To address the severe lack of three-dimensional structural information for eukaryotic transmembrane proteins (TMPs), Dr. Mark Dumont and his colleagues at the University of Rochester are developing protocols for the expression and purification of TMPs in the yeast Saccharomyces cerevisiae. Initial efforts have been focused on a set of endogenous yeast TMPs that are the highest expressing reading frames in a previously constructed genomic collection of Saccharomyces cerevisiae expression clones, targeting reading frames for which there are established biochemical assays for determining whether the protein is maintained in a native state in the presence of detergents. Genes encoding the target TMPs are transferred via ligation-independent cloning procedures to a series of vectors that allow galactose-controlled expression of reading frames fused to C-terminal His6, His10, and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease. Several TMP targets expressed from these vectors have been purified via affinity chromatography and gel filtration chromatography at levels and purities sufficient for ongoing crystallization trials. Single chain antibodies (scFvs) recognizing several targets have been developed as aids to crystallization and purification. Current efforts are focused on overcoming bottlenecks in protein production and crystallization by introducing the following improvements in the production pipeline: 1) improving overall levels of cellular expression of TMPs by altering protocols for cell growth and induction of expression; 2) increasing efficiency of cell lysis; 3) increasing the efficiency of detergent solubilization; 4) increasing the efficiency of proteolytic removal of affinity tags; 5) developing protocols for obtaining highly concentrated protein preparations that do not also contain high detergent concentrations; 6) optimizing the amount of residual lipid purifying with the TMP; 7) reducing the number of steps required for effective purification; 8) testing the use of additives such as lipids and enzyme inhibitors to stabilize purified proteins.

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