Miniaturized fluid array for high-throughput protein expression.
We describe a miniaturized fluid array device for high-throughput cell-free protein synthesis (CFPS), aiming to match the throughput and scale of gene discovery. Current practice of using E. coli cells for production of recombinant proteins is difficult and cost-prohibitive to implement in a high-throughput format. As more and more new genes are being identified, there is a considerable need to have high-throughput methods to produce a large number of proteins for studying structures and functions of the corresponding genes. The device consists of 96 units and each unit is for expression of one protein; thus up to 96 proteins can be produced simultaneously. The function of the fluid array was demonstrated by expression of a variety of proteins, with more than two orders of magnitude reduction in reagent consumption compared with a commercially available CFPS instrument. The protein expression yield in the device was up to 87 times higher for β-glucoronidase than that in a conventional microplate. The concentration of β-galactosidase expressed in the device was determined at 5.5 μg/μL. The feasibility of using the device for drug screening was demonstrated by measuring the inhibitory effects of mock drug compounds on synthesized β-lactamase without the need for harvesting proteins, which enabled us to reduce the analysis time from days to hours.
Khnouf
Ruba
R
Dept. of Biomedical Engineering, University of Florida, PO Box 116131, Gainesville, FL 32611, USA.
Olivero
Daniel
D
Jin
Shouguang
S
Fan
Z Hugh
ZH
D000469:Alkaline Phosphatase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D002439:Cefotaxime / Q000494:pharmacology; D019818:Clavulanic Acid / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D057166:High-Throughput Screening Assays / Q000379:methods*; D008156:Luciferases / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D040081:Protein Array Analysis / Q000379:methods*; D013329:Structure-Activity Relationship; D013407:Sulbactam / Q000494:pharmacology; D001616:beta-Galactosidase / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000235:genetics; D065093:beta-Lactamase Inhibitors; D001618:beta-Lactamases / Q000096:biosynthesis* / Q000235:genetics
D004791:Enzyme Inhibitors; D065093:beta-Lactamase Inhibitors; D019818:Clavulanic Acid; D008156:Luciferases; D000469:Alkaline Phosphatase; D001616:beta-Galactosidase; D001618:beta-Lactamases; D002439:Cefotaxime; D013407:Sulbactam