Selecting optimum eukaryotic integral membrane proteins for structure determination by rapid expression and solubilization screening.
A medium-throughput approach is used to rapidly identify membrane proteins from a eukaryotic organism that are most amenable to expression in amounts and quality adequate to support structure determination. The goal was to expand knowledge of new membrane protein structures based on proteome-wide coverage. In the first phase, membrane proteins from the budding yeast Saccharomyces cerevisiae were selected for homologous expression in S. cerevisiae, a system that can be adapted to expression of membrane proteins from other eukaryotes. We performed medium-scale expression and solubilization tests on 351 rationally selected membrane proteins from S. cerevisiae. These targets are inclusive of all annotated and unannotated membrane protein families within the organism's membrane proteome. Two hundred seventy-two targets were expressed, and of these, 234 solubilized in the detergent n-dodecyl-beta-D-maltopyranoside. Furthermore, we report the identity of a subset of targets that were purified to homogeneity to facilitate structure determinations. The extensibility of this approach is demonstrated with the expression of 10 human integral membrane proteins from the solute carrier superfamily. This discovery-oriented pipeline provides an efficient way to select proteins from particular membrane protein classes, families, or organisms that may be more suited to structure analysis than others.
Li
Min
M
Membrane Protein Expression Center, University of California at San Francisco, San Francisco, CA 94158-2517, USA.
Hays
Franklin A
FA
Roe-Zurz
Zygy
Z
Vuong
Linda
L
Kelly
Libusha
L
Ho
Chi-Min
CM
Robbins
Renée M
RM
Pieper
Ursula
U
O'Connell
Joseph D
JD
Miercke
Larry J W
LJ
Giacomini
Kathleen M
KM
Sali
Andrej
A
Stroud
Robert M
RM
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D006801:Humans; D008565:Membrane Proteins / Q000737:chemistry* / Q000302:isolation & purification; D010957:Plasmids; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000302:isolation & purification; D012995:Solubility
D008565:Membrane Proteins; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins