Novel system for in vivo biotinylation and its application to crab antimicrobial protein scygonadin

Yifeng Li, Rui Sousa

Research output: Contribution to journalArticlepeer-review

6 Scopus citations


BirA is a biotin ligase from Escherichia coli that specifically biotinylates a lysine side-chain within a 15-amino acid acceptor peptide (also known as Avi-tag). We developed a protocol for producing recombinant BirA ligase in E. coli for in vitro biotinylation (Li and Sousa, Prot Expr Purif, 82:162-167, 2012) in which the target protein was expressed as both thioredoxin and MBP fusions, and was released by TEV protease-mediated cleavage. The liberated ligase and the fusion proteins were enzymatically active. Based on that observation, we have now developed a novel system for in vivo biotinylation by co-expressing the Avi-tagged target protein with the MBP-BirA fusion. The effectiveness of this system was demonstrated by the successful in vivo labeling of antimicrobial protein, scygonadin. This new system shows improved efficiency compared with pre-existing one and this is likely attributed to the high expression level and solubility of the co-expressed MBP-BirA.

Original languageEnglish (US)
Pages (from-to)1629-1635
Number of pages7
JournalBiotechnology Letters
Issue number9
StatePublished - Oct 2012


  • Biotinylation
  • BirA
  • Co-expression
  • Fusion protein
  • Thioredoxin
  • Thrombin cleavage

ASJC Scopus subject areas

  • Biotechnology
  • Bioengineering
  • Applied Microbiology and Biotechnology


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