Abstract
Cell-free systems represent a promising approach to quickly and easily produce preparative amounts of proteins. However, it is still challenging to obtain high volumetric yields (>mg ml−1) of proteins from the present cell-free systems. This protocol presents a cell-free protein synthesis method using a novel DNA gel that dramatically increases protein yield compared with current systems. This protein-producing gel (termed 'P-gel system' or 'P-gel'), which consists of genes as part of the gel scaffolding, can produce mg ml−1 amounts of functional proteins. This protocol describes steps pertaining to plasmid design, fabrication of P-gel molds, formation of P-gel micropads and cell-free protein expression with an expected yield of up to 5 mg ml−1 of functional Renilla luciferase (Rluc). This entire process can take 1–3 d, depending on the desired quantity of protein.
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Acknowledgements
The work is partially supported by a NYSTAR Faculty Development Program Award, NYSTAR CAT grant, US National Science Foundation's CAREER award (grant number: 0547330) and a USDA NRI grant. We acknowledge the use of the facilities of the Cornell Centre for Materials Research, which is supported through NSF Grant DMR 0520404, part of the NSF MRSEC Program.
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N.P., S.H.U., H.F., J.X. and D.L. developed the original technique. N.P., J.S.K., E.J.R., M.R.H. and D.L. organized the procedure and cowrote the manuscript.
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Park, N., Kahn, J., Rice, E. et al. High-yield cell-free protein production from P-gel. Nat Protoc 4, 1759–1770 (2009). https://doi.org/10.1038/nprot.2009.174
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DOI: https://doi.org/10.1038/nprot.2009.174
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