title: Regio- and chemoselective covalent immobilization of proteins through unnatural amino acids.
abstract: A general approach was developed for the regio- and chemoselective covalent immobilization of soluble proteins on glass surfaces through an unnatural amino acid created by post-translationally modifying the cysteine residue in a CaaX recognition motif with functional groups suitable for "click" chemistry or a Staudinger ligation. Farnesyl diphosphate analogues bearing omega-azide or omega-alkyne moieties were attached to the cysteine residue in Cys-Val-Ile-Ala motifs at the C-termini of engineered versions of green fluorescent protein (GFP) and glutathione S-transferase (GST) by protein farnesyltransferase. The derivatized proteins were attached to glass slides bearing linkers containing azide ("click" chemistry) or phosphine (Staudinger ligation) groups. "Click"-immobilized proteins were detected by fluorescently labeled antibodies and remained attached to the slide through two cycles of stripping under stringent conditions at 80 degrees C. GFP immobilized by a Staudinger ligation was detected by directly imagining the GFP fluorophore over a period of 6 days. These methods for covalent immobilization of proteins should be generally applicable. CaaX recognition motifs can easily be appended to the C-terminus of a cloned protein by a simple modification of the corresponding gene, and virtually any soluble protein or peptide bearing a CaaX motif is a substrate for protein farnesyltransferase.
authors.affiliation: Department of Chemistry, University of Utah, 315 South 1400 East, Room 2020, Salt Lake City, Utah 84112, USA.
authors.forename: Cécile
authors.initials: C
authors.lastname: Gauchet
authors.forename: Guillermo R
authors.initials: GR
authors.lastname: Labadie
authors.forename: C Dale
authors.initials: CD
authors.lastname: Poulter
mesh_terms: D000596:Amino Acids / Q000737:chemistry*; D008958:Models, Molecular; D040081:Protein Array Analysis / Q000379:methods*; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*
chemical_list: D000596:Amino Acids; D011506:Proteins