Expression and characterization of hydrophobin HGFI fused with the cell-specific peptide TPS in Pichia pastoris

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• 作者：Baolong Niu ; Yujian Huang ; Suai Zhang ; Dandan Wang ; Haijin Xu ; Deling Kong ; ^{delingkong@126.com} ; Mingqiang Qiao ; ^{Mingqiangqiao@live.com}
• 关键词：Hydrophobin ; TPS ; Fusion expression ; Self-assembly ; Cell adhesion
• 刊名：Protein Expression and Purification
• 出版年：2012
• 期刊代码：184_10465928
• 类别：bio
• 出版时间：May, 2012
• 卷：83
• 期：1
• 页码：92-97
• 文件大小：524 K

摘要

The cell-specific peptide TPS (TPSLEQRTVYAK) has been proposed as a potential candidate for fabricating tissue engineering scaffolds based on its ability of binding to human endothelial progenitor cells (EPC) with high affinity and specificity.

In this study, the class I hydrophobin hgfI gene from Grifola frondosa and the tps were fused and cloned into pPIC9. The fusion gene was expressed in Pichia pastoris under the control of alcohol oxidase 1 promoter. Tricine-SDS-PAGE and Western blotting confirmed that the fusion protein TPS-linker-HGFI (TLH) was successfully secreted into the culture medium. The fusion protein TLH was purified by ultrafiltration and reverse-phase high performance liquid chromatography (RP-HPLC). Water contact angle (WCA) demonstrated that similar to recombinant HGFI (rHGFI), the purified TLH could convert the surface wettability of polystyrene and mica. X-ray photoelectron spectroscopy (XPS) measurements indicated that the purified TLH could form stable films on the hydrophobic siliconized glass surface.

The cell adhesion examination showed that the TLH modified poly(蔚-caprolactone) (PCL) could specially facilitate the EPC (particularly EPC derived from human) binding, while rHGFI modified PCL could nonselectively enhance cells adhesion. To the best of our knowledge, this is the first report that demonstrates that the TPS peptide was immobilized on biomaterial-PCL surface by fusion with hydrophobin. The potential application of this finding in combination with biomedical devices for EPC culture, will facilitate the current techniques used for cell-based therapies.