A novel two-step extraction method with detergent/polymer systems for primary recovery of the fusion protein endoglucanase I–hydrophobin I

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• 作者：Collé ; n ; Anna ; Persson ; Josefine ; Linder ; Markus ; Nakari-Setä ; lä ; Tiina ; Penttilä ; Merja ; et. al.
• 关键词：Aqueous two-phase system ; Detergent removal ; Extraction ; Fusion protein ; Hydrophobin ; Protein purification
• 刊名：Biochimica et Biophysica Acta (BBA)/General Subjects
• 出版年：2002
• 出版时间：January 15, 2002
• 年：2002
• 卷：1569
• 期：1-3
• 页码：139-150
• 全文大小：408 K

文摘

Extraction systems for hydrophobically tagged proteins have been developed based on phase separation in aqueous solutions of non-ionic detergents and polymers. The systems have earlier only been applied for separation of membrane proteins. Here, we examine the partitioning and purification of the amphiphilic fusion protein endoglucanase I_core–hydrophobin I (EGI_core–HFBI) from culture filtrate originating from a Trichoderma reesei fermentation. The micelle extraction system was formed by mixing the non-ionic detergent Triton X-114 or Triton X-100 with the hydroxypropyl starch polymer, Reppal PES100. The detergent/polymer aqueous two-phase systems resulted in both better separation characteristics and increased robustness compared to cloud point extraction in a Triton X-114/water system. Separation and robustness were characterized for the parameters: temperature, protein and salt additions. In the Triton X-114/Reppal PES100 detergent/polymer system EGI_core–HFBI strongly partitioned into the micelle-rich phase with a partition coefficient (K) of 15 and was separated from hydrophilic proteins, which preferably partitioned to the polymer phase. After the primary recovery step, EGI_core–HFBI was quantitatively back-extracted (K_{EGIcore–HFBI}