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.

  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: