Research and Development for Purification of Membrane Proteins

膜蛋白的结晶需要艰苦的工作，主要是因为很难制备稳定、高纯度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，通过诱导生物膜中的自结合和随后的膜泡化来选择性分离膜蛋白。细菌视紫红质是一种发现于盐生细菌中的跨膜蛋白，在生理条件下形成二维晶体(称为紫色膜)。当紫膜在高温下与少量洗涤剂(辛基硫代葡萄糖苷)在高浓度沉淀剂存在下孵育时，产生了大小均匀的球形细菌视紫红质小泡(多面体组装)。低温下多面体组装的稳定性降低。通过在低温下促进多面体组装的融合过程，我们获得了一种新的…更多的三维绕射X射线的绕射超过3.0埃。该晶体属于P622空间群，晶胞尺寸a=b=104.7*，c=114.1*，由层叠的平面膜组成，每个细菌视紫红质三聚体排列在蜂窝状晶格上。晶体含有天然脂类(每个BR 5个磷脂)，其中一个磷脂牢固地结合在三聚单元中相邻单体之间的缝隙上。豌豆捕光的叶绿素-蛋白质复合体在结晶条件下形成直径为27 nm的多面体结构。它被组装成一个八面体晶体，属于P2_13空间群，晶胞尺寸为a=b=c=390*。另一种牛视紫红质蛋白的纯化方法是建立另一种具有7个跨膜α螺旋的蛋白质。光感受器细胞的盘膜用密度梯度离心法纯化，纯化后的膜在高浓度二价阳离子存在下用去污剂(烷基葡萄糖苷)处理。对混合物进行单步离心法，即可得到高纯度的视紫红质样品。利用纯化后的样品，采用气相扩散悬滴法制备了视紫红质的3D晶体。较少

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