毕赤酵母Kar2p过表达对假黑盘菌素表达量的影响

The Effect of Kar2p Overexpression on Plectasin Expression in Pichia pastoris

Kar2p是一种分子伴侣蛋白,为研究其对外源蛋白在毕赤酵母中表达的影响,从毕赤酵母菌株GS115中克隆出编码该蛋白的基因,利用其对毕赤酵母转化子P-Ple进行二次转化,获得了能够同时过表达假黑盘菌素和Kar2p的二次转化子P-Ple Kar;采用荧光定量PCR方法,对P-Ple和P-Ple Kar两个转化子中的Kar2p转录水平进行了测定发现,在P-Ple Kar中Kar2p的转录水平比P-Ple上调了约18倍。对P-Ple和P-Ple Kar两个转化子的生长曲线进行比较,发现Kar2p过表达对宿主细胞的生长无明显影响,但对外源蛋白假黑盘菌素的表达确实产生了一定的抑制作用,受Kar2p的影响,在诱导培养48 h时,假黑盘菌素的最高抑菌活性明显降低。这种抑制表达不单针对假黑盘菌素,对毕赤酵母的蛋白酶表达和分泌也同样起到了一定的抑制作用,具体表现在分泌到培养液中的假黑盘菌素在发酵后期仍然能保持较高抑菌活性,没有被发酵液中的累积的蛋白酶快速降解。与P-Ple相比,P-Ple Kar假黑盘菌素的表达量虽略有降低,但可以维持更长时间的稳定表达。

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-Ple Kar that simultaneously over-expressed plectasin and Kar2 p was obtained. The transcription levels of Kar2 p in P-Ple and P-Ple Kar were analyzed by real-time PCR. Kar2 p transcription in P-Ple Kar was up-regulated about 18 folds as in P-Ple. Comparing the growth curves between P-Ple and P-Ple Kar, the overexpression of Kar2 p 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 Kar2 p slightly reduced the expression level of plectasin in P-Ple Kar, but kept the expression level stable for a longer time.