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An Intro to Cell-free Protein synthesis

Cell-free protein synthesis (aka In vitro translation) refers to protein production in vitro using lysates generated that provide the cellular machinery necessary for synthesis. The lysates can be of bacterial or eukaryotic origin.

It is a useful alternative to in vivo synthesis for generating protein for the study of things like:

  • protein:protein interactions (pull-down assays, co-immunoprecipitation, post-translation modifications)
  • protein:DNA interactions (Gel shift assay or EMSA)
  • or protein:RNA interactions

So what are the advantages of cell free protein synthesis, as opposed to synthesising in vivo?

Here are a few, taken from reference 1:
1. Improved protein yields: Since there are no cellular processes going on so virtually all of the resources available in the extract go towards the production of your protein of interest.

2. An optimal environment for the production of your protein. An open system, with no cell walls (like a super enriched prebiotic soup!). You can add components to create the most optimal environment for your protein production.

3. No cell viability concerns if the protein expressed is toxic. Since no cell growth is needed.

You can synthesis your protein in vitro either from a suitably constructed DNA template (plasmid, oligo, cDNA or PCR product) using a coupled system that will perform in vitro trancription and translation in tandem (coupled systems), or you can do the two steps separately. Either way you can make lots of protein made in just a few hours. There are number of companies that offer invitro coupled systems and invitro transcription or in vitro translations systems.

Transcription is done using the phage RNA polymerases T3, T7 or SP6 so the coding sequence you want to express must be flanked by the promoters for one of these polymerases. In vitro transcription kits contain the polymerase ribonucleotides, buffers and magnesium required for transcription.

Translation is an altogether more complicated affair, with ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation/elongation/termination factors, GTP, ATP, Mg2+ and K+ among the requirements. These are provided by lysates, which can be from prokaryotic or eukaryotic sources, depending on your requirements.

Typically, E.Coli S30 fraction is used for prokaryotic expression (Check out a home-brew version of S12 extract with claims that it works better J. Biotech. 2006 126(4) 554-561).

Rabbit Reticulocyte lysate (RRL) and wheat germ extract (WGE) are the most popular lysates for eukaryotic expression.

The choice of the system should be determined not just by origin but also by the biological nature of the protein and the requirements of downstream applications. The protein yields from an E.coli based system can be much greater than eukaryotic-based systems. The yields can be as high as a few mg/mL depending on protein and reaction format. However, RRL and WGE are better platforms for functional studies as post-translational modifications (2).

If you are having trouble will cell free protein synthesis, or have any questions, we are happy to answer your questions in our discussion forum. You can ask your question using the form below, or start a new topic in the forum itself.

References:
1. Madina B. Iskakova et al (2006). Troubleshooting coupled in vitro transcription-translation system derived from E.coli cells: synthesis of high-yield fully active proteins. Nucleic Acids Research 34 (19).
2. Katzen F et al (2005). The past, present and future of cell-free protein synthesis. Trends in Biotechnology 23(3) 150-156.

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