Selective immobilization of proteins to self-assembled monolayers presenting active site-directed capture ligands

Abstract

This paper describes a method for the selective and covalent immobilization of proteins to surfaces with control over the density and orientation of the protein. The strategy is based on binding of the serine esterase cutinase to a self-assembled monolayer presenting a phosphonate ligand and the subsequent displacement reaction that covalently binds the ligand to the enzyme active site. Surface plasmon resonance (SPR) spectroscopy showed that cutinase binds irreversibly to a monolayer presenting the capture ligand at a density of 1% mixed among tri(ethylene glycol) groups. The covalent immobilization is specific for cutinase, and the glycol-terminated monolayer effectively prevents unwanted nonspecific adsorption of proteins. To demonstrate that the method could be used to immobilize proteins of interest, a cutinase-calmodulin fusion protein was constructed and immobilized to the monolayer. SPR showed that calcineurin selectively associated with the immobilized calmodulin. This capture ligand immobilization method combines the advantages that the immobilization reaction is highly selective for the intended protein, the tether is covalent and, hence, stable, and the method avoids the need for synthetic modification and rigorous purification of proteins before immobilization. These characteristics make the method well suited to a range of applications and, in particular, for constructing protein microarrays.

Figure 1

(A) Strategy for protein immobilization. The protein of interest is fused to a capture protein, which specifically binds a capture ligand of the substrate to give covalent immobilization of proteins while maintaining activity and orientation. The density of immobilized protein can be controlled by adjusting the density of the ligand. (B) Structure of F. solani cutinase bound to an n-undecyl o-methyl chlorophosphonate ester inhibitor (11). The inhibitor is covalently bound through an active-site serine residue.

Scheme 1

Figure 2

Synthetic scheme for the phosphonate disulfide (6) used to prepare self-assembled monolayers. (a) CDI, CH₂Cl₂; (b) oxalyl chloride, CH₂Cl₂; 4-nitrophenol, Et₃N, CH₂Cl₂; (c) 5, Et₃N, DMF.

Figure 3

Structure of a monolayer designed to covalently immobilize cutinase. The phosphonate capture ligand is present at a density of ≈1% of total alkanethiolate. After cutinase binds to the phosphonate capture ligand, an active-site serine residue displaces p-nitrophenol, resulting in irreversible covalent immobilization of the protein. The tri(ethylene glycol) groups prevent nonspecific protein adsorption to the monolayer.

Figure 4

SPR spectroscopy data showing the biospecific and irreversible immobilization of cutinase to the phosphonate-tethered SAM shown in Fig. 3. Conditions are described in Materials and Methods. The change in resonance angle (Δθ) is plotted on the vertical axis. The scale bar applies to all data, which are offset for clarity. (A) Cutinase (25 μM) was irreversibly immobilized to the monolayer. (B) Incubation of cutinase with four equivalents of soluble inhibitor 1 before immobilization completely inhibited immobilization of the protein. (C) Cutinase within crude E. coli periplasmic lysate also underwent efficient immobilization, demonstrating that the protein does not need to be purified before immobilization. (D) An analogous experiment using periplasmic lysate without cutinase resulted in no immobilization and demonstrates that the monolayer is resistant to nonspecific protein adsorption.

Figure 5

SPR data for binding of calcineurin to surfaces presenting calmodulin (CaM). (A) The Cut-CaM fusion (1 μM) irreversibly immobilized to SAMs presenting the phosphonate capture ligand. (B) Calcineurin (0.7 μM, 0.2 mM CaCl₂) binds to immobilized CaM in the presence of Ca²⁺ ions. (C) Calcineurin is unable to bind CaM in the presence of the divalent ion chelator EGTA (2 mM), demonstrating the known requirement of Ca²⁺ for binding. (D) Incubation of calcineurin with soluble CaM (2 μM) also prevents binding of calcineurin, showing that the interaction between immobilized CaM and calcineurin is biospecific. (E) calcineurin does not bind to a surface presenting cutinase alone, demonstrating that calcineurin binding is not mediated by cutinase.