A generic tool for biotinylation of tagged proteins in transgenic mice.
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.
Driegen
Siska
S
Department of Cell Biology and Genetics, Erasmus MC, Rotterdam, The Netherlands.
Ferreira
Rita
R
van Zon
Arend
A
Strouboulis
John
J
Jaegle
Martine
M
Grosveld
Frank
F
Philipsen
Sjaak
S
Meijer
Dies
D
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
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