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To address the severe lack of three-dimensional structural information for eukaryotic transmembrane proteins (TMPs), Dr. Mark Dumont is developing protocols for the expression and purification of TMPs in baker’s yeast, Saccharomyces cerevisiae. Working with colleagues at the University of Rochester and Yale University, the Dumont laboratory began by creating a library of nearly 6,000 plasmids, each containing a tagged, sequence-verified clone of a different yeast gene . The expression clones included both soluble proteins and TMPs, and the expression of each gene was evaluated by immunoblotting. The library was created using C-terminal tagging, which was believed would be minimally detrimental to TMP function, and expression testing was conducted under conditions that would allow detection of TMPs. Unexpectedly, the rate of expression that was actually observed for predicted TMPs (88%) (Fig. 1) was only slightly less than that for the overall population of ORFs (95%). Indeed, these clones have proved to be extremely useful in providing information about the extent to which particular genes, especially those encoding TMPs, can be overproduced .
Initially, target selection from the plasmid library has been focused on the set of highest expressing reading frames that were predicted to contain at least two transmembrane segments. Targets have also been further selected based on the availability of an established biochemical assay that can be used to determine whether the protein is maintained in a native state in the presence of detergent. Genes encoding a target TMP are transferred via LIC cloning to a series of vectors that were constructed so as to allow galactose-controlled expression of reading frames fused to C-terminal His10, and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease (Fig. 2). To date, several TMP targets have been purified via affinity and gel filtration chromatography at levels and purities sufficient for ongoing crystallization trials.
Successful purification of yeast TMPs for the MPSBC project has required the Dumont group to overcome a number of difficulties including dealing with endogenous yeast proteases, achieving efficient binding of solubilized yeast proteins to affinity matrices, obtaining efficient proteolytic removal of affinity tags, and developing procedures for concentrating detergent-solubilized proteins without unduly concentrating detergents. Current efforts are focused on producing target TMPs for crystallization and on overcoming the most significant bottlenecks in protein production and crystallization by: 1) improving overall levels of cellular expression of TMPs through optimization of protocols for cell culture and induction of expression; 2) decreasing protein losses during cell lysis and affinity purification; 3) increasing the yield of 3C protease cleavage; 4) reducing the number of steps required for effective purification; 5) optimizing the amount of residual lipid and detergent purifying with the TMP; and 6) using additives such as lipids and known ligands to stabilize purified proteins. [ Learn More ]
1. Gelperin DM, White MA, Wilkinson ML, Kon Y, Kung LA, Wise KJ, Lopez-Hoyo
N, Jiang L, Piccirillo S, Yu H, Gerstein M, Dumont ME, Phizicky EM, Snyder
M, Grayhack EJ. Biochemical and genetic analysis of the yeast
proteome with a movable ORF collection. Genes Dev. 2005 Dec