Directed Supramolecular Surface Assembly of SNAP‐tag Fusion Proteins

Abstract

Supramolecular assembly of proteins on surfaces and vesicles was investigated by site-selective incorporation of a supramolecular guest element on proteins. Fluorescent proteins were site-selectively labeled with bisadamantane by SNAP-tag technology. The assembly of the bisadamantane functionalized SNAP-fusion proteins on cyclodextrin-coated surfaces yielded stable monolayers. The binding of the fusion proteins is specific and occurs with an affinity in the order of 10(6) M(-1) as determined by surface plasmon resonance. Reversible micropatterns of the fusion proteins on micropatterned cyclodextrin surfaces were visualized by using fluorescence microscopy. Furthermore, the guest-functionalized proteins could be assembled out of solution specifically onto the surface of cyclodextrin vesicles. The SNAP-tag labeling of proteins thus allows for assembly of modified proteins through a host-guest interaction on different surfaces. This provides a new strategy in fabricating protein patterns on surfaces and takes advantage of the high labeling efficiency of the SNAP-tag with designed supramolecular elements.

Immobilization of Ferrocene-Modified SNAP-Fusion Proteins

“…CFP was expressed as a C -terminal SNAP-tag fusion protein in E. coli (see the Experimental Section). This fluorescent SNAP-fusion protein was labeled with the supramolecular moieties (see Section 2.1) by reacting it with a 5 to 6-fold excess of either of the benzylguanine derivatives [26]. Within two hours at 37 °C the reactions had reached completion as determined by LC/MS (Figures 6 and 7).…”

“…CD-vesicles were prepared as described in the literature [26]. Fluorescence microscopy was done with a CKX41 inverted microscope from Olympus equipped with a Hg-lamp (U-RFL-T from Olympus).…”

“…SNAP-tag technology is a promising candidate for in vivo functionalization of proteins expressed within cells or for pull-down assays. Recently, we have adopted this strategy to label fluorescent proteins with adamantanes [26]. Here we want to combine the high chemical specificity and reactivity of the SNAP-tag fusion proteins with the β-cyclodextrin (β-CD)- and cucurbit [7]uril (CB [7])-ferrocene host-guest interaction for rapid and facile entries for site-selective and reversible protein immobilization.…”

Redox-active host-guest supramolecular assemblies of peptides and proteins at surfaces

“…For single, site-specific labelling we employed SNAP-tag technology, which is a genetically encoded protein tag, to efficiently label proteins with guest moieties featuring an O 6 -benzylguanine moiety such as adamantane [14] and redoxactive ferrocene [15]. In the case of the ferrocene, ferrocene-substituted benzylguanine was covalently attached to a cysteine residue within the guanine binding pocket of the SNAP-tag (Fig.…”

Engineered SNAP-MBD2b proteins for specific recognition of methylated DNA

“…It is an engineered variant based on human repair protein O 6 -alkylguanine-DNA-alkyltransferase (hAGT) that covalently reacts with O 6 -benzyl guanine (BG) derivatives [16,17]. SNAP-tag technique has been described not only for the study of the interactions between proteins, immobilization on solid surface, and pull-down assays, but also for the subcellular localizing such as the nucleus, endoplasmic reticulum [17][18][19][20][21].…”