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Updated: Aug 18, 2026

Conjugative Mating Assays for Sequence-specific Analysis of Transfer Proteins Involved in Bacterial Conjugation
Published on: January 4, 2017
Site-specific conjugation of a temperature-sensitive polymer to a genetically-engineered protein
A Chilkoti1, G Chen, P S Stayton
1Center for Bioengineering, University of Washington, Seattle 98195.
Abstract:
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.
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