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.

Figure 1

Plate: Fluorescence intensities of immobilized proteins and controls. GFP-C₂ – 1 µM (2f), 5 µM (1e, 1f), 10 µM (1c, 1d), 20 µM (1a, 1b); GFP-F – 15 µM (2a, 2b, 2d, 2e); GST-C₂ - 1 µM (3f), 5 µM (4e, 4f), 10 µM (4c, 4d), 20 µM (4a, 4b); GST-F – 15 µM (3a, 3b, 3d, 3e); GFP antibody (2c); GST antibody (3c). Plot: Average values of relative fluorescence intensity versus protein concentration. GFP proteins (); GST proteins (); controls ().

Figure 2

Fluorescence intensities of GFP-N₃ () immobilized by Staudinger ligation and the GFP-F () control after storage in buffer.

Scheme 1

Modification of proteins with CaaX Recognition Motifs

Scheme 2

Preparation of glass slides for protein immobilization by “click chemistry”