In vivo biotinylated proteins as targets for phage-display selection experiments
Section snippets
Reagents
All plasmids were propagated and proteins were expressed in the E. coli strains XL1 Blue (Stratagene, La Jolla, CA) and BL21(DE3) (Novagen, Madison, WI), respectively. The pBirA plasmid was purchased from Avidity (Denver, CO) and E. coli XL1 Blue F′ tet host cells were from Stratagene (La Jolla, CA). Nickel–NTA agarose was purchased from Qiagen (Germantown, MD). Restriction endonucleases, T4 DNA polymerase, and Advantage cDNA polymerase were purchased from New England Biolabs (Beverly, MA),
Construction of pMCSG16 and pMCSG15
Two expression vectors were constructed to generate N- and C-terminal fusions for in vivo biotinylation. Vectors were generated by incorporating double-stranded oligonucleotides encoding the AviTag, a 15 amino acid long substrate for BirA, into the pMCSG7 plasmid [19]. The resulting recombinant, named pMCSG16 (Fig. 1A), encodes an N-terminal six histidine tag, AviTag, a (Gly–Ser)2 linker, the seven amino acid TEV protease cleavage site (ENLYFQS), and a LIC site. A second vector was constructed
Discussion
Biotin (vitamin H) is a small coenzyme that is synthesized by plants, most bacteria, and some fungi, and is primarily bound to protein in the cell. The BirA protein (biotin ligase) transfers biotin to the ε-amino group of specific lysine residue in an ATP-dependent process [29]. To define the substrate specificity of the BirA enzyme, Schatz [30] used phage display to find a 13-residue consensus sequence that is recognized and biotinylated by the enzyme. This sequence, and a 15-residue sequence
Acknowledgements
We acknowledge the editorial comments of Drs. John Kehoe, Ushma Kriplani, and Zhao Zhong Han. A special thanks to Dr. David Waugh (National Cancer Institute, Frederick, MD) for the TEV protease expression vector. This work was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences, under contract W-31-109-Eng-38.
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