The SNAP-tag technology revised: an effective chemo-enzymatic approach by using a universal azide-based substrate

Abstract

SNAP- tag ® is a powerful technology for the labelling of protein/enzymes by using benzyl-guanine (BG) derivatives as substrates. Although commercially available or ad hoc produced, their synthesis and purification are necessary, increasing time and costs. To address this limitation, here we suggest a revision of this methodology, by performing a chemo-enzymatic approach , by using a BG-substrate containing an azide group appropriately distanced by a spacer from the benzyl ring. The SNAP- tag ® and its relative thermostable version ( Ss OGT- H 5 ) proved to be very active on this substrate. The stability of these tags upon enzymatic reaction makes possible the exposition to the solvent of the azide-moiety linked to the catalytic cysteine, compatible for the subsequent conjugation with DBCO-derivatives by azide-alkyne Huisgen cycloaddition. Our studies propose a strengthening and an improvement in terms of biotechnological applications for this self-labelling protein-tag .

First thermostable CLIP-tag by rational design applied to an archaeal O-alkyl-guanine-DNA-alkyl-transferase

“…First, by using fluorescent substrates in competition with customised non-fluorescent nucleobases (BG-1 and BC-2, Fig. S2 and S4 ) [15] , [27] , IC 50 values were determined. As shown in Table 2 , all enzymes particularly prefer fluorescent substrates respect to our customised product ( Fig.…”

“…Subsequently, it was treated in ice with lysozyme and DNAse for 60 min and sonicated as described [14] . After a centrifugation of 30 min at 60,000 × g , the cell extract was recovered and applied to a Protino Ni-NTA Column 1 mL (Macherey-Nagel) for His 6 -tag affinity chromatography, accordingly to the procedure previously described [27] . The eluted protein fractions were collected, dialysed against phosphate buffered saline (PBS 1×, 20 mM phosphate buffer, NaCl 150 mM, pH 7.3) and confirmed by SDS-PAGE analysis.…”

“…The substrate specificity of CLIP- tag , Ss OGT- H 5 and Ss OGT- MC 8 proteins were evaluated on BG-N 3 and BC-N 3 substrates by the competitive inhibition assay performed as described [15] , [27] , [29] . By using fixed concentrations of the fluorescent BC-TMR substrate (5 μM) and enzymes (5 μM), an increasing concentration of guanine/cytosine-azide derivatives (0–1 mM) was added to the mixtures.…”

Folding‐upon‐Repair DNA Nanoswitches for Monitoring the Activity of DNA Repair Enzymes

“…The measured inactivation, as expected, follows a concentration‐dependent behaviour with a measured IC 50 =3.5±2 μM (Figure 5 c). Moreover, our platform is able to measure the inactivation efficiency of different molecules including 4‐azido‐ N ‐(4‐(hydroxymethyl) benzyl) butanamide (BGN3), ( N ‐(4‐(((2‐amino‐9 H ‐purin‐6‐yl)oxy)methyl)benzyl)‐4‐azidobutanamide (BGSN), Lomeguatrib, [40, 41] O 6 ‐Benzylguanine ( O 6 ‐BG), [42] demonstrating once again its versatility (Figure 5 d).…”

“…[38,39] Therelative FRET signal of the nanoswitch incubated with hMGMT and O 6 -BG (Figure 5b,g rey line) is,a t0 .8 AE 0.1, similar to the values observed in the absence of enzyme (Figure 5b,b lack line) an effect consistent with the inactivation of hMGMT activity by O 6 -BG.T he measured inactivation, as expected, follows ac oncentration-dependent behaviour with am easured IC 50 = 3.5 AE 2 mM( Figure 5c). Moreover,o ur platform is able to measure the inactivation efficiency of different molecules including 4-azido-N-(4-(hydroxymethyl) benzyl) butanamide (BGN3), (N-(4-(((2-amino-9H-purin-6-yl)oxy)methyl)benzyl)-4-azidobutanamide (BGSN), Lomeguatrib, [40,41] O 6 -Benzylguanine (O 6 -BG), [42] demonstrating once again its versatility (Figure 5d).…”