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

DIR@IMTECH: CSIR-Institute of Microbial Technology

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Title Molecular cloning, expression, purification and characterization of truncated forms of human plasminogen in Pichia pastoris expression system
 
Creator Joshi, Kishore K.
Sahni, Girish
 
Subject QD Chemistry
 
Description 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
 
Publisher Elsevier Science
 
Date 2010
 
Type Article
PeerReviewed
 
Format application/pdf
 
Identifier http://crdd.osdd.net/open/1109/1/sahni2010.pdf
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
 
Relation http://dx.doi.org/10.1016/j.procbio.2010.04.014
http://crdd.osdd.net/open/1109/