Researchers have developed a new microfluidic device that enables high-throughput (HT), cell-free protein synthesis for genomic and proteomic functional analysis.
This latest research has built on the use of in vitro transcription and translation (IVT) techniques to bypass problems with traditional host cell protein synthesis techniques while being incorporated into a system that can be used on a high-throughput scale to make proteins for functional analysis studies.
The new device, described in an early access article in the journal Biotechnology Progress by researchers from the University of Florida, US, uses microfluidic channels to increase the yield of the reaction by ensuring nutrients are constantly supplied while by-products are removed.
With the completion of the human genome project the interest in identifying the functions of newly discovered genes and the proteins they encode for has intensified. HT means of producing large numbers of proteins in parallel are necessary in order to match the HT discovery of genes and ascertain their roles.
Traditional biological synthesis of a protein proceeds via gene transcription and protein translation in a host cell such as the bacterium Escherichia coli (E. coli).
However, recombinant protein production in such host systems has several limitations such as the formation of insoluble protein aggregates, intracellular protein degradation, lack of post-translational modifications and low expression of genes that code for proteins that are toxic to the host.
The recovery of the proteins can often be problematic as the cells often need to be broken up by a lysis step before the proteins can be purified from 'genomic DNA'.
According to the authors, recombinant protein production using host cells is difficult to implement in an HT format.
The IVT method avoids these problems by conducting the reactions in the absence of a host cell and instead using a DNA template coding sequence that is transcribed into messenger RNA (mRNA) that translates the proteins.
This technique has been commercialised by Roche Applied Science in its Rapid Translation System (RTS) and the researchers have 'borrowed' the concept in designing the nested well microfluidic array systems for continuous-expression cell-free (CECF) protein synthesis lab-on-a-chip.
The researchers tested the device by comparing its production yields of three different proteins, synthesising green fluorescent protein (GFP), chloramphenicol acetyl-transferase (CAT) and luciferase, with yields gained when conducting the reactions in conventional microwell vessels.
Duration of protein expression was found to be increased between 5 and 10 times using the microfluidic device due to the continuous feeding and by-product removal regime.
This led to yields that were between 13 and 22 times higher than those observed in the traditional vessels.
In addition, the researchers found that a positive hydrostatic flow of the nutrients into the reaction vessel was essential to obtaining these high yields.
"It is possible to integrate the array device with assay and detection elements so that the expressed proteins can be analysed in the device," wrote the researchers.
Such integration would remove the need to harvest proteins before analysis; in addition they could be fused to other proteins to enable enzymatic inhibition assays for drug screening process.