Cell-free expression of soluble and membrane proteins in an array device for drug screening.
Enzymes and membrane protein receptors represent almost three-quarters of all current drug targets. As a result, it would be beneficial to have a platform to produce them in a high-throughput format for drug screening. We have developed a miniaturized fluid array device for cell-free protein synthesis, and the device was exploited to produce both soluble and membrane proteins. Two membrane-associated proteins, bacteriorhodopsin and ApoA lipoprotein, were coexpressed in an expression medium in the presence of lipids. Simultaneous expression of ApoA lipoprotein enhanced the solubility of bacteriorhodopsin and would facilitate functional studies. In addition, the device was employed to produce two enzymes, luciferase and beta-lactamase, both of which were demonstrated to be compatible with enzyme inhibition assays. Beta-lactamase, a drug target associated with antibiotic resistance, was further used to show the capability of the device for drug screening. Beta-lactamase was synthesized in the 96 units of the device and then assayed by a range of concentrations of four mock drug compounds without harvesting and purification. The inhibitory effects of these compounds on beta-lactamase were measured in a parallel format, and the degree in their drug effectiveness agreed well with the data in the literature. This work demonstrated the feasibility of the use of the fluid array device and cell-free protein expression for drug screening, with advantages in less reagent consumption, shorter analysis time, and higher throughput.
Khnouf
Ruba
R
Department of Biomedical Engineering, University of Florida, P.O. Box 116131, Gainesville, Florida 32611, USA.
Olivero
Daniel
D
Jin
Shouguang
S
Coleman
Matthew A
MA
Fan
Z Hugh
ZH
D001054:Apolipoproteins A / Q000235:genetics / Q000378:metabolism*; D001436:Bacteriorhodopsins / Q000235:genetics / Q000378:metabolism*; D002474:Cell-Free System; D004353:Drug Evaluation, Preclinical; D004791:Enzyme Inhibitors / Q000737:chemistry; D007700:Kinetics; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D040081:Protein Array Analysis / Q000379:methods*; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000235:genetics / Q000378:metabolism
D001054:Apolipoproteins A; D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D001436:Bacteriorhodopsins; D008156:Luciferases; D001618:beta-Lactamases