Development of a small peptide tag for covalent labeling of proteins

Abstract

A 21-mer peptide that can be used to covalently introduce synthetic molecules into proteins has been developed. Phage-displayed peptide libraries were subjected to reaction-based selection with 1,3-diketones. The peptide was further evolved by addition of a randomized region and reselection for improved binding. The resulting 21-mer peptide had a reactive amino group that formed an enaminone with 1,3-diketone and was used as a tag for labeling of maltose binding protein. Using this peptide tag and 1,3-diketone derivatives, a variety of molecules such as reporter probes and functionalities may be covalently introduced into proteins of interest.

Figure 1

Selection of peptides that form enaminones with diketones. (a) 1,3-Diketones and enaminone formation. (b) Peptide libraries and selected peptide sequences. Selection implies selection using phage display with 1-BSA immobilized on wells in a microtiter plate.

Figure 2

Binding assays of rpf1368-MBP to diketones. (a) ELISA with 1-BSA. The cell lysate of E. coli cells expressing the fusion protein (labeled as cell lysate) was evaluated, as were purified proteins. Data of binding to 1-BSA and to BSA (background) are shown: 1-BSA (gray), BSA (white). Deviations were within ±10% of the indicated values. (b) ELISA with 1-biotin. Data of binding to 1-biotin supported on NeutrAvidin and to NeutrAvidin (background) are shown: 1-Biotin/NeutrAvidin (gray), NeutrAvidin (white). Deviations were within ±10% of the indicated values.

Figure 3

Time course of enaminone formation of rpf1368-MBP with 2,4-pentanedione. Increase in absorption at 318 nm, characteristic of the enaminone, was monitored. Conditions: [rpf1368-MBP] 2 μM and [2,4-pentanedione] 500 μM in 2.5% CH₃CN/PBS. The second order rate constant k in this reaction {V (M/min) = k[2,4-pentanedione (M)][peptide rpf1368 (M)]} was 6.0 M^-1min^-1.