Miniaturized fluid array for high‐throughput protein expression

Abstract

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.

Bottom-Up Construction of Complex Biomolecular Systems With Cell-Free Synthetic Biology

“…Successive work aimed to improve protein synthesis yield for cell-free TX-TL reactions by using a dialysis membrane to separate the reaction from the feeding solution of amino acids and energy sources, leading to a semi-continuous reaction (Kim and Choi, 1996;Madin et al, 2000). This idea was then extended to be compatible with standard micro-well plate systems that could be used for higher throughput applications (Mei et al, 2006(Mei et al, , 2007Khnouf et al, 2009Khnouf et al, , 2010. Following upon the same principles of continuous exchange cell-free reactions, a passive PDMS microreactor was built which separated the feeding and reaction chambers with a dialysis membrane, enabling protein synthesis for up to 15 h (Hahn et al, 2007) (Figure 3B).…”

Optimization of a miniaturized fluid array device for cell‐free protein synthesis

“…To achieve high‐throughput protein expression and reduce reagent volumes and cost, CFPS is often performed in miniaturized or microfluidic devices (Angenendt et al, ; Agresti et al, ; Biyani et al, ; Fallah‐Araghi et al, ; Hahn et al, ; Jackson et al, ; Jackson and Fan, ; Khnouf et al, ; Khnouf et al, ; Mei et al, ; Okano et al, ; Osaki et al, ; Park et al, ; Teh et al, ; Wu et al, ; Wu et al, ). However, in the CECF format, miniaturization is generally restricted due to the necessity for a porous membrane that allows passive chemical diffusion between reaction and feeding solutions.…”

“…However, in the CECF format, miniaturization is generally restricted due to the necessity for a porous membrane that allows passive chemical diffusion between reaction and feeding solutions. In miniaturized systems, the CECF format has been accomplished through the use of microdroplets (Agresti et al, ; Fallah‐Araghi et al, ; Osaki et al, ; Teh et al, ; Wu et al, ; Wu et al, ), a protein‐producing gel (Park et al, ), and microfluidic devices (Hahn et al, ; Jackson and Fan, ; Jackson et al, ; Khnouf et al, ; Mei et al, ). In these microfluidic devices, an array of protein expression microreactors are typically created for the simultaneous expression of numerous proteins with the reaction solution being isolated from the feeding solution by a dialysis or nanoporous membrane (Hahn et al, ; Jackson and Fan, ; Jackson et al, ; Khnouf et al, ; Mei et al, ).…”

Cell-Free Protein Synthesis in Miniaturized Array Devices and Effects of Device Orientation

“…These goals have been accomplished in the form of droplets, [21][22][23][24] protein-producing gel, 25 and microfluidic devices. 14,[26][27][28] With the droplets, a pseudo-filtration membrane is formed through oil-in-water emulsions, and the platform offers an ultra-high-throughput screening method because a large number of droplets can be easily and rapidly created. [21][22][23][24] For the protein-producing gel, the isolation of the genetic template and reaction solution is achieved through a hydrogel, and the large surface-area-to-volume ratio of the reaction vessel enhances protein synthesis yield.…”

“…14,[26][27][28] In this work, we designed and fabricated two miniaturized array devices for continuous-exchange CFPS and investigated the effect of membrane orientation on protein synthesis yield. Compared with the devices reported previously, 14 the membrane in this work is oriented vertically in reference to the table surface to reduce or eliminate possible membrane clogging (due to possible sedimentation of large molecules such as aggregated proteins or ribosomes). We optimized these devices by studying the effects of hydrostatic pressure and reaction times on protein synthesis yield.…”