Eukaryotic Membrane Protein Folding in E. coli

描述(申请人提供)：正确折叠的真核膜蛋白的异源表达是目前对其结构/功能研究的主要限制。缺乏合适的表达技术最严重地限制了通过结晶学进行结构测定，这只可能是那些从自然来源获得的高丰度的真核膜蛋白。大肠杆菌是应用最广泛的原核膜蛋白异源表达载体，但普遍发现真核蛋白在大肠杆菌膜上不能正确折叠。这一应用旨在开发一种在大肠杆菌中正确折叠的真核膜蛋白生产的新技术。我们将利用最近的发现，即大肠杆菌细胞中的脂类通过与折叠中间体以类似于蛋白质分子伴侣的方式与特定的相互作用来帮助膜蛋白折叠。我们的方法是对脂类类型和组成接近于要表达的目标蛋白来自的真核生物的菌株进行基因工程。因此，“外来的”膜蛋白将被表达并组装成更能代表其天然脂质环境的膜。首先，我们将构建模拟真核藻衣藻质膜脂类的菌株，并表达编码在该生物体基因组中的微生物视紫红质载脂蛋白。视紫红质脱辅基蛋白形成一个膜嵌入的视网膜结合袋，由它们的七个跨膜螺旋中的每一个残基组成，视网膜络合作用产生一种具有可见吸收和光化学活性的色素。微生物视紫红质对本研究的独特价值在于，通过激光闪光动力学光谱测量其在高度分散的细胞材料(如全细胞悬浮液)的大背景下的光化学反应循环，能够快速和定量地检测它们的适当折叠。其次，我们将构建模拟哺乳动物血浆和内质网膜脂质的菌株。目标蛋白将是人视杆和视锥色素、细胞色素P450亚型和12-螺旋人腺苷酸环化酶亚型，每一种都有有效的折叠方法。如果脂质伴侣功能的开发成功，将在真核生物膜蛋白急需的表达技术上实现突破。应PA-03-100高影响/高风险研究和PA-02-060膜蛋白结构生物学的要求提交。

DESCRIPTION (provided by applicant): Heterologous expression of correctly folded eukaryotic membrane proteins is currently the primary limitation to their structure/function study. The lack of suitable expression techniques most severely limits structural determination by crystallography, which has been possible only for those eukaryotic membrane proteins that are available in high abundance from natural sources. Escherichia coli has been the most extensively used heterologous expression vehicle for prokaryotic membrane proteins, but a general finding is that eukaryotic proteins improperly fold in the E. coli membrane. This application is to develop a novel technology for correctly folded eukaryotic membrane protein production in E. coli. We will exploit the recent discovery that lipids in E. coli cells assist membrane protein folding by specific interactions with folding intermediates in a manner analogous to protein molecular chaperones. Our approach is to genetically engineer strains with lipid types and compositions approximating that of the eukaryotic organism from which the target protein to be expressed derives. The "foreign" membrane proteins will therefore be expressed and assembled into membranes that more closely represent their native lipid environment. First, we will construct strains mimicking the lipids of the eukaryotic alga Chlamydomonas reinhardtii plasma membrane, and express microbial rhodopsin apoproteins encoded in the genome of that organism. Rhodopsin apoproteins form a membrane-embedded retinal-binding pocket comprised of residues from each of their seven transmembrane helices, and retinal-complexation produces a pigment with visible absorption and characteristic photochemical activity. The unique value of the microbial rhodopsins to this study is their ability to be assayed rapidly and quantitatively for proper folding by laser flash kinetic spectroscopic measurement of their photochemical reaction cycles in a large background of highly scattering cell material, such as whole cell suspensions. Second, we will construct strains mimicking the lipids of mammalian plasma and endoplasmic reticulum membranes. Target proteins will be human rod and cone visual pigments, cytochrome P450 isoforms and 12-helix human adenylyl cyclase isoforms, each having efficient assays for their proper folding. If exploiting the lipid chaperone function is successful, a breakthrough in the much-needed expression technology for eukaryotic membrane proteins will be achieved. Submitted in response to PA-03-100 High Impact/High Risk Research and PA-02-060 Structural Biology of Membrane Proteins.