When you think about the 'front end' of a protein crystallization project, E.coli is front and center. These bacteria are used to create designed vector constructs and are the standard vehicle for heterologous protein over expression. However, one could also chemically synthesize the protein from scratch - a notorious example is Lysozyme, the 1.04 Å X-ray structure of which has been obtained from crystals grown from a sample that had been obtained by total chemical synthesis. An earlier example for such a path is the structure of the anti-HIV protein AOP-RANTES.
Chemical synthesis is a tour de force and not a practicable path for most of us. Now though, protein synthesis without involving a chemical fume hood nor culturing E.coli, or any living organisms for that matter, can be done in a standard lab. There are several reports of crystallographic structures that were produced from crystals that had been grown from protein material obtained in cell-free systems. The protein producing machinery is still of biological origin, though. So technically, living organisms are involved - but the timing of their cultivation and the protein production is uncoupled. This circumvents the need to cultivate cells in your own lab and could be done away with altogether when the DNA is of synthetic origin as well.
Here are two X-ray crystallographic protein structure reports that are based on crystals grown from in-vitro expressed protein:
A. Deniaud, L. Liguori, I. Blesneac, J.L. Lenormand, E. Pebay-Peyroula Crystallization of the membrane protein hVDAC1 produced in cell-free system Biochimica et Biophysica Acta (BBA) - Biomembranes, Volume 1798, Issue 8, August 2010, Pages 1540-1546
Miyazono, K.I., Watanabe, M., Kosinski, J., Ishikawa, K., Kamo, M., Sawasaki, T., Nagata, K., Bujnicki, J.M., Endo, Y., Tanokura, M., and Kobayashi, I. (2007) Novel protein fold discovered in the PabI family of restriction enzymes Nucleic Acids Res., 35, 1908-1918.
The case for retiring E.coli and utilizing cell-free systems can be made especially for those proteins that are expressed at low yield or that are toxic to E.coli or any other cells (i.e. DNA modifying enzymes) or when specific labels need to be introduced into the protein. Direct access to the protein synthesis machinery is unique and allows tackling difficult targets, such as membrane proteins. A recent summary of such ongoing research is to be published here:
Emily T.Beebe,Shin-ichiMakino,AkiraNozawa,YukoMatsubara, Ronnie O.Frederick,JohnG.Primm,MichaelA.GorenandBrianG.Fox Robotic large-scaleapplicationofwheat cell-freetranslationtostructuralstudies including membrane proteins New Biotechnology July 2010
And of course, one of the reasons I mention this synthetic biology route has to do with the fact that we offer via Emerald BioSystems the wheat-germ based protein expression system. Our partner in Japan, Cell Free Sciences has developed reagents and a sophisticated robot that enables researchers to produce milligram amounts of protein. The robot is called Protemist DTII. All you need to do is load the instrument with target-DNA and reagents, klick a button on the screen and walk away. When you're back after one and a half days the instrument has produced (via transcription, translation and affinity purification) your purified target protein. Pretty convenient, isn't it? The instrument that may be most interesting to protein crystallographers though, is the new Protemist XE, shown below. Its capacity is designed to produce tens of milligrams of protein within a one or two day campaign.
No living cells involved: ten milligram of GFP produced with the Protemist XE using the wheat germ cell-free expression system.
Drop us a note (sales@emeraldbiosystems.com) if you're interested in more information about this protein production system (and tell Frank that Peter sent you :)
Regards,
Peter
P.S. I just saw this comprehensive review article in Nature Biotech, covering the subject of cell-free protein synthesis for functional and structural analysis of membrane proteins:
Junge F, Haberstock S, Roos C, Stefer S, Proverbio D, Dötsch V, Bernhard F.
Advances in cell-free protein synthesis for the functional and structural analysis of membrane proteins.
N Biotechnol. 2010 Jul 15. [Epub ahead of print]