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.
Chilkoti
A
A
Center for Bioengineering, University of Washington, Seattle 98195.
Chen
G
G
Stayton
P S
PS
Hoffman
A S
AS
D000180:Acrylic Resins / Q000737:chemistry*; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry; D015786:Cytochromes b5 / Q000737:chemistry* / Q000235:genetics; D008301:Maleimides / Q000737:chemistry; D013058:Mass Spectrometry; D016297:Mutagenesis, Site-Directed; D011108:Polymers / Q000737:chemistry*; D015202:Protein Engineering; D013696:Temperature
D000180:Acrylic Resins; D008301:Maleimides; D011108:Polymers; C052970:poly-N-isopropylacrylamide; D015786:Cytochromes b5; D003545:Cysteine