Song Dongmin, Gao Zhendong, Zhao Liqiang, Wang Xiangxiang, Xu Haijin, Bai Yanling, Zhang Xiuming, Linder Markus B, Feng Hui, Qiao Mingqiang
The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.
School of Chemical Technology, Aalto University, P.O. Box 16100, 00076 Aalto, Finland.
Protein Expr Purif. 2016 Dec;128:22-8. doi: 10.1016/j.pep.2016.07.014. Epub 2016 Jul 26.
Hydrophobins are proteins produced by filamentous fungi with high natural-surfactant activities and that can self-assemble in interfaces of air-water or solid-water to form amphiphilic membranes. Here, we reported a high-yield fermentation method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris, attaining production of 300 mg/L by keeping the dissolved oxygen level at 15%-25% by turning the methanol-feeding speed. We also developed a novel HGFI-purification method enabling large-scare purification of HGFI, with >90% recovery. Additionally, we observed that hydrophobin HGFI in fermentation broth precipitated at pH < 7.0 and temperatures >90 °C. We also identified the structure and properties of proteins purified by this method through atomic force microscopy, circular dichroism, X-ray photoelectron spectroscopy, and water-contact angle measurement, which is similar to protein purification by ultrafiltration without heating treatment that enables our method to maintain native HGFI structure and properties. Furthermore, the purification method presented here can be applied to large-scale purification of other type I hydrophobins.
疏水蛋白是丝状真菌产生的具有高天然表面活性剂活性的蛋白质，能够在气-水或固-水界面自组装形成两亲性膜。在此，我们报道了一种在毕赤酵母中高产发酵来自灰树花的疏水蛋白HGFI的方法，通过调节甲醇进料速度将溶解氧水平保持在15%-25%，实现了300 mg/L的产量。我们还开发了一种新型的HGFI纯化方法，能够大规模纯化HGFI，回收率>90%。此外，我们观察到发酵液中的疏水蛋白HGFI在pH < 7.0和温度>90 °C时沉淀。我们还通过原子力显微镜、圆二色性、X射线光电子能谱和水接触角测量确定了用该方法纯化的蛋白质的结构和性质，这与未经加热处理的超滤蛋白质纯化相似，使我们的方法能够保持天然HGFI的结构和性质。此外，本文提出的纯化方法可应用于其他I型疏水蛋白的大规模纯化。
