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Research and Development for Purification of Membrane Proteins

膜蛋白纯化的研究与开发

基本信息

批准号：
07458176
负责人：
KOUYAMA Tsutomu
金额：
$ 4.54万
依托单位：
Nagoya University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
1995
资助国家：
日本
起止时间：
1995 至 1996
项目状态：
已结题

项目摘要

Crystallization of membrane proteins requires hard work, primarily because of difficulty in preparation of a stable, highly-purified and concentrated protein sample. In order to simplify the purification step of membrane proteins, we have tried to develop a novel procedure for selective isolation of a membrane protein by inducing self-association in a biological membrane and subsequent membrane vesicularization.Bacteriorhodopsin, a transmembrane protein found in halobacterium halobium, forms a two-dimensional crystal (called purple mebrane) under physiological condition. When purple membrane was incubated at high temperature with a small amount of detergent (octylthioglucoside) in the presence of a high concentration of precipitant, uniformly-sized spherical vesicles (polyhedral assembly) of bacteriorhodopsin was produced. The stability of the polyhedral assembly decreased at low temperature. By promoting fusion processes of the polyhedral assembly at low temperature, we obtained a new … More three-dimensional that diffracts X-ray diffract beyond 3.0 angstrom. This crystal belongs to the space group P622 with cell dimensions of a=b=104.7*, and c=114.1*, and it is shown to be made up of stacked planar membranes, in each of bacteriorhodopin trimers are arranged on a honey-comb lattice. The crystal contains native lipids (5 phospholipids per bR) and one phospholipid is bound firmly to a crevice between adjacent monomers in the trimeric unit. This lipid is suggested to act as a glue for formation of the trimeric structure.Light-harvesting chlorophyll-protein complex from pea is shown to form a polyhedral structure with a diameter of 27 nm under crystallization condition. It is assembled into an octahedral crystal that belongs to the space group of P2_13 with cell dimensions of a=b=c=390*.Another purification procedure was developed for bovine rhodopsin, a protein with a 7-fold transmembrane alpha-helices. The disk membrane of the photoreceptor cell was purified by a density-gradient centrifugation and the purified membrane was treated with detergent (alkylglucoside) in the presence of a high concentration of divalent cation. A single step of centrifugation of the mixture yielded a highly-purified sample of rhodopsin. Using this purified sample, we obtained 3D crystals of rhodopsin by the vapor diffusion hanging drop method. Less

膜蛋白的结晶需要艰苦的工作，主要是因为难以制备稳定、高度纯化和浓缩的蛋白质样品。为了简化膜蛋白的纯化步骤，我们试图开发一种新的方法，通过诱导生物膜的自结合和随后的膜泡化来选择性分离膜蛋白。细菌视紫红质是在盐细菌中发现的一种跨膜蛋白，在生理条件下形成二维晶体（称为紫膜）。当紫色膜与少量洗涤剂（辛基硫糖苷）在高浓度沉淀剂的存在下在高温下孵育时，产生大小均匀的细菌视紫红质球形囊泡（多面体组合）。在低温下，多面体组装体的稳定性下降。通过促进多面体组件在低温下的融合过程，我们获得了一种新的更三维的x射线衍射超过3.0埃。该晶体属于P622空间群，细胞尺寸为a=b=104.7*, c=114.1*，由堆叠的平面膜组成，在每个细菌视多巴胺三聚体中排列成蜂窝状晶格。该晶体含有天然脂质（每个溴含5个磷脂），其中一个磷脂与三聚体单元中相邻单体之间的缝隙紧密结合。这种脂质被认为是形成三聚体结构的粘合剂。在结晶条件下，豌豆光能叶绿素-蛋白复合物形成了直径为27 nm的多面体结构。它被组装成一个八面体晶体，属于P2_13的空间群，胞元尺寸为a=b=c=390*。另一种纯化方法是开发牛视紫红质，一种具有7倍跨膜α -螺旋的蛋白质。光感受器的圆盘膜通过密度梯度离心纯化，纯化后的膜在高浓度二价阳离子存在下用洗涤剂（烷基糖苷）处理。对混合物进行一步离心，就得到了高纯度的视紫红质样品。利用该纯化样品，采用气相扩散挂滴法获得了三维视紫红质晶体。少