A versatile system for site-specific enzymatic biotinylation and regulated expression of proteins in cultured mammalian cells.
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 10ng 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 5ng 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.
Kulman
John D
JD
Department of Biochemistry, University of Washington, Seattle, WA 98195, USA. jkulman@u.washington.edu
Satake
Masanobu
M
Harris
Jeff E
JE
D000595:Amino Acid Sequence; D000818:Animals; D001360:Avidin / Q000378:metabolism; D001710:Biotin / Q000378:metabolism*; D019921:Biotinylation; D016466:CHO Cells; D002478:Cells, Cultured; D006224:Cricetinae; D003412:Cricetulus; D004318:Doxycycline / Q000494:pharmacology; D005786:Gene Expression Regulation / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D014162:Transfection; D044767:Ubiquitin-Protein Ligases / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D012097:Repressor Proteins; D001360:Avidin; D001710:Biotin; C432966:ITCH protein, human; D044767:Ubiquitin-Protein Ligases; D004318:Doxycycline