Site-Specific Conjugation of a Temperature-Sensitive Polymer to a Genetically Engineered Protein

A genetically-engineered mutant of cytochrome b5, incorporating a unique cysteine residue, was conjugated to maleimide-terminated oligo(N-isopropylacrylamide). The conjugation of the protein by reaction of the cysteine residue, precisely positioned by site-directed mutagenesis techniques, with an activated oligomer containing only one reactive end group in the oligomer chain permits the site-specific and stoichiometric conjugation of the oligomer with the protein. The protein-oligomer conjugate was shown to exhibit lower critical solution temperature (LCST) behavior, similar to the free oligomer. Furthermore, the LCST behavior of the protein-oligomer conjugate is reversible and allows selective precipitation of the conjugate above its LCST.