herein constructed a recombinant gloshedobin without His-tag and developed a novel egg yolk antibody (IgY)-immunoaffinity chromatography for its purification in a higher activity yield. The purification process involving Octyl Sepharose FF, IgY-immunoaffinity chromatography and Source Q, yielded 454.7U mg1 protein of interest and 34.8% activity recovery. The anti-gloshedobin IgY was obtained from eggs by injecting diluted gloshedobin into the breast muscle of laying hens and then purified by several steps including 3.5%, 8.5% and 12% polyethylene glycol-6000 precipitation and affinity chromatography using gloshedobin-coupled agarose gel. The obtained IgY was covalently linked to CNBr pre-activated agarose gel, Sepharose 4B FF, to yield immunoaffinity adsorbents. Both immunological and enzymatic activities of the purified enzyme were determined by western-blotting analysis and fibrinogen clotting assay, respectively.

American Journal of Biochemistry and Biotechnology
Volume 1 No. 1, 2005, 17-21

DOI: https://doi.org/10.3844/ajbbsp.2005.17.21

Submitted On: 24 March 2005 Published On: 31 March 2005

How to Cite: Yang, Q., Lei, X., Xu, J. & An, L. (2005). Purification of A Recombinant Thrombin-like Enzyme, Gloshedobin by Egg Yolk Antibody-Coupled Adsorbents. American Journal of Biochemistry and Biotechnology, 1(1), 17-21. https://doi.org/10.3844/ajbbsp.2005.17.21

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Keywords

  • Escherichia coli
  • expression
  • egg yolk
  • Immunoaffinity chromatography
  • purification
  • thombin-like enzyme