Conformational antibody binding to a native, cell-free expressed GPCR in block copolymer membranes.

G-protein coupled receptors (GPCRs) play a key role in physiological processes and are attractive drug targets. Their biophysical characterization is, however, highly challenging because of their innate instability outside a stabilizing membrane and the difficulty of finding a suitable expression system. We here show the cell-free expression of a GPCR, CXCR4, and its direct embedding in diblock copolymer membranes. The polymer-stabilized CXCR4 is readily immobilized onto biosensor chips for label-free binding analysis. Kinetic characterization using a conformationally sensitive antibody shows the receptor to exist in the correctly folded conformation, showing binding behaviour that is commensurate with heterologously expressed CXCR4

Sensorgrams of mAb 12G5 binding (100 nM) to CXCR4-ACMs immobilized at low RU (ca. 1400) via biotin/spteptavidin immobilization of the embedding polymersome matrix.

<p>The analyte was injected in triplicate, at cycle 7, 14, and 21. Intermediate cycles involved blank injections. The blue line shows the fit to the curve assuming 1∶1 binding kinetics. The inset shows the relative decrease in binding activity of the surface as measured by the binding level 4 s before the end of the injection.</p

Kinetic parameters extracted from fitting the binding curves of concentration series of three different mAbs against a single preparation of immobilized CXCR4 ACMs.

[a]<p>KD for 12G5 binding to CXCR4-ACMS shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0110847#pone-0110847-g002" target="_blank">Figure 2</a> was 60.2±17 nM.</p><p>Kinetic parameters extracted from fitting the binding curves of concentration series of three different mAbs against a single preparation of immobilized CXCR4 ACMs.</p

Kinetic screening of 12G5 mAb binding to CXCR4-ACMs immobilized onto biosensor chips.

<p>A: Ab was injected at increasing concentrations (6.25–400 nM) over 100 s, followed by a buffer wash (without regeneration) between injections (immobilization level: ca. 5000 RU; biotin/streptavidin immobilization). B. Saturation binding of 125-I SDF1α to CXCR4-ACMs. A dissociation constant of 8.4 nM was determined. C. The same series of measurements as shown in Fig. 2 A, conducted using immobilized VLPS (immobilization level: 5000 RU).</p