Refolding, purification, and activation of miniplasminogen and microplasminogen isolated from E. coli inclusion bodies》.pdfVIP

Protein Expression and PuriWcation 52 (2007) 395–402 /locate/yprep Refolding, puriWcation, and activation of miniplasminogen and microplasminogen isolated from E. coli inclusion bodies Dan Medynski 1, Michael Tuan 1, Wayne Liu, Shili Wu, Xinli Lin ¤ Proteomtech Inc., 3505 Cadillac Ave., Building F7, Costa Mesa, CA 92626, USA Received 12 September 2006, and in revised form 14 October 2006 Available online 26 October 2006 Abstract Two des-kringle derivatives of human plasminogen, microplasminogen and miniplasminogen, have been expressed at high levels as inclusion bodies in Escherichia coli using a T7 expression system. In each case, the isolated inclusion bodies were refolded and puriWed. A Wnal yield of »10% of total refolded protein was observed in each case. Both refolded molecules were successfully activated to their func- tional forms, microplasmin and miniplasmin, by the plasminogen activator urokinase. The kinetic properties of the refolded microplas- min and miniplasmin were comparable to full length, native plasmin. © 2006 Elsevier Inc. All rights reserved. Keywords: Miniplasmin; Microplasmin; E. coli expression; Inclusion bodies; Refolding; Activation 2 domains (approximately 78–80 amino acids each), while the Plasmin (Plm) is a serine protease with several impor- tant physiological roles, including dissolving blood clots. B chain contains a “linker” region of 20 amino acids and a Plasmin is normally found circulating inertly in the blood serine