SUMO Fusions to Enhance Expression of Membrane Proteins

SUMO 融合增强膜蛋白的表达

• 批准号: 7555633
• 负责人: Tauseef R. Butt
• 金额: $ 58.98万
• 依托单位: LIFESENSORS, INC.
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2010-01-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7555633
• 关键词: Binding; Cellular Membrane; Cleaved cell; Cloning; Commit; Databases; Detergents; Escherichia coli; Figs - dietary; Glycoproteins; Goals; Hand; Life; Marketing; Membrane Proteins; Metabolic; Methods; Molecular; Molecular Chaperones; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Pichia; Procedures; Production; Progress Reports; Property; Proteins; Resolution; Role; Structure; System; Technology; Ubiquitin; Work; Yeasts; commercialization; improved; meetings; novel; protein expression; success; vector

DESCRIPTION (provided by applicant): Rather modest success has yet been achieved in expressing and purifying membrane proteins as a prerequisite to obtain high resolution structures. Only 124 unique structures for membrane proteins have been resolved out of > 30,000 soluble proteins. This "bottleneck" can be divided into at least three major parts: i) the difficulty to express quantitatively and qualitatively sufficient amounts, ii) the requirement for detergent-dependent solubilization procedures prior to purification, which can substantially reduce specific binding or catalytic activities and iii) the high sensitivity to degradation during purification resulting in instable material of short shelf-life. LifeSensors has developed a novel SUMO fusion technology that will be improved and applied to enhance the expression and purification of a wide variety of membrane proteins in Pichia pastoris (P. pastoris). The key features of this technology are: 1) the fusion of the C-terminus of SUMO to the N-terminus of a membrane protein; 2) the enhanced qualitative and quantitative membrane protein expression by the inherent chaperoning effect of SUMO; 3) the exploitation of the robust properties of SUMO protease to generate a desired N-terminus. Phase I goals for this project were met using E.coli as the expression host; however, the detected expression levels of SUMO-fused eukaryotic membrane proteins with more than three transmembrane helices, such as GPCRs, were not very satisfactory. The yeast P. pastoris has been shown to be generally more compatible with the metabolic needs for efficient functional production of difficult-to-express eukaryotic proteins, such as glycoproteins and membrane proteins. The benefits of the SUMO fusion technology will be combined with that of the P. pastoris expression system to quantitatively and qualitatively enhance the expression of membrane proteins. First, the utility of the SUMO fusion technology will be established by cloning and expressing 25 structurally and/or therapeutically important membrane proteins in P. pastoris; next, the system will be validated by the demonstration of an at least 5-fold enhancement in the functional production of SUMO-fused compared to unfused membrane protein derivatives. Finally, the best SUMO-tag facilitating enhanced quality and quantity of membrane proteins in P. pastoris will be identified. Appropriate P. pastoris vectors and strains will be marketed in a kit format as a novel SUMO-enhanced membrane protein expression system.Membrane proteins have been extremely difficult to produce in quantities sufficient for structural studies. One of the principal reasons for this deficit is that lack of a method of expression and purification that works well for this class of protein. LifeSensors has developed a promising approach to this problem (called SUMO fusion) and has demonstrated in Phase I that membrane proteins can be expressed using it. For the most difficult groups of membrane proteins, however, called GPCRs, the bacterial system is unsatisfactory. In Phase II it is proposed to apply the SUMO technology in a yeast system (Pichia pastoris) to express a group of GPCRs and to develop the optimized system as a kit for general use in expressing GPCRs.

描述（由申请人提供）：作为获得高分辨率结构的先决条件，在表达和纯化膜蛋白方面尚未取得相当程度的成功。在超过 30,000 种可溶性蛋白质中，仅解析出 124 种独特的膜蛋白结构。该“瓶颈”可分为至少三个主要部分：i) 难以定量和定性表达足够的量，ii) 纯化前需要依赖去污剂的溶解程序，这会大大降低特异性结合或催化活性， iii) 纯化过程中对降解的高度敏感性导致材料不稳定且保质期短。 LifeSensors 开发了一种新型 SUMO 融合技术，该技术将得到改进并应用于增强毕赤酵母 (P. Pastoris) 中多种膜蛋白的表达和纯化。该技术的主要特点是：1）SUMO的C端与膜蛋白的N端融合； 2) SUMO固有的陪伴效应增强膜蛋白的定性和定量表达； 3) 利用 SUMO 蛋白酶的强大特性来生成所需的 N 末端。使用大肠杆菌作为表达宿主实现了该项目的第一阶段目标；然而，检测到的具有三个以上跨膜螺旋的SUMO融合真核膜蛋白（例如GPCR）的表达水平并不十分令人满意。毕赤酵母已被证明通常更能满足难以表达的真核蛋白（如糖蛋白和膜蛋白）的有效功能性生产的代谢需求。 SUMO融合技术的优势将与毕赤酵母表达系统的优势相结合，定量和定性地增强膜蛋白的表达。首先，将通过在巴斯德毕赤酵母中克隆和表达 25 种结构上和/或治疗上重要的膜蛋白来建立 SUMO 融合技术的实用性；接下来，该系统将通过证明与未融合的膜蛋白衍生物相比，SUMO 融合的功能生产至少提高 5 倍来验证。最后，将确定促进毕赤酵母膜蛋白质量和数量提高的最佳 SUMO 标签。合适的巴斯德毕赤酵母载体和菌株将以试剂盒形式作为新型 SUMO 增强膜蛋白表达系统进行销售。膜蛋白的生产量极其难以满足结构研究的需要。造成这种缺陷的主要原因之一是缺乏适合此类蛋白质的表达和纯化方法。 LifeSensors 开发了一种有前途的方法来解决这个问题（称为 SUMO 融合），并在第一阶段证明可以使用它来表达膜蛋白。然而，对于最困难的膜蛋白组（称为 GPCR），细菌系统却不能令人满意。在第二阶段，建议在酵母系统（毕赤酵母）中应用SUMO技术来表达一组GPCR，并开发优化的系统作为表达GPCR的通用试剂盒。