载G蛋白偶联受体APJ的磷脂纳米盘的研制
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摘要
以G蛋白偶联受体APJ(即血管紧张素受体AT1相关的受体蛋白)、膜骨架蛋白(MSP)和磷脂为原料制备载APJ磷脂纳米盘。考察了不同投料比(APJ∶MSP分别为1∶2、1∶4和1∶8)和加入Bio-BEADs树脂(聚苯乙烯二乙烯基苯树脂)后4℃孵育时间(2、4和6 h)对APJ组装入磷脂纳米盘的效率和单体比例的影响。结果表明,APJ∶MSP采用1∶4,加入Bio-BEADs后4℃孵育2 h,所得的APJ磷脂纳米盘直径约10 nm,高度约5 nm。组装入该磷脂纳米盘的APJ仍具有与其拮抗剂ML221结合的能力,提示磷脂纳米盘能保留APJ的生物活性。
The nanodiscs loaded with G protein coupled receptor APJ, the putative receptor protein related to the angiotensin receptor AT1, were prepared with membrane scaffold protein(MSP) and phospholipid. The effects of molar ratio of APJ to MSP(1∶2, 1∶4 and 1∶8) and incubation time(2, 4 and 6 h) after adding Bio-BEADs resin [poly(styrene-divinylbenzene) resin] at 4 ℃ on assembly efficiency and the proportion of monomers were investigated. The optimal conditions were as follows: the molar ratio of APJ to MSP was 1∶4, and the incubation time was 2 h. The average diameter and thickness of the AJP-loaded nanodiscs were approximately 10 nm and 5 nm, respectively. The AJP reconstituted into the nanodiscs remained the ability to bind its antagonist, ML221, which indicated that the nanodiscs could maintain the biological activity of AJP.
The nanodiscs loaded with G protein coupled receptor APJ, the putative receptor protein related to the angiotensin receptor AT1, were prepared with membrane scaffold protein(MSP) and phospholipid. The effects of molar ratio of APJ to MSP(1∶2, 1∶4 and 1∶8) and incubation time(2, 4 and 6 h) after adding Bio-BEADs resin [poly(styrene-divinylbenzene) resin] at 4 ℃ on assembly efficiency and the proportion of monomers were investigated. The optimal conditions were as follows: the molar ratio of APJ to MSP was 1∶4, and the incubation time was 2 h. The average diameter and thickness of the AJP-loaded nanodiscs were approximately 10 nm and 5 nm, respectively. The AJP reconstituted into the nanodiscs remained the ability to bind its antagonist, ML221, which indicated that the nanodiscs could maintain the biological activity of AJP.
引文
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[2]Marchese A,George SR,Kolakowski Jr.LF,et al.Novel GPCRs and their endogenous ligands:expanding the boundaries of physiology and pharmacology[J].Trends Pharmacol Sci,1999,20(9):370—375.
[3]Milligan G.Strategies to identify ligands for orphan G-protein-coupled receptors[J].Biochem Soc Transact,2002,30(4):789—793.
[4]Hu Y,Li Y,Zhang X,et al.Solution structures and backbone dynamics of a flavodoxin Mio C from Escherichia coli in both Apo-and Holo-forms:implications for cofactor binding and electron transfer[J].J Biol Chem,2006,281(46):35454—35466.
[5]Benovic JL,Shorr RG,Caron MG,et al.The mammalian beta 2-adrenergic receptor:purification and characterization[J].Biochemistry,1984,23(20):4510—4518.
[6]Kurstjens NP,Fr?hlich M,Dees C,et al.Binding of alphaand beta gamma-subunits of Go to beta 1-adrenoceptor in sealed unilamellar lipid vesicles[J].Eur J Biochem,1991,197(1):167—176.
[7]Jean-Fran?ois M,Ali S,Stéphane A,et al.Quantitative assessment of beta 1-and beta 2-adrenergic receptor homoand heterodimerization by bioluminescence resonance energy transfer[J].J Biol Chem,2002,277(47):44925—44931.
[8]Seddon AM,Curnow P,and Booth PJ.Membrane proteins,lipids and detergents:not just a soap opera[J].Biochim Biophys Acta,2004,1666(1-2):105—117.
[9]O'Dowd BF,Heiber M,Chan A,et al.A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11[J].Gene,1993,136(1-2):355—360.
[10]Tatemoto K,Hosoya M,Habata Y,et al.Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor[J].Biochem Biophys Res Commun,1998,251(2):471—476.
[11]Lee DK,Saldivia VR,Nguyen T,et al.Modification of the terminal residue of apelin-13 antagonizes its hypotensive action[J].Endocrinology,2005,146(1):231—236.
[12]Lee DK,George SR,O'Dowd BF.Unravelling the roles of the apelin system:prospective therapeutic applications in heart failure and obesity[J].Trends Pharmacol Sci,2006,27(4):190—194.
[13]Page RC,Moore JD,Nguyen HB,et al.Comprehensive evaluation of solution nuclear magnetic resonance spectroscopy sample preparation for helical integral membrane proteins[J].J Struct Funct Genom,2006,7(1):51—64.