A green fluorescent protein screen for identification of well-expressed membrane proteins from a cohort of extremophilic organisms.
Green fluorescent protein (GFP) fusion proteins provide a potentially facile tool for identification of well expressed, properly behaved membrane proteins for biochemical and structural study. Here, we present a GFP-expression survey of >300 membrane proteins from 18 bacterial and archaeal extremophiles, organisms expected to be rich sources of membrane proteins having robust biophysical properties. We find that GFP-fusion fluorescence intensity is an excellent indicator of over-expression potential. By employing a follow-up optimization protocol using a suite of non-GFP constructs and different expression temperatures, we obtain 0.5-15 mg L(-1) expression levels for 90% of the tested candidate proteins that pass the GFP screen. Evaluation of the results suggests that certain organisms may serve as better sources of well-expressed membrane proteins than others, that the degree to which codon usage matches the expression host is uncorrelated with success rate, and that the combination of GFP screening and expression optimization is essential for producing biochemically tractable quantities of material.
Hammon
Justus
J
Cardiovascular Research Institute, Department of Biochemistry and Biophysics, Department of Cellular and Molecular Pharmacology, California Institute for Quantitative Biosciences, University of California, San Francisco, California 94158-2330, USA.
Palanivelu
Dinesh V
DV
Chen
Joy
J
Patel
Chintan
C
Minor
Daniel L
DL
D001419:Bacteria / Q000235:genetics / Q000378:metabolism*; D002850:Chromatography, Gel; D003001:Cloning, Molecular / Q000379:methods*; D003062:Codon; D003902:Detergents; D004926:Escherichia coli / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D040901:Proteomics; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism
D003062:Codon; D003902:Detergents; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins