Cell-free protein synthesis (CFPS) is favored by researchers as it allows them to quickly generate a protein independent of cell culture. Eukaryotic lysates can carry out post-translational modifications of the protein, but the nuances of different translation systems can also lead to changes in protein production. So, which cell-free expression system is better? A study recently published in the journal BioTechniques offered an answer.
For the current cell-free protein synthesis, the E. coli is still the first choice. However, due to the growing interest in human and mammalian proteins and their associated post-translational modifications, eukaryotic systems are also gradually welcomed by researchers. In addition, for certain technologies, such as mRNA display and nucleic acid programmable protein arrays (NAPPA), researchers tend to use eukaryotic expression systems when they study protein-protein interaction
In this study, researchers of Arizona State University compared three commonly used cell-free translation systems on the market: wheat germ extract (WGE, Promega), rabbit reticulocyte lysate (RRL, Promega) and HeLa cell lysates (HCL, Thermo Fisher).
For each system, researchers have quantified the amount of the luciferase protein generated from circular plasmid and PCR-generated linear DNA that contains three different translation enhancing elements in the 5 ‘ untranslated region (UTR) with or without a 62-nucleotide poly-A tail in the 3 ‘ UTR. The 5′ UTR sequences includes 500-nucleotide encephalomyocarditis (EMCV) internal ribosome entry site (IRES) , as well as two minimal translation enhancing elements derived from the tobacco mosaic virus (TMV) and alfalfa mosaic virus (AMV).
The researchers found that the protein produced by plasmid DNA is approximately 500 times than that of the linear DNA. For the element on the template, 5 ‘UTR sequence preferences have a significant impact on the amount of expression, in contrast, 3′ poly-A tail is not so important.
They believe that the most effective protein translation system are HeLa cell lysates with plasmids containing the EMCV IRES in the 5 ‘UTR. Linear template is not so effective, probably because it is degraded in the cell lysates. While linear template cyclization can prevent degradation and improve yield.
In some applications, you must use a specific lysate or DNA template. To this end, the researchers give some advice. For wheat germ extract, when the 5 ‘ UTR contains AMV or TMV, the plasmid and linear DNA template can play the best role. For HeLa cell lysates, the situation is just the opposite: it exhibits a strong preference for EMCV.
In addition, if the end of the poly-A tail in the 3 ‘ UTR appears, translation of a linear DNA template with EMCV increased by 10 times. Rabbit reticulocyte lysate shows no clear preference to the sequence of plasmid 5′ UTR, but prefer to linear template AMV and TMV.
To illustrate the results are not luciferase unique, the researchers also tested several other human proteins, and reached similar conclusions. In order to maximize protein production, they recommend using using HCL with commercial plasmids that carry an EMCV IRES in the 5′ UTR. Despite the relatively high cost of the system, the amount of protein it produced is 20-50 times that that of other systems. This makes it ideal for high-throughput protein expression analysis.
Source: Comparative analysis of eukaryotic cell-free expression systems, BioTechniques, Vol. 59, No. 3, September 2015, pp. 149–151