Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation

Yifeng Li, Rui Sousa

Research output: Contribution to journalArticlepeer-review

42 Scopus citations

Abstract

The extremely tight binding between biotin and avidin or streptavidin makes labeling proteins with biotin a useful tool for many applications. BirA is the Escherichia coli biotin ligase that site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (also known as Avi-tag). As a complementary approach to in vivo biotinylation of Avi-tag-bearing proteins, we developed a protocol for producing recombinant BirA ligase for in vitro biotinylation. The target protein was expressed as both thioredoxin and MBP fusions, and was released from the corresponding fusion by TEV protease. The liberated ligase was separated from its carrier using HisTrap HP column. We obtained 24.7 and 27.6 mg BirA ligase per liter of culture from thioredoxin and MBP fusion constructs, respectively. The recombinant enzyme was shown to be highly active in catalyzing in vitro biotinylation. The described protocol provides an effective means for making BirA ligase that can be used for biotinylation of different Avi-tag-bearing substrates.

Original languageEnglish (US)
Pages (from-to)162-167
Number of pages6
JournalProtein Expression and Purification
Volume82
Issue number1
DOIs
StatePublished - Mar 2012

Keywords

  • Biotinylation
  • BirA
  • Fusion expression
  • Maltose-binding protein
  • TEV protease cleavage
  • Thioredoxin

ASJC Scopus subject areas

  • Biotechnology

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