title: A generic tool for biotinylation of tagged proteins in transgenic mice.
abstract: The remarkable high affinity (Kd approximately 10(-15) M) of avidin/streptavidin for biotin has been extensively exploited in purification methodologies. Recently a small peptide sequence (Avi-tag) has been defined that can be specifically and efficiently biotinylated by the bacterial BirA biotin ligase. Fusion of this small peptide sequence to a protein of interest and co-expression with the BirA gene in mammalian cells allowed purification of the biotinylated protein together with its associated proteins and other molecules. Ideally, one would like to apply these technologies to purify tagged proteins directly from mouse tissues. To make this approach feasible for a large variety of proteins we developed a mouse strain that expresses the BirA gene ubiquitously by inserting it in the ROSA26 locus. We demonstrate that the BirA protein is indeed expressed in all tissues tested. In order to demonstrate functionality we show that it biotinylates the transgene-encoded Avi-tagged Gata1 and Oct6 transcription factors in erythroid cells of the foetal liver and Schwann cells of the peripheral nerve respectively. Therefore, this mouse can be crossed to any transgenic mouse to obtain efficient biotinylation of an Avi-tagged protein for the purpose of protein (complex) purification.
authors.affiliation: Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands.
authors.forename: Siska
authors.initials: S
authors.lastname: Driegen
authors.forename: Rita
authors.initials: R
authors.lastname: Ferreira
authors.forename: Arend
authors.initials: A
authors.lastname: van Zon
authors.forename: John
authors.initials: J
authors.lastname: Strouboulis
authors.forename: Martine
authors.initials: M
authors.lastname: Jaegle
authors.forename: Frank
authors.initials: F
authors.lastname: Grosveld
authors.forename: Sjaak
authors.initials: S
authors.lastname: Philipsen
authors.forename: Dies
authors.initials: D
authors.lastname: Meijer
mesh_terms: D000818:Animals; D019921:Biotinylation / Q000379:methods*; D019731:Carbon-Nitrogen Ligases / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D005260:Female; D050982:GATA1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D005822:Genetic Vectors; D008099:Liver / Q000166:cytology / Q000196:embryology / Q000378:metabolism; D051379:Mice; D008822:Mice, Transgenic; D050816:Octamer Transcription Factor-6 / Q000235:genetics / Q000378:metabolism; D011506:Proteins / Q000235:genetics*; D022661:RNA, Untranslated; D012097:Repressor Proteins / Q000235:genetics* / Q000378:metabolism; D012583:Schwann Cells / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
chemical_list: D029968:Escherichia coli Proteins; D050982:GATA1 Transcription Factor; C494689:Gata1 protein, mouse; C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D011506:Proteins; D022661:RNA, Untranslated; D012097:Repressor Proteins; D014157:Transcription Factors; D050816:Octamer Transcription Factor-6; D019731:Carbon-Nitrogen Ligases; C058125:birA protein, E coli