I had previously talked about the basics of cell-free protein synthesis and ways to improve yields from the technique. But recently I came across an article describing a technology that promises to dramatically improve the efficiency of cell free protein synthesis.
The article, published in Nature Materials by Dan Lou et al from Cornell University, shows a novel way to produce proteins cell-free using DNA hydrogels.
What are P-gels?
Protein-producing DNA hydrogels (P-gels) contain the template plasmid DNA (from which the protein will be synthesised) assembled into a large scaffold structure by ligation to x-shaped pieces of cross-linker DNA, and embedded into a blob of a polymer called PDMS. The P-gels are molded into micropads of defined dimensions during the gelling process and after gelation, the PDMS forms a spongy, jelly-like matrix that absorbs water, but does not dissolve in it.
Several P-gel micropads can then be transferred into a coupled cell-free extract reaction mix for protein production (see part B of the figure on the right).
How good are they?
P-gels have shown very promising results;They can produce several-times times greater protein than regular solutions base cell-free protein synthesis methods. The results were consistently good for the 16 different proteins, including membrane proteins and toxic proteins, that the authors tried.
Why are they better?
Several factors unique to the DNA hydrogels make the system very efficient. Firstly, the plasmid DNA is physically cross-linked to the scaffold, which protects the genes from damage, such as degradation by nucleases.
Secondly, the gel matrix compresses the template into a smaller area makeing the effective concentration of the DNA, lysate and the other factors required for protein synthesis higher — and higher concentration results in a more efficient reaction. This is similar to the way that PEG works to improve the efficiency of ligation reactions (see 5 DNA Ligation Tips).
Finally, the transcription efficiency is greater than solutions-based system due to the increase in enzyme turnover during transcription. Since the genes are “locked’ in place, they are readily available for use by the phage polymerases.
Where can you buy them?
Unfortunately, the hydrogels are not very easy to make or in commercial production yet. They require a clean room and specialized micro-moulds. So for now, we just need to keep improving our solution-based methods.
For more information on DNA hydrogels, checknews.cornell.edu/stories/April09/ProteinGel
References:
1. Nature Materials 8, 432 – 437 (2009)
2. Nature Methods 6 (5), 326 (2009)
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