基于多重非共价相互作用的荧光融合蛋白质微米环
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摘要
利用蛋白质融合技术,将绿色荧光蛋白(GFP)与可二聚化的链酶亲和素突变体(SA)融合,形成二聚化融合蛋白质GFP-SA,并以此作为生物大分子自组装的构筑基元.同时,设计并合成了相应的配体分子RhYBio2,该配体中的2个生物素分子能特异性结合相邻融合蛋白中的SA,并将2个以上的二聚化融合蛋白GFP-SA排列成纳米线,随后该纳米线再通过配体中的罗丹明B分子二聚,进一步组装形成荧光蛋白质微米环.采用动态光散射(DLS)对其粒径及分布进行了表征,并利用透射电子显微镜(TEM)和激光共聚焦显微镜(confocal microscope)观察了组装体的形貌,即蛋白质微米环,其形貌规整并具有较强的荧光.
Proteins are attractive building blocks for construction of variant functional materials because of their chemical and structural diversities,and intrinsic functions.As the industry of biotechnology continues to expand,so does the expression of recombinant proteins with wide varieties.In this work,we adopted the recombinant protein technique to construct a new fusion protein,GFP-SA,as the building block of self-assemblies.The purification of GFP-SA was characterized by Superdex 75 size exclusive chromatography,SDS-PAGE,and MALDI-TOF.Then,GPC and native-PAGE were used to characterize the dimerization of GFP-SA based on the hydrogen bonds between neighboring SAs.Furthermore,ITC was employed to test the binding ability between GFP-SA and biotin,which revealed K_D=0.24μmol/L.In this study,we also designed and successfully synthesized the ligandwhich is composed of two biotin molecules and one rhodamine B molecule.The size of GFP-SA increased rapidly to 370 nm within one minute after mixing withWe measured the particle size ofmixture every few minutes until the size stabilized at around 1300 nm 2 h later.However,size variation was barely observed for the controlled samples of SA/RhYBio_2(controlled protein)and GFP-SA/YBio(controlled ligand).We hypothesized that the two biotin molecules ofcould bind specifically with SA and aligninto nanowires,which assembled further into micro rings.Their size was measured by dynamic light scattering(DLS)while the morphology was observed intuitively on a transmission electron microscope(TEM)and a confocal microscope(CM).The characteristic results from TEM and CM suggested an uneven size distribution of the micro rings prepared,which might be attributable to the flexibility of the fusion protein GFP-SA.These micro rings ofwith fluorescence has great potential for biological applications.
Proteins are attractive building blocks for construction of variant functional materials because of their chemical and structural diversities,and intrinsic functions.As the industry of biotechnology continues to expand,so does the expression of recombinant proteins with wide varieties.In this work,we adopted the recombinant protein technique to construct a new fusion protein,GFP-SA,as the building block of self-assemblies.The purification of GFP-SA was characterized by Superdex 75 size exclusive chromatography,SDS-PAGE,and MALDI-TOF.Then,GPC and native-PAGE were used to characterize the dimerization of GFP-SA based on the hydrogen bonds between neighboring SAs.Furthermore,ITC was employed to test the binding ability between GFP-SA and biotin,which revealed K_D=0.24μmol/L.In this study,we also designed and successfully synthesized the ligandwhich is composed of two biotin molecules and one rhodamine B molecule.The size of GFP-SA increased rapidly to 370 nm within one minute after mixing withWe measured the particle size ofmixture every few minutes until the size stabilized at around 1300 nm 2 h later.However,size variation was barely observed for the controlled samples of SA/RhYBio_2(controlled protein)and GFP-SA/YBio(controlled ligand).We hypothesized that the two biotin molecules ofcould bind specifically with SA and aligninto nanowires,which assembled further into micro rings.Their size was measured by dynamic light scattering(DLS)while the morphology was observed intuitively on a transmission electron microscope(TEM)and a confocal microscope(CM).The characteristic results from TEM and CM suggested an uneven size distribution of the micro rings prepared,which might be attributable to the flexibility of the fusion protein GFP-SA.These micro rings ofwith fluorescence has great potential for biological applications.
引文
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20Qi W J,Zhang Y F,Kochovski Z,Wang J,Lu Y,Chen G S,Jiang M.Nano Res,2018,10:5566-5572
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23Qureshi M H,Yeung J C,Wu S C,Wong S L.J Biol Chem,2001,49:46422-46428
2 Ringler P,Schulz G E.Science,2003,5642:106-109
3 Padilla J E,Colovos C,Yeates T O.Proc Natl Acad Sci USA,2001,5:2217-2221
4 Lai Y T,King N P,Yeates T O.Trends Cell Biol,2012,12:653-661
5 Bai Y,Luo Q,Liu J.Chem Soc Rev,2016,10:2756-2767
6 Lin Mingchang(林铭昌),Chen Guosong(陈国颂).Acta Polymerica Sinica(高分子学报),2017,(7):1114-1120
7 Meng Jianqiang(孟建强),Dong Yongquan(董永全),Li Zichen(李子臣).Acta Polymerica Sinica(高分子学报),2010,(5):550-555
8 Oohora K,Burazerovic S,Onoda A,Wilson Y M,Ward T R,Hayashi T.Angew Chem Int Ed,2012,16:3818-3821
9 Kostiainen M A,Kasyutich O,Cornelissen J J,Nolte R J.Nat Chem,2010,5:394-399
10 Kostiainen M A,Hiekkataipale P,Laiho A,Lemieux V,Seitsonen J,Ruokolainen J,Ceci P.Nat Nanotechnol,2013,1:52-56
11 Miao L,Han J,Zhang H,Zhao L,Si C,Zhang X,Hou C,Luo Q,Xu J,Liu J.ACS Nano,2014,4:3743-3751
12Salgado E N,Radford R J,Tezcan F A.Acc Chem Res,2010,5:661-672
13Brodin J D,Ambroggio X I,Tang C,Parent K N,Baker T S,Tezcan F A.Nat Chem,2012,5:375-382
14Biswas S,Kinbara K,Oya N,Ishii N,Taguchi H,Aida T.J Am Chem Soc,2009,22:7556-7557
15Bai Y,Luo Q,Zhang W,Miao L,Xu J,Li H,Liu J.J Am Chem Soc,2013,30:10966-10969
16Sakai F,Yang G,Weiss M S,Liu Y,Chen G,Jiang M.Nat Commun,2014:4634
17Yang G,Zhang X,Kochovski Z,Zhang Y F,Dai B,Sakai F J,Jiang L,Lu Y,Ballauff M,Li X M,Liu C,Chen G S,Jiang M.J Am Chem Soc,2016,6:1932-1937
18Yang G,Ding H M,Kochovski Z,Hu R T,Lu Y,Ma Y Q,Chen G S,Jiang M.Angew Chem Int Ed,2017,36:10691-10695
19Yang G,Hu R T,Ding H M,Kochovski Z,Mei S L,Lu Y,Ma Y Q,Chen G S,Jiang M.Mat Chem Front,2018,9:1642-1646
20Qi W J,Zhang Y F,Kochovski Z,Wang J,Lu Y,Chen G S,Jiang M.Nano Res,2018,10:5566-5572
21Xu M,Liu L,Yan Q.Angew Chem Int Ed,2018,18:5029-5032
22O'Sullivan V J,Barrette-Ng I,Hommema E,Hermanson G T,Schofield M,Wu S C,Honetschlaeger C,Ng K K S,Wong S L.Plos One,2012,4:e35203
23Qureshi M H,Yeung J C,Wu S C,Wong S L.J Biol Chem,2001,49:46422-46428