Purification of transmembrane proteins from Saccharomyces cerevisiae for X-ray crystallography.
To enhance the quantity and quality of eukaryotic transmembrane proteins (TMPs) available for structure determination by X-ray crystallography, we have optimized protocols for purification of TMPs expressed in the yeast Saccharomyces cerevisiae. We focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays. Genes encoding the target TMPs are transferred via ligation-independent cloning to a series of vectors that allow 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. 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. Initial purifications were based on expression of the genes under control of a galactose-inducible promoter, but higher cell densities and improved expression have been obtained through use of the yeast ADH2 promoter. Wide variations have been observed in the behavior of different TMP targets during purification; some can be readily purified, while others do not bind efficiently to affinity matrices, are not efficiently cleaved from the matrices, or remain tightly associated with the matrices even after cleavage of the affinity tags. The size, oligomeric state, and composition of purified protein-detergent complexes purified under different conditions were analyzed using a colorimetric assay of detergent concentrations and by analytical size-exclusion chromatography using static light scattering, refractive index, and UV absorption detection to monitor the elution profiles. Effective procedures were developed for obtaining high concentrations of purified TMPs without excessively concentrating detergents.
Clark
Kathleen M
KM
Department of Pediatrics, University of Rochester School of Medicine and Dentistry, Rochester, NY 14642, USA.
Fedoriw
Nadia
N
Robinson
Katrina
K
Connelly
Sara M
SM
Randles
Joan
J
Malkowski
Michael G
MG
DeTitta
George T
GT
Dumont
Mark E
ME
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D018360:Crystallography, X-Ray / Q000379:methods*; D056890:Eukaryota / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005822:Genetic Vectors; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D014965:X-Rays
D008565:Membrane Proteins; D029701:Saccharomyces cerevisiae Proteins