Postdoctoral Mobile Station of Biology, Genetic Engineering Research Center, College of Life Sciences, Chongqing University, Chongqing, 400030, China.
College of Biological and Food Engineering, Huaihua University, Huaihua, 418008, China.
Microb Cell Fact. 2018 Sep 14;17(1):145. doi: 10.1186/s12934-018-0992-x.
The fungal ribotoxin hirsutellin A (HtA) exhibits strong insecticidal activity; however, efficient systems for expressing recombinant HtA (rHtA) are lacking. Here, we established an efficient heterologous expression system to produce large amounts of rHtA.
Recombinant Pichia pastoris transformants with high levels of secretory rHtA were screened, and in a fed-batch reactor, rHtA was secreted at levels up to 80 mg/l following methanol induction, which was more than sixfold higher than that in shake flasks. Approximately 7 mg of highly pure rHtA was obtained from 300 ml of fed-batch culture supernatant by Ni-nitriloacetic acid affinity chromatography and CM Sepharose ion-exchange chromatography. Mass spectrometry results revealed rHtA as a native N-terminal non-glycosylated monomeric protein with a molecular weight of 15.3 kDa. Purified rHtA exhibited excellent thermal and protease stability and dose-dependent cytotoxicity to Sf9 insect cells and insecticidal activity against Galleria mellonella larvae.
This is the first report of rHtA expression in P. pastoris. The rHtA was expressed at a high level under high-cell-density fed-batch fermentation and was efficiently purified using a two-step purification method. Purified rHtA exhibited thermal and protease stability, as well as appropriate bioactivities. Our results indicate that fed-batch production by P. pastoris is an efficient method to produce functional rHtA.
真菌核糖核酸酶 hirsutellin A（HtA）具有很强的杀虫活性；然而，缺乏有效的表达重组 HtA（rHtA）的系统。在这里，我们建立了一个高效的异源表达系统来大量生产 rHtA。
筛选出高水平分泌 rHtA 的重组毕赤酵母转化体，在分批补料发酵罐中，甲醇诱导后 rHtA 的分泌量高达 80mg/L，是摇瓶中的六倍以上。通过 Ni-亚氨二乙酸亲和层析和 CM Sepharose 离子交换层析，从 300ml 分批补料培养上清液中获得约 7mg 高纯度的 rHtA。质谱结果表明 rHtA 是一种天然的 N 端非糖基化的单体蛋白，分子量为 15.3kDa。纯化的 rHtA 表现出极好的热稳定性和蛋白酶稳定性，对 Sf9 昆虫细胞具有剂量依赖性细胞毒性，并对家蚕幼虫具有杀虫活性。
这是 rHtA 在毕赤酵母中表达的首次报道。rHtA 在高密度细胞分批补料发酵下以高水平表达，并通过两步纯化方法高效纯化。纯化的 rHtA 表现出热稳定性和蛋白酶稳定性，以及适当的生物活性。我们的结果表明，毕赤酵母的分批补料生产是生产功能性 rHtA 的有效方法。
