Cloning and expression of antifreeze protein from Antarctic psychrophilic prasinophyte, Pyramimonas gelidicola

Abstract

Antarctic psychrophilic prasinophyte, Pyramimonas gelidicola isolated from Antarctic ocean was cultivated in culture collection of Korea Polar Research Institute (KOPRI) and was identified by morphological and molecular biological approaches. We had screened the ice binding and antifreeze activities of concentrated culture media of this microorganism by cold-finger method and found that this microorganism had strong ice activities. In order to discover the genes for antifreeze and other proteins of P.gelidicola, the cDNA library construction was performed. Over 2,000 ESTs were collected and categorized genetic functions by orthologous groups for eukaryotes (KOGs). It was revealed that 14.4% of ESTs was related to function of posttranslational modification, protein turnover and chaperones (J code of KOG category). Among the ESTs of this species, it was shown that six ESTs consisted of contiguous groups for the gene of antifreeze protein (AFP). The sequences of ORF and parts of 5'-UTR and 3'-UTR were analyzed. The ORF was composed of 275 amino acids and the deduced protein was predicted as 28.5 kDa in size. The N-terminal 22 amino acids were assumed as a signal peptide. The gene for mature AFP was amplified and inserted into pCold1 expression vector containing N-terminal His-Tag sequences. The recombinant plasmid was transformed to BL21 (DE3). The transformant was induced for 24 hours at 15℃ using 1mM isopropyl-β-D-thiogalactopyranoside (IPTG). The expressed AFP was solubilized in 20mM Trisㆍ HCl buffer by sonification at 4℃. The solubilized recombinant AFP in natural condition was purified by Ni affinity chromatography. The ice binding and antifreeze activities of purified recombinant AFP were measured by ice-pitting instrument and nanolitre-osmometer. The recombinant AFP was shown to have strong ice binding activity according to observation of pitting shapes on ice surfaces. The ice crystal morphology and thermal hysteresis (TH) activity was analyzed by nanolit