Sequential Peptide Affinity (SPA) system for the identification of mammalian and bacterial protein complexes

J Proteome Res. May-Jun 2004;3(3):463-8. doi: 10.1021/pr034084x.

Abstract

A vector system is described that combines reliable, very low level, regulated protein expression in human cells with two affinity purification tags (Sequential Peptide Affinity, or SPA, system). By avoiding overproduction of the target protein, this system allows for the efficient purification of natural protein complexes and their identification by mass spectrometry. We also present an adaptation of the SPA system for the efficient purification and identification of protein complexes in E. coli and, potentially, other bacteria.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cells, Cultured
  • Electrophoresis, Polyacrylamide Gel
  • Escherichia coli / genetics
  • Fluorescent Antibody Technique
  • Humans
  • Mass Spectrometry
  • Multiprotein Complexes / genetics*
  • Multiprotein Complexes / isolation & purification
  • Multiprotein Complexes / metabolism
  • Plasmids / genetics*
  • Promoter Regions, Genetic / genetics
  • Protein Binding / physiology
  • Recombinant Proteins / genetics*
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Transcription Factors, TFII / genetics
  • Transcription Factors, TFII / isolation & purification
  • Transcription Factors, TFII / metabolism*

Substances

  • Multiprotein Complexes
  • Recombinant Proteins
  • Transcription Factors, TFII
  • transcription factor TFIIF