Abstract: Kar2p is one of the molecular chaperones. In order to investigate its effect on expression of heterologous proteins in Pichia pastoris, the gene encoding the protein was cloned from P. pastoris GS115 and transformed into the P. pastoris transformant P-Ple. The twice transformant P-PleKar that simultaneously over-expressed plectasin and Kar2p was obtained. The transcription levels of Kar2p in P-Ple and P-PleKar were analyzed by real-time PCR. Kar2p transcription in P-PleKar was up-regulated about 18 folds as in P-Ple. Comparing the growth curves between P-Ple and P-PleKar, the overexpression of Kar2p had no effect on cell growth of host, but inhibited the expression of heterologous protein plectasin. Duo to the Kar2p’s effect, the highest antibacterial activity of plectasin significantly decreased at the 48 h of induction. This did not only inhibit the expression of plectasin, but also the expression and secretion of protease in P. pastoris. It was evidenced that plectasin secreted into the culture medium maintained high antibacterial activity in the last stage of fermentation, and were not rapidly degraded by protease accumulated in culture. Compared with P-Ple, the overexpression of Kar2p slightly reduced the expression level of plectasin in P-PleKar, but kept the expression level stable for a longer time.

Key words: Kar2p, plectasin, Pichia pastoris, transformation, expression