Molecular cloning, expression, purification and characterization of truncated forms of human plasminogen in Pichia pastoris expression system

Joshi, Kishore K. and Sahni, Girish (2010) Molecular cloning, expression, purification and characterization of truncated forms of human plasminogen in Pichia pastoris expression system. Process Biochemistry, 45 (8). pp. 1251-1260. ISSN 13595113

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Abstract

Human plasminogen (HPG), which contains a catalytic domain together with five kringle domains, can be readily purified from blood plasma by chromatography on lysine-agarose. However, its truncated derivatives are needed for various important therapeutic applications. The proteolytic digestion of plasminogen in vitro yields several low molecular weight variants viz. the kringle-less catalytic domain, known as micro-plasminogen (microPG), or miniplasminogen (miniPG), which consists of microPG with an intact kringle-5 domain. However, this method is extremely cumbersome due to a requirement of stringent control on the limited proteolysis process, which often leads to very poor recoveries of the desired product/s, apart from the potentially serious safety-regulatory issues associated with blood-derived therapeutic products. Here, we describe the high-level secretory expression of these important plasminogen derivatives employing Pichia pastoris as the expression host with an engineered alpha-mating factor signal sequence. The purified proteins were found to be functionally comparable with human blood plasminogen-derived ‘native’ forms in terms of their N-terminal amino acid sequences and molecular mass, as well as functional properties. This study paves the way for the facile large-scale production of recombinant human plasminogen derivatives required for thrombolytic and other life-saver therapies

Item Type:	Article
Additional Information:	Copyright of this article belongs to Elsevier Science.
Uncontrolled Keywords:	Plasminogen; Expression; Microplasminogen; Miniplasminogen; Purification; Pichia pastoris
Subjects:	Q Science > QD Chemistry
Depositing User:	Dr. K.P.S.Sengar
Date Deposited:	28 Feb 2012 16:08
Last Modified:	28 Feb 2012 16:08
URI:	http://crdd.osdd.net/open/id/eprint/1109

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