Wheat germ cell-free translation, purification, and assembly of a functional human stearoyl-CoA desaturase complex

Protein Expr Purif. 2008 Dec;62(2):171-8. doi: 10.1016/j.pep.2008.08.002. Epub 2008 Aug 15.


A wheat germ cell-free extract was used to perform in vitro translation of human stearoyl-CoA desaturase in the presence of unilamelar liposomes, and near complete transfer of the expressed integral membrane protein into the liposome was observed. Moreover, co-translation of the desaturase along with human cytochrome b(5) led to transfer of both membrane proteins into the liposomes. A simple, single step purification via centrifugation in a density gradient yielded proteoliposomes with the desaturase in high purity as judged by capillary electrophoresis. After in vitro reconstitution of the non-heme iron and heme active sites, the function of the reconstituted enzyme complex was demonstrated by conversion of stearoyl-CoA to oleoyl-CoA. This simple translation approach obviates the use of detergents or other lipids to stabilize and isolate a catalytically active integral membrane enzyme. The applicability of cell-free translation to the assembly and purification of other integral membrane protein complexes is discussed.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Catalysis
  • Cell-Free System / drug effects
  • Cytochromes b5 / metabolism
  • Detergents / pharmacology
  • Electrophoresis, Capillary
  • Genetic Vectors / genetics
  • Humans
  • Isoenzymes / isolation & purification
  • Liposomes / isolation & purification
  • Mice
  • Molecular Sequence Data
  • Mycobacterium / enzymology
  • Plant Extracts / analysis
  • Protein Biosynthesis* / drug effects
  • Stearoyl-CoA Desaturase / biosynthesis*
  • Stearoyl-CoA Desaturase / isolation & purification*
  • Triticum / metabolism*


  • Detergents
  • Isoenzymes
  • Liposomes
  • Plant Extracts
  • Cytochromes b5
  • Stearoyl-CoA Desaturase