Effective reduction of truncated expression of gloshedobin in Escherichia coli using molecular chaperone ClpB

Abstract

Snake venom thrombin-like enzymes (TLEs) have been widely studied for potential therapeutic applications as anti-coagulants in the treatment of blood clotting disorders. However, due to the cysteine-rich nature of these proteins, their expressions in Escherichia coli were often impeded by inclusion body (IB) formation. The formation of truncated expression products significantly complicated the production of gloshedobin, a recently isolated TLE from the snake venom of Gloydius shedaoensis [Yang, Q., Li, M., Xu, J.Q., Bao, Y.M., Lei, X.Y., An, L.J., 2003a. Expression of gloshedobin, a thrombin-like enzyme from the venom of Gloydius shedaoensis, in Escherichia coli. Biotechnology Letters 25, 101–104; Yang, Q., Xu, J.Q., Li, M., Lei, X.Y., An, L.J., 2003b. High-level expression of a soluble snake venom enzyme, gloshedobin, in E. coli in the presence of metal ions. Biotechnology Letters 25, 607–610]. In this work, it was found that the expression of gloshedobin was strongly dependent on the expression host. The truncated expression was reduced by 25% when the protein was expressed in E. coli BL21(DE3)pLysS instead of BL21(DE3). It was also demonstrated that co-expression of ClpB (a molecular chaperone) in BL21(DE3) enabled the expression of gloshedobin mostly in intact form without compromising expression level, while almost completely eliminating its truncation products. This suggests a new simple strategy to significantly improve the quality of protein expression in TLE production and may find its useful application for many other recombinant proteins whose expressions and/or purifications are hindered by the formation of truncation products.