A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells

John D. Kulman, Masanobu Satake, Jeff E. Harris

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)


We have developed a system for producing biotinylated recombinant proteins in mammalian cells. The expression construct consists of an inducible tetracycline response element (TRE) that drives expression of a bicistronic cassette comprising a biotin acceptor peptide (BioTag) fused to either terminus of the target protein, the gene for Escherichia coli biotin ligase (BirA), and an intervening internal ribosome entry site (IRES). By either transient or stable transfection of Chinese hamster ovary (CHO) Tet-On cells, we successfully expressed, detected, and immobilized biotinylated human Itch, a pleiotropic multi-domain ubiquitin-protein ligase, as well as Gla-RTK, a putative vitamin K-dependent receptor tyrosine kinase. The biotinylation of recombinant Itch in transiently transfected CHO Tet-On cells required biotin supplementation and coexpression of BirA, occurred quantitatively and specifically on the lysine residue of the BioTag, and enabled detection of Itch by Western blot in as little as 10 ng of total lysate protein. Stably selected clones were rapidly pre-screened for doxycycline (dox)-inducible BirA expression by ELISA, and subsequently screened for dox-inducible expression of biotinylated Itch. Biotinylated Gla-RTK was detectable in as little as 5 ng of total lysate protein from transiently transfected CHO Tet-On cells, and exhibited pronounced tyrosine phosphorylation. In stable clones however, constitutive phosphorylation was prevented by reducing the expression level of Gla-RTK through the titration of dox. These results demonstrate the utility of this system for the expression of 'difficult' proteins, particularly those that are cytotoxic or those that may require lower expression levels to ensure appropriate post-translational modification.

Original languageEnglish
Pages (from-to)320-328
Number of pages9
JournalProtein Expression and Purification
Issue number2
Publication statusPublished - 2007 Apr
Externally publishedYes


  • Avidin
  • Biotin
  • Doxycycline-regulated expression
  • Receptor tyrosine kinase
  • Streptavidin
  • Ubiquitin-protein ligase

ASJC Scopus subject areas

  • Biotechnology


Dive into the research topics of 'A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells'. Together they form a unique fingerprint.

Cite this