Extended Data Figure 7. “On-column” glutaraldehyde cross-linking of the pre-formed complex.
a. Schematic representation of the “on-column” cross-linking strategy. A glutaraldehyde solution is injected to a size exclusion chromatography column, followed by injection of the purified complex protein. As the complex protein passes through the glutaraldehyde bolus, the receptor and the βarr components of the complex are cross-linked through proximal primary amine groups. This procedure allows only brief exposure of the complex to glutaraldehyde and serves as an “in-line” purification of homogenously cross-linked protein from any aggregation that may arise from non-specific cross-linking. b. “On-column” cross-linking of the T4L-β2V2R-βarr1-ScFv30 complex. Purified complex (approximately 20μM) was injected on to a 24mL Superdex 200 gel filtration column after a pre-injection of 200μL of 0.25% glutaraldehyde bolus. Individual fractions were collected and analyzed by “Simplyblue” stained SDS-PAGE. c. “On-column” cross-linking of the T4L-β2V2R-βarr1-Fab30 complex performed as described for the ScFv complex above.