Protein Affinity Purification using Intein/Chitin Binding Protein Tags

Sarah F. Mitchell, Jon R. Lorsch

Research output: Contribution to journalArticle

Abstract

Isolation of highly purified recombinant protein is essential for a wide range of biochemical and biophysical assays. Affinity purification in which a tag is fused to the desired protein and then specifically bound to an affinity column is a widely used method for obtaining protetin of high purity. Many of these methods have the drawbacks of either leaving the recombinant tag attached to the protein or requiring the addition of a protease which then must be removed by further chromatographic steps. The fusion of a self-cleaving intein sequence followed by a chitin-binding domain (CBD) allows for one-step chromatographic purification of an untagged protein through the thiol-catalyzed cleavage of the intein sequence from the desired protein. The affinity purification is highly specific and can yield pure protein without any undesired N- or C-terminal extensions. This protocol is based on the IMPACT™-System (intein mediated purification with an affinity chitin-binding tag) marketed by New England Biolabs.

Original languageEnglish (US)
Pages (from-to)111-125
Number of pages15
JournalMethods in Enzymology
Volume559
DOIs
StatePublished - Jun 20 2015
Externally publishedYes

Fingerprint

Inteins
Chitin
Purification
Carrier Proteins
Proteins
New England
Recombinant Proteins
Sulfhydryl Compounds
Assays
Peptide Hydrolases
Fusion reactions

Keywords

  • CBD
  • Cell harvesting
  • Cell lysis
  • Chitin beads
  • IMPACT™-System
  • Intein/Chitin binding protein tags
  • Protein affinity purification

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Protein Affinity Purification using Intein/Chitin Binding Protein Tags. / Mitchell, Sarah F.; Lorsch, Jon R.

In: Methods in Enzymology, Vol. 559, 20.06.2015, p. 111-125.

Research output: Contribution to journalArticle

Mitchell, Sarah F. ; Lorsch, Jon R. / Protein Affinity Purification using Intein/Chitin Binding Protein Tags. In: Methods in Enzymology. 2015 ; Vol. 559. pp. 111-125.
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