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. ? ? ?
