by Greg Kilmer, B.S.; Brian Webb, Ph.D.; Atul Deshpande, Ph.D.; Krishna Vattem, Ph.D. - 01/10/14
Cell-free, in vitro translation (IVT) systems enable small amounts of proteins to be produced quickly. Protein expression protocols that can be adapted to high-throughput (HTP) techniques provide an efficient means for producing a large number of recombinant proteins for use in screening and other research applications. Here we demonstrate the speedy and efficient simultaneous expression of several proteins using the Thermo Scientific 1-Step Human High-Yield IVT System applied to 96-well microdialysis plates.
The Mini IVT Kit (Part No. 88892) contains separate microdialysis devices to perform individual 100µL reactions, where each device is incubated in a separate 2mL microcentrifuge tube. Thermo Scientific Pierce 96-well Microdialysis Plates (Part No. 88260) contain 8-segment strips of these same microdialysis devices (Figures 1 and 2), enabling protein expression in an automation-compatible, 96-well format.
We validated the 96-well HTP format by performing replicate IVT reactions in eight wells of a microdialysis plate (two 4-segment cartridges). We expressed TurboGFP (green fluorescent protein) using the positive control DNA (pCFE-GFP) that is supplied in the IVT kits. Our protocol was identical to the kit procedure (Part No. 88891) for four 100µL reactions. We incubated the reactions for 18 hours at 30°C.
Expression was favorable and consistent in all eight test wells, producing in an average of 461µg/mL GFP per well (Figure 3). Even before removing sample for quantitation in an assay plate, GFP expression is visually evident in the microdialysis devices (Figure 4). These values are comparable to results obtained with the default IVT kit procedure, in which the devices are incubated in 2mL microcentrifuge tubes (data not shown).
We have successfully demonstrated the use of Pierce 96-well Microdialysis Plates for high-throughput expression of proteins with the 1-Step High-Yield Mini IVT Kits. We conclude that switching from microcentrifuge tubes to the microdialysis plate for IVT reactions in the microdialysis devices facilitates rapid and consistent expression of up to 96 proteins at a time.
The protocol was identical to the default procedure for the 1-Step Human High-Yield Mini IVT Kit (Part No. 88891) for four 100µL reactions, except that the microdialysis devices containing the reactions were incubated in a Pierce 96-well Microdialysis Plate (Part No. 88260) instead of 2mL microcentrifuge tubes. Reactions were incubated for 18 hours at 30°C.
For quick visual detection, the microdialysis devices containing the GFP reactions were viewed using a Molecular Imager VersaDoc™ MP 4000 System (Bio-Rad Laboratories, Inc.) with a FITC filter (ex/em: 482/512nm), 0.1 sec exposure, aperture 4.
Quantitation using fluorescent plate reader: 10µL aliquots of each reaction were diluted 15-fold with phosphate-buffered saline (PBS) containing 0.1% BSA to yield 150µL test samples. A set of standards was prepared from recombinant tGFP protein of known concentration (Part No. 88899) by serial dilution (50µg/mL to 0.78µg/mL) in 0.1% BSA-PBS. Triplicates of test samples and standards (25µL) were pipetted into wells of a black 384-well plate and the fluorescence values measured by a Tecan™ Safire™ Fluorescent Plate Reader at ex/em: 482/512nm. Concentrations of test samples were calculated by reference to the standard curve.
Additional methods for detecting and measuring expression of GFP from the control DNA (pCFE-GFP) supplied in the IVT Kits are described in the product instructions. Be aware that the tGFP control protein is from the copepod Pontellina plumata. This GFP is not reactive to antibodies generated against Aequorea victoria GFP (i.e., EGFP or other EGFP mutants). Instead, use polyclonal antibodies to TurboGFP (Ab. No. PA5-22688) for Western blot detection.
The Thermo Scientific 1-Step Human High-Yield In Vitro Translation (IVT) Kits enable the expression of functional proteins with 10 to 100 times greater yield per mL than other mammalian IVT systems.
The 1-Step High-Yield IVT System uses modified HeLa cell extracts to take advantage of the robust human translation machinery and generate functional full-length proteins. In this system, protein expression is performed in a proprietary dialysis device that allows a continuous supply of nucleotides, amino acids and energy generating substrates into the reaction while removing inhibitors of proteins synthesis. This continuous-exchange cell-free (CECF) system enables protein expression in an overnight incubation of up to 750 µg/mL. The complete mini-scale kit includes all the components required for transcription and translation of a recombinant gene, including an optimized expression vector.
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