Chemoenzymatic Site-Specific ReversibleImmobilization and Labeling of Proteinsfrom Crude Cellular Extract WithoutPrior Purification Using Oxime andHydrazine Ligation

摘要

In a facile and potentially general method for protein modification at the C-terminus,aldehyde-modified proteins, obtained from enzymatic protein prenylation, react rapidlywith hydrazide and aminooxy surfaces and fluorophores at neutral pH and in micromolarconcentration ranges of reagents. This strategy was used for fluorescent labeling ofeGFP-CVIA, as a model protein, with aminooxy and hydrazide fluorophores or PEGs, andimmobilization onto and subsequent release of the protein from hydrazide-functionalizedagarose beads using hydrazone-oxime exchange. This method is described in detail hereand provides site-specifically PEGylated or fluorescently labeled proteins starting fromcrude cellular extract in three steps: prenylation, capture, and release.Curr.Protoc.Chem.