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Cell-free protein expression, PURE joy! 

The previous post of this series hinted at an efficient way of getting around problems with in vivo protein expression; to use a cell-free, in vitro, expression system. Cell-free protein expression offers a way to minimize the time and workload required to produce your peptide of interest and prepare it for downstream applications, at high concentration and requiring less effort to purify.  

In such a system the components necessary for translation are sourced either directly from lysates or reconstituted from isolates from recombinant sources. These components are then fed with genetic material (DNA or mRNA) encoding a peptide of interest, allowing tight regulation of amounts of starting material, as well as reaction conditions. Further customization can also be made by addition of additional components such as co-factors, custom tRNAs, (synthetic) lipid bilayers and detergents, to mention a few. 

One of the first cell free expression systems to gain commercial success is known as PURE (Protein synthesis Using Recombinant Elements1), and includes all components necessary for protein translation. This has been further improved over time, as in e.g. PURExpress2, where additional component optimizations have been performed and several variants are offered to ensure full customizability. A common attribute among PURE-based systems is that the constituent proteins (excluding the ribosomes) are all His-tagged to enable reverse-purification of the synthesized peptide. 

In contrast to extract or lysate derived-expression systems, PURE largely avoids the “black-box” nature of the former by utilizing only known concentrations of well-defined components.  

The ability to utilize mRNA, plasmid or linear DNA, means a number of source materials and methods for producing them can be employed, all to suit each specific use case. A multitude of uses are possible, beyond mere protein synthesis studies of protein-protein interactions, activity, and folding – assays for synthetic biology and directed evolution have been performed. However, perhaps one of the more common applications however is expression of membrane, or toxic, proteins which can be highly problematic in vivo. But the minimalistic composition of the PURE system means that it is less susceptible to interference by the produced peptide, while addition of specific detergents or lipid bilayers can allow for the proper folding and export of membrane-integrated proteins, which also means they are sequestered from the synthesis machinery, further reducing the risk of toxicity.  

The ability to translate proteins directly from templates of choice can enable co-expression of multiple peptides in a single reaction, and optimization of the reaction by alteration of component concentrations.  

And beyond enzymes and oligos, other components, such as detergents and (synthetic) lipids, can play a major role in the production of, particularly, membrane proteins and allow the user to isolate them in their native and active forms. 

In the next part, we take a closer look at tools for isolation and purification of proteins, regardless of the expression system used. 

Until then, if you want to know more about how cell free expression can help you produce your peptide of interest, don’t hesitate to contact us 

Learn more about the NEBExpress system.