A cell-free protein synthesis system for high-throughput proteomics

Proc Natl Acad Sci U S A. 2002 Nov 12;99(23):14652-7. doi: 10.1073/pnas.232580399. Epub 2002 Oct 30.


We report a cell-free system for the high-throughput synthesis and screening of gene products. The system, based on the eukaryotic translation apparatus of wheat seeds, has significant advantages over other commonly used cell-free expression systems. To maximize the yield and throughput of the system, we optimized the mRNA UTRs, designed an expression vector for large-scale protein production, and developed a new strategy to construct PCR-generated DNAs for high-throughput production of many proteins in parallel. The resulting system achieves high-yield expression and can maintain productive translation for 14 days. Additionally, in the integration of a PCR-directed system for template creation, at least 50 genes can be translated in parallel, yielding between 0.1 and 2.3 mg of protein by one person within 2 days. Assessment of correct protein folding by the products of this high-throughput protein-expression system were performed by enzymatic assays of kinases and by NMR spectroscopic analysis. The cell-free system, reported here, bypasses many of the time-consuming cloning steps of conventional expression systems and lends itself to a robotic automation for the high-throughput expression of proteins.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell-Free System
  • DNA Primers
  • Databases, Protein
  • Enzymes / genetics*
  • Genetic Vectors
  • Humans
  • Kinetics
  • Plant Proteins / genetics*
  • Polymerase Chain Reaction / methods
  • Proteins / genetics*
  • Proteomics / methods*
  • RNA, Messenger / genetics
  • Triticum


  • DNA Primers
  • Enzymes
  • Plant Proteins
  • Proteins
  • RNA, Messenger