TITLE:
Effect of 4% glycerol and low aeration on result of expression in Escherichia coli of Cin3 and three Venturia inaequalis EST’s recombinant proteins
AUTHORS:
Taha H. Al-Samarrai, William T. Jones, Dawn Harvey, Christopher A. Kirk, M. Templtone
KEYWORDS:
Venturia inaequalis; Expressed Sequence Tag (ESTs); Phytopathogenic Fungus; Appressorium; A Stroma
JOURNAL NAME:
American Journal of Molecular Biology,
Vol.3 No.1,
January
23,
2013
ABSTRACT: The phytopathogenic fungus Venturia inaequalis causes scab of apple. Once this fungus penetrates the plant surface, it forms a specialized body called a stroma between the inner cuticle surface and the epidermal cell wall. A novel Venturia inaequalis 5704 (Cin3) and three expressed sequence tags (ESTs); 38, 6987, and 4010 are strongly up-regulated in the early stages of infection. The CIN3 and three ESTs using two vectors pMAL-c2 and pET 21 were expressed in Escherichia coli. Recombinant proteins expression, solubility and yields were analyzed. 38, 5704 (Cin3) and 6987 re- combinant proteins were expressed in soluble form and while 4010 was expressed in inclusion bodies. Re- solution on native-PAGE, the recombinant proteins; 38, 5704 (Cin3), 6987 were shown to be present in dimmer, tetramer and polymer. A method was de- veloped, consisting of induction of expression at va- rious temperatures, and using enriched broth with 4% glycerol together with slow shaking, led to a decrease in concentration of nascent polypeptide and production of soluble recombinant proteins of; 38, 5704 (Cin3), 6987 and 4010. Resolution on native- PAGE, the recombinant proteins were shown to be present as monomer.