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取得了相当小的成功。在30,000种可溶性蛋白质中，只有124种独特的膜蛋白质结构被解析出来。这一“瓶颈”至少可分为三个主要部分：i)难以定量和定性地表达足够的量，ii)在提纯之前需要依赖洗涤剂的增溶程序，这可能会大大降低特定的结合或催化活性，以及iii)提纯过程中对降解的高度敏感性，导致材料不稳定，货架期短。Lifesensors开发了一种新的相扑融合技术，该融合技术将被改进和应用于提高多种膜蛋白在巴斯德毕赤酵母中的表达和纯化。这项技术的主要特点是：1)将相扑的C-端融合到膜蛋白的N-端；2)通过相扑固有的伴侣效应增强膜蛋白的定性和定量表达；3)利用相扑酶的强健特性来产生所需的N-端。该项目的第一阶段目标是使用大肠杆菌作为表达宿主；然而，检测到的具有三个以上跨膜螺旋的SUMO融合真核膜蛋白的表达水平并不是很令人满意，例如GPCRs。酵母菌已被证明与高效功能性生产难于表达的真核蛋白的代谢需求更相容，如糖蛋白和膜蛋白。相扑融合技术的优点将与巴斯德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种结构和/或治疗上重要的膜蛋白来确定相扑融合技术的实用性；接下来，将通过证明与未融合的膜蛋白衍生物相比，相扑融合蛋白的功能生产至少提高5倍来验证该系统。最后，将确定有助于提高巴斯德毕赤酵母膜蛋白质量和数量的最佳相扑标签。合适的巴斯德毕赤酵母载体和菌株将以试剂盒的形式作为一种新的相扑增强型膜蛋白表达系统上市。膜蛋白的生产已经非常困难，很难产生足够的数量来进行结构研究。造成这一缺陷的主要原因之一是缺乏一种对这类蛋白质起良好作用的表达和纯化方法。Lifesensors已经开发出一种很有前途的方法来解决这个问题(称为相扑融合)，并在第一阶段证明了可以使用它来表达膜蛋白。然而，对于最困难的膜蛋白质组，称为GPCRs，细菌系统并不令人满意。在第二阶段，建议将相扑技术应用于酵母系统(巴斯德毕赤酵母)中表达一组GPCRs，并开发优化后的系统作为表达GPCRs的通用试剂盒。

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.