American Journal of Biochemistry and Biotechnology

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

Qing Yang, Xuyu Lei, Jianqiang Xu and Lijia An

DOI : 10.3844/ajbbsp.2005.17.21

American Journal of Biochemistry and Biotechnology

Volume 1, Issue 1

Pages 17-21


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.


© 2005 Qing Yang, Xuyu Lei, Jianqiang Xu and Lijia An. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.