Cell-free protein synthesis (aka: in vitro translation) refers to protein production in vitro using lysates that provide the cellular machinery necessary for synthesis. Ribosomes, tRNAs, aminoacyl-tRNA synthetases, initiation/elongation/termination factors, GTP, ATP, Mg2+ and K+ are among the requirements for a translation system. These are provided by lysates, which can be from prokaryotic or eukaryotic sources, depending on your requirements.
Cell-free protein synthesis is most commonly used for generating protein for study of things like:
• protein:protein interactions (pull-down assays and co-immunoprecipitation)
• protein:DNA interactions (Gel shift assay or EMSA)
• protein:RNA interactions
• protein function studies (incorporating modified or unnatural amino acids, post-translation modifications)
Cell-free protein synthesis is a useful alternative to in vivo synthesis for the analysis and production of proteins. Some of them include:
1. Better protein yields: In invivo protein expression, a large part of the metabolic resources are dedicated towards basic cellular processes. However, in an invitro production system, all those resources are directed only towards the production of your protein of interest.
2. An optimal environment: There are no cell walls in invitro expression, i.e. it is an open system (if you wish, like a super enriched prebiotic soup!). You can add components to create the most optimal environment for your protein production.
3. Cell viability concerns: There is no cell growth involved in invitro expression and hence even proteins considered toxic to the host can be expressed (1).
4. Ease of use: No elaborate setup is required to get started. Special skill in cell culture or microbiology technique is not a requirement either. It is facile.
5. Time saving: In most cases enough protein can be produced in a few hours.
A couple of things to consider when choosing an invitro system for your protein needs:
A. Choice of template: Template can be DNA (plasmid, oligo, cDNA or PCR product) or RNA. DNA templates are convenient and can be used in a coupled system that will perform in vitro transcription and translation in tandem, in the same tube, commonly called coupled in vitro transcription/translation. Or, the two steps can be carried out separately.
Transcription is typically done using the phage RNA polymerases T3, T7 or SP6. 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. RNA made using an invitro transcription system can be purified and used in an invitro translation system.
B. Choice of System: 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, 554–61)].
Rabbit Reticulocyte lysate (RRL) and wheat germ extract (WGE) are the most popular lysates for eukaryotic expression. Other expression systems include lysates from insect cells, fungi and yeast (3).
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).
There are number of companies that offer invitro coupled systems and invitro transcription or in vitro translations systems. Some even carry a sample set of all the systems.
If you have questions, comments, tips or experience using these systems, feel free to drop us a line in our comments section.
References:
1. Iskakova, Madina, B. 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) 150–6.
3. Falzon, L. et al. (2006) Finding one of a kind: advances in single-protein production. Current Opinion in Biotechnology 17 347–52.
Hello, I am interested to hear anyone’s opinion on using the WG system on the robot manufactured by Cell free sciences in Japan.
Is there similar machine made in the US? Who else produces the WG reagent in this country – I only know it is imported from Japan.
Thanks,
Li
Li
I believe this system now can be purchased from Cambridge Isotopes.
The best source of information regarding use of the system
is the Center for Eukaryotic Structural Genomics
http://www.uwstructuralgenomics.org/cellfree.htm
gary
I’m interested in protein:DNA interactions. I use TNT® T7 Quick Coupled Transcription/Translation System for this purpose. I test this DNA-protein binding using electro mobility shift assay. But I have a problem with master mix contained in the kit. Probably, my biotin labeled oligonucleotids bind with some protein from master mix. Because, when I put together free master mix and my DNA, I get very hight signal. So that I can’t see my band (my protein:DNA interaction, specific competition..).Do you have some usefull protocol, which I could use regarding this application?
Thanks,
Anet
Anet,
We have forwarded this to Technical Services. You should be contacted within 24 hours. Let us know if not and we will follow up.
Michele
The following article contains useful information regarding optimization of gel shift assays using in vitro-expressed proteins. While the article focuses on fluorescent gel shift assays, many of the suggestions for reducing nonspecific bands would apply to any gel shift assay system.
Wieczorek D and Schagat T. Cell-Free Expressed Protein in Fluorescent Gel Shift Assays. [Internet] 2009. [cited: year, month, date]; 1, tpub_019. Available from:
http://www.promega.com/pubs/tpub_019.htm