Single-column Purification of Free Recombinant Proteins Using a Self-Cleavable Affinity Tag Derived From a Protein Splicing Element

Gene. 1997 Jun 19;192(2):271-81. doi: 10.1016/s0378-1119(97)00105-4.

Abstract

A novel protein purification system has been developed which enables purification of free recombinant proteins in a single chromatographic step. The system utilizes a modified protein splicing element (intein) from Saccharomyces cerevisiae (Sce VMA intein) in conjunction with a chitin-binding domain (CBD) from Bacillus circulans as an affinity tag. The concept is based on the observation that the modified Sce VMA intein can be induced to undergo a self-cleavage reaction at its N-terminal peptide linkage by 1,4-dithiothreitol (DTT), beta-mercaptoethanol (beta-ME) or cysteine at low temperatures and over a broad pH range. A target protein is cloned in-frame with the N-terminus of the intein-CBD fusion, and the stable fusion protein is purified by adsorption onto a chitin column. The immobilized fusion protein is then induced to undergo self-cleavage under mild conditions, resulting in the release of the target protein while the intein-CBD fusion remains bound to the column. No exogenous proteolytic cleavage is needed. Furthermore, using this procedure, the purified free target protein can be specifically labeled at its C-terminus.

MeSH terms

  • Affinity Labels
  • Amino Acid Sequence
  • Base Sequence
  • Carrier Proteins / chemistry
  • Chitin
  • Genetic Vectors*
  • Hydrogen-Ion Concentration
  • Maltose-Binding Proteins
  • Methods
  • Molecular Sequence Data
  • Protein Processing, Post-Translational*
  • Protein Splicing*
  • Proton-Translocating ATPases / metabolism
  • Recombinant Proteins / isolation & purification*
  • Temperature
  • Vacuolar Proton-Translocating ATPases*

Substances

  • Affinity Labels
  • Carrier Proteins
  • Maltose-Binding Proteins
  • Recombinant Proteins
  • Chitin
  • Vacuolar Proton-Translocating ATPases
  • Proton-Translocating ATPases