Wang Zefang, Feng Shuren, Huang Yujian, Li Shan, Xu Haijin, Zhang Xiuming, Bai Yanling, Qiao Mingqiang
The Key Laboratory of Bioactive Materials, Ministry of Education, College of Life Sciences, Nankai University, No. 94 Weijin Road, Tianjin 300071, PR China.
Protein Expr Purif. 2010 Jul;72(1):19-25. doi: 10.1016/j.pep.2010.03.017. Epub 2010 Mar 27.
Hydrophobins are small secreted proteins produced by filamentous fungi. Being amphipathic and self-assembling, hydrophobins have drawn great attention since their discovery. The increase of production can reduce the cost and open up several new applications of hydrophobins. We successfully expressed recombinant Class I hydrophobin HGFI (rHGFI) by using pPIC9 vector with an alcohol oxidase 1 promoter in Pichia pastoris. Tricine-SDS-PAGE and Western blotting demonstrated that rHGFI, an 8 kDa protein, was secreted into the culture medium. The culture conditions of the transformant strain were optimized by controlling the methanol concentration and induction time. Ultrafiltration and reverse-phase high performance liquid chromatography were used to perform a large-scale purification of rHGFI. A stable production of rHGFI around 86 mg/L was achieved after the two-step purification. X-ray photoelectron spectroscopy and water contact angle measurements indicated that the functional rHGFI could self-assemble on hydrophobic siliconized glass and Teflon as well as on hydrophilic mica surfaces. A methylthiazol tetrazolium assay showed that rHGFI film could facilitate human aortic smooth muscle cell proliferation due to its cytocompatibility.
疏水蛋白是丝状真菌分泌的小分子蛋白质。由于具有两亲性和自组装特性，疏水蛋白自发现以来就备受关注。提高产量可以降低成本并开拓疏水蛋白的多种新应用。我们通过在毕赤酵母中使用带有醇氧化酶1启动子的pPIC9载体成功表达了重组I类疏水蛋白HGFI（rHGFI）。Tricine-SDS-PAGE和Western印迹表明，8 kDa的rHGFI分泌到了培养基中。通过控制甲醇浓度和诱导时间对转化菌株的培养条件进行了优化。采用超滤和反相高效液相色谱对rHGFI进行大规模纯化。经过两步纯化后，实现了rHGFI约86 mg/L的稳定产量。X射线光电子能谱和水接触角测量表明，功能性rHGFI可以在疏水的硅化玻璃和聚四氟乙烯以及亲水的云母表面上自组装。甲基噻唑四氮唑试验表明，rHGFI膜因其细胞相容性可促进人主动脉平滑肌细胞增殖。
