Cell-free protein synthesis for functional and structural studies

Methods Mol Biol. 2014;1091:161-78. doi: 10.1007/978-1-62703-691-7_11.

Abstract

Recent advances in cell-free protein expression systems have made them reliable and practical for functional and structural studies of a wide variety of proteins. In particular, wheat germ cell-free translation can consistently produce target proteins in microgram quantities from relatively inexpensive, small-scale reactions. Here we describe our small-scale protein expression method for rapidly producing proteins for functional assay and techniques for determining if the target is suitable for scale-up to amounts potentially needed for structure determination. The cell-free system is versatile and can be easily customized with the inclusion of additives. We describe simple modifications used for producing membrane proteins.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell-Free System*
  • Dialysis
  • Endopeptidase K / metabolism
  • Liposomes
  • Membrane Proteins / biosynthesis
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Membrane Proteins / isolation & purification
  • Plant Proteins / biosynthesis
  • Plant Proteins / chemistry
  • Plant Proteins / genetics
  • Plant Proteins / isolation & purification
  • Protein Biosynthesis*
  • Proteins / chemistry
  • Proteins / genetics*
  • Proteins / isolation & purification
  • Proteins / metabolism
  • Solubility

Substances

  • Liposomes
  • Membrane Proteins
  • Plant Proteins
  • Proteins
  • Endopeptidase K