Figure 2.

Overview of two schemes for polyprotein functionalization and surface attachment based upon (a) a directional and (b) a dual-aldehyde construct. The polyprotein consisted of two repeats of two NuG2 (green circles) flanking the protein of interest (blue star). To generate an aldehyde, a 6-amino-acid tag (LCTPSR, purple rectangle) was introduced into the DNA coding for the polyprotein (not shown). After expression and purification, the polyprotein was reacted overnight (o/n) with formylglycine-generating enzyme (FGE), which converts the cysteine only in the short tag into a formylglycine (fGly), an aldehyde-containing amino acid. Next, the aldehyde at one or both ends of the polyprotein was functionalized with a HIPS-based reagent. In both constructs, one end was labelled with a biotin and, in the right column, the other end with a DBCO, a copperless click chemistry reagent that reacts with azide (N₃) moieties. If desired, the directional construct could be directly linked to a maleimide functionalized surface using a terminal cysteine or subsequently DBCO-labelled for linkage to an azide-functionalized surface. Note that while the dualaldehyde construct only yields a fraction of polyproteins with both DBCO and biotin labels, only such heterobifunctionally labeled proteins are efficiently stretched between an azide-functionalized surface and a streptavidin-coated tip.