Development of Methodology for Medium Throughput Cloning,
Expression Analysis, and Purification of Protein:Protein Complexes from Yeast

To expedite cloning and expression analysis for protein:protein complexes, Drs. Eric Phizicky and Elizabeth Grayhack have developed a suite of vectors for use in yeast strains with commonly found genetic markers.  Each vector features high-throughput, ligation-independent cloning and is designed to express genes under control of the strong, regulatable galactose promoter (PGAL), thereby permitting the cloning of genes that are otherwise toxic as well as high level expression of the corresponding proteins.
 
An initial set of vectors express individual ORFs (Open Reading Frames) with a tri-partite affinity tag on their C-terminus.  This tag is comprised of a protease 3C site, an HA epitope, a His6 tag for immobilized metal ion affinity chromatography (IMAC) purification, and the ZZ domain of protein A for purification on IgG Sepharose. Binary protein complexes are made using two otherwise identical vectors (one with a LEU2 marker and one with a URA3 marker), each expressing different genes of the complex.
 
More recent vectors allow for expression of two proteins simultaneously, using a bi-directional PGAL promoter, with different tags on each ORF of a pair. Three such sets of vectors, containing either a LEU2 marker or a URA3 marker, have been constructed for expression of an ORF-3C-HA-His6-ZZ fusion together with an untagged ORF, a His6-tagged ORF, or a His10-tagged ORF.  These vectors have three important uses.  First, vectors with different tags can be used to unequivocally demonstrate the existence of a complex, by co-purification of ORFs using only one tag on one ORF.   Second, use of a vector with a non-cleavable His6 or His10-tag on one ORF, and a cleavable ZZ tag on the other ORF, allows for efficient purification using two affinity steps, each directed at one ORF of the complex. Third, use of pairs of vectors, each with a bi-directional PGAL promoter, allows for purification of complexes comprised of as many as four protein subunits. Expression with these vectors can be as high as 15 mg/l in the best cases.
           
Several medium-throughput methods for analysis of expression and purification of protein complexes have also developed.  These employ use of small cultures, rapid analysis of expression using stick-and-strip binding to either IgG Sepharose or IMAC followed by analysis of proteins on SDS-PAGE, and rapid analysis of authentic complexes employing differentially tagged proteins. 
           
Major ongoing research efforts in the lab are focused on development of methods to further enhance expression of proteins in yeast, on further documentation of the utility of yeast for expression of exogenous proteins and complexes of proteins, and on development of medium-throughput methods for detection of complexes suitable for purification and subsequent structural analysis.

Reference
           
Phizicky, E. M. and Grayhack, E. J. (2006). Proteome-scale analysis of biochemical activity.  Crit. Rev. Biochem. Mol. Biol. 41:315-327. [PubMed]