Protein synthesis in a device with nanoporous membranes and microchannels

Abstract

Cell-free protein synthesis (CFPS) is an alternative approach to cell-based recombinant protein production. It involves in vitro transcription and translation in a cell-free medium. In this work, we implemented CFPS in a plastic array device. Each unit in the array consisted of an inner well and an outer well. Two synthesis steps, gene transcription and protein translation, took place in the inner well, in which a cell-free medium was used to provide ribosomes and additional components necessary for protein synthesis. The outer well was concentric to the inner well and it functioned as a nutrient reservoir. A nanoporous membrane was sandwiched between the inner and outer wells for retaining the synthesized proteins and removing the reaction byproducts. A microfluidic channel was employed to connect these two wells for supplying fresh nutrients for longer reaction time and higher expression yield. Synthesis of luciferase was shown to last 8 times longer and yield 10 times more proteins than in a conventional container. The device also enables more than 2 orders of magnitude reduction in reagent consumption compared to a bench-top instrument. The effects of the membrane pore size and microfluidic channel on the protein production yield were also studied. The array device has potential to become a platform for parallel protein expression for proteomics applications, matching high-throughput gene discovery.