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.
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.
We have developed a new mass spectrometry (MS) based approach for continuous, spatially resolved in vitro biochemical detection and demonstrated its utility in a 3-D cell culture system. Extracellular liquid is passively extracted at a low flow rate (~10 nL/s) through a small bore silica capillary (ID 50 μm); inline microdialysis (MD) removes ions that would interfere with mass spectrometric analysis, and the sample is ionized by nanoelectrospray ionization (nano-ESI) and mass analyzed in a time-of-flight mass spectrometer. The system successfully detects low-volume, low-concentration releases of a small protein (8 μL of 5 μM cytochrome-c, molecular mass ~12 kDa) and exhibits ~1 min temporal resolution. The system also displays sensitivity to probe proximity to the sample release point. Due to the sensitivity of ESI-MS and its ability to simultaneously detect and identify multiple unanticipated biochemicals, this approach shows considerable potential as a biomarker discovery tool.
We present our investigation of using microfluidic devices for rapid protein separation. The devices were made from cyclic olefin copolymers that have high optical clarity and high glass transition temperature. Protein separation was achieved by using isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). A laser-induced fluorescence (LIF) imaging system was developed to detect proteins while they migrated under an electric field. IEF was carried out in a separation medium consisting of carrier ampholytes and a mixture of linear polymers. Dynamic coating of the linear polymers prevented proteins from adsorption and suppressed electroosmotic flows. PAGE was achieved in twenty-nine parallel channels. In addition, we integrated IEF with PAGE in a microfluidic device for two-dimensional protein separation.
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