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High Throughput Protein Expression and Purification

Volume 498 of the series Methods in Molecular Biology pp 185-198

High-Throughput Biotinylation of Proteins

  • Brian K. KayAffiliated withDepartment of Biological Sciences, University of Illinois at Chicago
  • , Sang ThaiAffiliated withDepartment of Biological Sciences, University of Illinois at Chicago
  • , Veronica V. VolginaAffiliated withDepartment of Biological Sciences, University of Illinois at Chicago

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Summary

One of the more useful tags for a protein in biochemical experiments is biotin, because of its femtomolar dissociation constant with streptavidin or avidin. Robust methodologies have been developed for other the in vivo addition of a single biotin to recombinant protein or the in vitro enzymatic or chemical addition of biotin to a protein. Such modified proteins can be used in a variety of experiments, such as affinity selection of phage-displayed peptides or antibodies, pull-down of interacting proteins from cell lysates, or displaying proteins on arrays. We present three complementary approaches for biotinylating proteins in vivo in Escherichia coli or in vitro using chemical or enzymatical reactions all of which can be scaled up to tag large numbers of proteins in parallel.

Key words:

Affinity selection Biotin Biotin ligase BirA E. coli Phage-display Protein labeling Protein—protein interactions Streptavidin Streptavidin coated magnetic beads