真菌疏水蛋白HGFI在酿酒酵母中的表达、纯化和性质测定
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摘要
真菌疏水蛋白是丝状真菌分泌的小分子量蛋白质,由于其独特的两亲性和自组装特性,近年来成为研究热点.利用pSP-G1作为表达载体,成功的在酿酒酵母中表达出疏水蛋白HGFI,并利用超滤结合HPLC的方法对该蛋白进行了纯化.此外,还对纯化的HGFI通过接触角,圆二色谱,X射线光电子能谱和原子力显微镜进行了性质测定,证实其性质与毕赤酵母表达系统表达的HGFI一致.该酿酒酵母表达系统与毕赤酵母表达系统相比,由于其不存在甲醇诱导和过度糖基化,从而具有更高的生物安全性,因而能拓展其在食品及药物传输系统方面的应用.
Hydrophobin, small protein secreted by filamentous fungi, has attracted increasing attention based on its unique character of amphipathy and self-assembly. In this article, plasmid p SP-G1 was applied to achieve the successful expression of Hydrophobin HGFI from Grifola frondosa in Saccharomyces cerevisiae. Then HGFI was purified by the method of ultrafiltration combined with HPLC. Moreover, the characterization of HGFI expressed in Saccharomyces cerevisiae were identified by WCA, CD, XPS and AFM,which proved that the property of Sc-HGFI was similar with HGFI expressed in Pichia pastoris. ScHGFI, compared with HGFI expressed in Pichia pastoris, might show higher biological safety since the expression of HGFI in Saccharomyces cerevisiae didn't need methanol induction and excessive glycosylation.The higher biological safety of Sc-HGFI may promote the application in area of drug delivery system and emulsion and preservation of food.
Hydrophobin, small protein secreted by filamentous fungi, has attracted increasing attention based on its unique character of amphipathy and self-assembly. In this article, plasmid p SP-G1 was applied to achieve the successful expression of Hydrophobin HGFI from Grifola frondosa in Saccharomyces cerevisiae. Then HGFI was purified by the method of ultrafiltration combined with HPLC. Moreover, the characterization of HGFI expressed in Saccharomyces cerevisiae were identified by WCA, CD, XPS and AFM,which proved that the property of Sc-HGFI was similar with HGFI expressed in Pichia pastoris. ScHGFI, compared with HGFI expressed in Pichia pastoris, might show higher biological safety since the expression of HGFI in Saccharomyces cerevisiae didn't need methanol induction and excessive glycosylation.The higher biological safety of Sc-HGFI may promote the application in area of drug delivery system and emulsion and preservation of food.
引文
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17 Niu B,Wang D,Yang Y,et al.Heterologous expression and characterization of the hydrophobin HFBI in Pichia pastoris and evaluation of its contribution to the food industry[J].Amino Acids,2012,43(2):763-771.
18 Linder M B,Qiao M,Laumen F,et al.Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems[J].Biochemistry,2004,43(37):11 873-11 882.
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20 Wang Z,Feng S,Huang Y,et al.Expression and characterization of a Grifola frondosa hydrophobin in Pichia pastoris[J].Protein Expression and Purification,2010,72(1):19-25.
21 Song D,Gao Z,Zhao L,et al.High-yield fermentation and a novel heat-precipitation purification method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris[J].Protein Expr Purif,2016,128:22-28.
22 Wang Z,Feng S,Huang Y,et al.Prokaryotic expression,purification,and polyclonal antibody production of a hydrophobin from Grifola frondosa[J].Acta Biochimica et Biophysica Sinica,2010,42(6):388-395.
23 Wang X,Mao J,Chen Y,et al.Design of antibacterial biointerfaces by surface modification of poly(epsiloncaprolactone)with fusion protein containing hydrophobin and PA-1[J].Colloids Surf B Biointerfaces,2017,151:255-263.
2 Hektor H J,Scholtmeijer K.Hydrophobins:proteins with potential[J].Current Opinion in Biotechnology,2005,16(4):434-439.
3 Linder M B.Hydrophobins:Proteins that self assemble at interfaces[J].Current Opinion in Colloid&Interface Science,2009,14(5):356-363.
4 Linder M B,Szilvay G Z R,Nakari-Set L T,et al.Hydrophobins:the protein-amphiphiles of filamentous fungi[J].FEMS Microbiology Reviews,2005,29(5):877-896.
5 Ren Q,Kwan A H,Sunde M.Two forms and two faces,multiple states and multiple uses:properties and applications of the self-assembling fungal hydrophobins[J].Biopolymers,2013,100(6):601-612.
6 Wosten H A B,Scholtmeijer K.Applications of hydrophobins:current state and perspectives[J].Applied Microbiology and Biotechnology,2015,99(4):1 587-1 597.
7 Khalesi M,Gebruers K,Derdelinckx G.Recent advances in fungal hydrophobin towards using in industry[J].Protein J,2015,34(4):243-255.
8 Piscitelli A,Pennacchio A,Cicatiello P,et al.Rapid and ultrasensitive detection of active thrombin based on the Vmh2 hydrophobin fused to a Green Fluorescent Protein[J].Biosensors&Bioelectronics,2016,87:816-822.
9 Wang X S,Wang H C,Huang Y J,et al.Noncovalently functionalized multi-wall carbon nanotubes in aqueous solution using the hydrophobin HFBI and their electroanalytical application[J].Biosensors&Bioelectronics,2010,26(3):1 104-1 108.
10 Zhao Z-X,Qiao M-Q,Yin F,et al.Amperometric glucose biosensor based on self-assembly hydrophobin with high efficiency of enzyme utilization[J].Biosensors and Bioelectronics,2007,22(12):3 021-3 027.
11 Zhao L,Ma S,Pan Y,et al.Functional modification of fibrous PCL scaffolds with fusion protein VEGF-HGFI enhanced cellularization and vascularization[J].Adv Healthc Mater,2016,5(18):2 376-2 385.
12 Zhang M,Wang Z,Wang Z,et al.Immobilization of anti-CD31 antibody on electrospun poly(?-caprolactone)scaffolds through hydrophobins for specific adhesion of endothelial cells[J].Colloids and Surfaces B:Biointerfaces,2011,85(1):32-39.
13 Fang G,Tang B,Liu Z,et al.Novel hydrophobin-coated docetaxel nanoparticles for intravenous delivery:In vitro characteristics and in vivo performance[J].European Journal of Pharmaceutical Sciences,2014,60:1-9.
14 Bimbo L M,M?kil?E,Raula J,et al.Functional hydrophobin-coating of thermally hydrocarbonized porous silicon microparticles[J].Biomaterials,2011,32(34):9 089-9 099.
15 Valo H K,Laaksonen P I H,Peltonen L J,et al.Multifunctional hydrophobin:toward functional coatings for drug nanoparticles[J].ACS nano,2010,4(3):1 750-1 758.
16 Bimbo L M,Sarparanta M,Santos H A,et al.Biocompatibility of thermally hydrocarbonized porous silicon nanoparticles and their biodistribution in rats[J].ACS nano,2010,4(6):3 023-3 032.
17 Niu B,Wang D,Yang Y,et al.Heterologous expression and characterization of the hydrophobin HFBI in Pichia pastoris and evaluation of its contribution to the food industry[J].Amino Acids,2012,43(2):763-771.
18 Linder M B,Qiao M,Laumen F,et al.Efficient purification of recombinant proteins using hydrophobins as tags in surfactant-based two-phase systems[J].Biochemistry,2004,43(37):11 873-11 882.
19 Yu L,Zhang B,Szilvay G R,et al.Protein HGFI from the edible mushroom Grifola frondosa is a novel 8k Da class I hydrophobin that forms rodlets in compressed monolayers[J].Microbiology,2008,154(6):1 677-1 685.
20 Wang Z,Feng S,Huang Y,et al.Expression and characterization of a Grifola frondosa hydrophobin in Pichia pastoris[J].Protein Expression and Purification,2010,72(1):19-25.
21 Song D,Gao Z,Zhao L,et al.High-yield fermentation and a novel heat-precipitation purification method for hydrophobin HGFI from Grifola frondosa in Pichia pastoris[J].Protein Expr Purif,2016,128:22-28.
22 Wang Z,Feng S,Huang Y,et al.Prokaryotic expression,purification,and polyclonal antibody production of a hydrophobin from Grifola frondosa[J].Acta Biochimica et Biophysica Sinica,2010,42(6):388-395.
23 Wang X,Mao J,Chen Y,et al.Design of antibacterial biointerfaces by surface modification of poly(epsiloncaprolactone)with fusion protein containing hydrophobin and PA-1[J].Colloids Surf B Biointerfaces,2017,151:255-263.