title: High-yield fermentation and a novel heat-precipitation purification method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris.
abstract: 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.
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.
authors.forename: Dongmin
authors.initials: D
authors.lastname: Song
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.
authors.forename: Zhendong
authors.initials: Z
authors.lastname: Gao
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.
authors.forename: Liqiang
authors.initials: L
authors.lastname: Zhao
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.
authors.forename: Xiangxiang
authors.initials: X
authors.lastname: Wang
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.
authors.forename: Haijin
authors.initials: H
authors.lastname: Xu
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.
authors.forename: Yanling
authors.initials: Y
authors.lastname: Bai
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China.
authors.forename: Xiuming
authors.initials: X
authors.lastname: Zhang
authors.affiliation: School of Chemical Technology, Aalto University, P.O. Box 16100, 00076 Aalto, Finland.
authors.forename: Markus B
authors.initials: MB
authors.lastname: Linder
authors.affiliation: Tianjin Environmental Protection Technical Development Center, Tianjin, China.
authors.forename: Hui
authors.initials: H
authors.lastname: Feng
authors.affiliation: The Key Laboratory of Molecular Microbiology and Technology, Ministry of Education, Nankai University, Tianjin, China. Electronic address: mingqiangqiao@aliyun.com.
authors.forename: Mingqiang
authors.initials: M
authors.lastname: Qiao
mesh_terms: D005656:Fungal Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D048691:Grifola / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics / Q000254:growth & development*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins
keywords: Characterization; Fed-batch fermentation; Hydrophobin HGFI; Purification