Abstract: Hipposin is the N-terminal derivative of Atlantic halibut (Hippoglossus hippoglossus L.) histone H2A with 51 amino acids, which has a good ability to inhibit gram-positive and gram-negative bacteria. The cDNA encoding hipposin (hip) was optimized and synthesized with reference to the codon preference of Pichia pastoris and its native cDNA sequence, respectively. In this synthesized fragment, nucleotides encoding recognition site for the enzyme (Kex2) cutting?α-factor signal peptide and 6×His tag were added to 5′ and 3′ terminals, respectively, and Xho I and Xba I restriction sites were added to the both ends. This fragment was ligated with pPICZαA to construct the recombinant expression vector pPICZαA-hip. The pPICZαA-hip was transformed into P. pastoris X-33. The yeast transformants containing multicopy gene insertion were selected by using Zeocin, and identified by PCR for yeast genomic DNA. Expression was induced by adding 1% methanol at 30℃ and 250 r/min. Immobilized metal affinity chromatography (IMAC) was used to purify the target protein. The most appropriate expression time was 96 h. Western Blot analysis demonstrated that the His monoclonal antibody could be specifically bound to expressed products. The highly purified target protein was obtained from the fermentation supernatant, but it was found that a part of the expressed product was hydrolyzed. The present results provide important initial values for preparation of hipposin by recombinant DNA expression.

Key words: Atlantic halibut, hipposin antimicrobial peptide, Pichia pastoris, recombinant DNA expression