SUMO Fusions to Enhance Expression of Membrane Proteins

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已经取得了相当有限的成功。在超过30，000种可溶性蛋白质中，只有124种膜蛋白的独特结构被解析。该“瓶颈”可分为至少三个主要部分：i）难以定量和定性地表达足够的量，ii）在纯化之前需要去污剂依赖性溶解程序，这可显著降低特异性结合或催化活性，以及iii）在纯化期间对降解的高敏感性，导致短保质期的不稳定材料。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巴斯德毕赤酵母（P. pastoris）中各种膜蛋白的表达和纯化。该技术的关键特征是：1）SUMO的C-末端与膜蛋白的N-末端融合; 2）通过SUMO的固有伴侣效应增强膜蛋白的定性和定量表达; 3）利用SUMO蛋白酶的稳健性质产生期望的N-末端。使用大肠杆菌作为表达宿主实现了该项目的I期目标;然而，检测到的具有三个以上跨膜螺旋的SUMO融合真核细胞膜蛋白（如GPCR）的表达水平不是很令人满意。酵母巴斯德毕赤酵母已被证明通常更适合于有效功能性生产难以表达的真核蛋白（如糖蛋白和膜蛋白）的代谢需求。SUMO融合技术的优点将与毕赤酵母表达系统的优点相结合，以定量和定性地提高膜蛋白的表达。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，将通过证明与未融合的膜蛋白衍生物相比，SUMO融合的膜蛋白衍生物的功能性生产至少增强5倍来验证该系统。最后，将鉴定促进提高巴斯德毕赤酵母中膜蛋白质的质量和数量的最佳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增强的膜蛋白表达系统上市。这种缺陷的主要原因之一是缺乏对这类蛋白质有效的表达和纯化方法。LifeSensors已经开发出一种很有前途的方法来解决这个问题（称为SUMO融合），并在第一阶段证明了膜蛋白可以使用它来表达。然而，对于最困难的膜蛋白组，称为GPCR，细菌系统是不令人满意的。在第二阶段，提出了在酵母系统（巴斯德毕赤酵母）中应用SUMO技术来表达一组GPCR，并将优化的系统开发为通用于表达GPCR的试剂盒。

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.