Cloning, expression and partial characterization of antifreeze protein from a psychrophilic yeast isolated from the Arctic

Abstract

A psychrophilic yeast having an extracellular antifreeze protein (AFP) was isolated from the Arctic. Its 18S and 28S ribosomal RNA sequences have a high similarity with Leucosporidium antarcticum but its ITS1, 5.8S, and ITS2 sequences are variable. We designated this isolate as L. sp. AY30. The semi-purified AFP from the culture medium of this yeast grown at 1 ℃ for 1 week has high antifreeze activity and is about 26 kDa in size on SDS-PAGE analysis. The genomic DNA sequence of AFP was obtained from a draft version of pyrosequencing data using local tBlastN. The candidate contig region of about 2 Kb in size containing the AFP gene was reamplified by PCR and sequenced. The forward upstream PCR primers for 3'-RACE were designed from the genomic DNA sequence. The largest 3'-RACE PCR product of about 1 Kb was sequenced. The nucleotide sequence contained 5'-UTR (119 bp) and 3'-UTR (40 bp). The ORF consists of 261 amino acids and the deduced protein was calculated as 26 kDa in size. The N-terminal 20 residues were predicted as a signal peptide. The mature AFP gene was inserted into pCold expression vector with N-terminal His-Tag and Factor Xa sequence. The recombinant plasmid was transformed into E. coli. The transformant was induced for 20 hours at 15℃ using 1 mM IPTG. The overexpressed AFP in the soluble faction of cell lysate was purified using Ni affinity chromatography. The ice crystal morphology and TH activity was analyzed using nanolitre osmometer. Its ice crystal shape and the burst shape revealed that it may bind to the basal plane of ice crystal, which is the characteristic of hyperactive AFPs. The TH activity was about 0.3 ℃ in the concentration of 3 ug/ml.