Protocol abstract


Nature Protocols 4, 1759 - 1770 (2009)
Published online: 12 November 2009 | doi:10.1038/nprot.2009.174

Subject Category: Biochemistry and protein analysis

High-yield cell-free protein production from P-gel

Nokyoung Park1, Jason S Kahn1, Edward J Rice1, Mark R Hartman1, Hisakage Funabashi1,2, Jianfeng Xu1,3, Soong Ho Um1,4 & Dan Luo1


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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  1. Department of Biological and Environmental Engineering, Cornell University, Ithaca, New York, USA.
  2. Current address: Department of Biotechnology and Life Science, Tokyo University of Agriculture and Technology, Naka-cho, Koganei, Tokyo, Japan.
  3. Current address: Arkansas Bioscience Institute, Arkansas State University, State University, Arkansas, USA.
  4. Current address: Department of Materials Science and Engineering, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts, USA.

Correspondence to: Dan Luo1 e-mail: dan.luo@cornell.edu