Extended Data Fig. 6. Separation of a 95:5 mixture of Nd:Dy using immobilized Hans-LanM.
The desorption scheme consisted of three stepped concentrations of malonate (30, 50, 90 mM; see right axis) followed by pH 1.5 HCl. The results revealed that slightly lower purity Dy was generated using Hans-LanM compared to the R100K variant (83.6% vs 98% Dy purity at similar yield, respectively; compare to Fig. 4d). While similar selectivity profiles were observed for the immobilized proteins for La through Gd in equilibrium binding experiments with La-Dy, the selectivity pattern diverged at Tb (Fig. 4c). The selectivity difference between Hans-LanM and the R100K variant was confirmed by using a Nd/Dy binary system, as the uncertainties in the distribution factor determination for Dy in the 9-element RE group precluded the ability to distinguish small differences in the Dy/Nd separation factor between proteins (Supplementary Tables S13–S14). In this binary Nd/Dy experiment (Extended Data Table 3), we determined a separation factor of 8.12 ± 0.40 for Hans-LanM and 12.7 ± 1.3 for the R100K variant, which is consistent with the improved Dy separation efficacy of R100K. While consistent with the values derived from the 9-element experiment, the results differ slightly from the equilibrium binding results with the free Hans-LanM and Hans-LanM(R100K) proteins, which revealed similarly high selectivity for Nd over Dy (Fig. 4a,b), likely reflecting weaker LRE-induced dimerization in the R100K variant at the low protein concentration (20 µM) of the solution experiments with free protein. The La/Nd selectivity on-column is also distinct from that observed with the apparent Kd values of the free proteins (wild-type and R100K) in solution, although the experiments with free proteins utilized single element solutions and effects from mixed metal binding may impact the on-column data. The R100K variant is also better behaved on the column, as evidenced by the 2:1 RE:protein stoichiometry. One possible explanation for these results could be that immobilization interferes with dimerization; however, Fig. 2b shows the N- and C-termini of the Hans-LanM dimer, indicating that the C-termini are ~20 Å from the nearest part of the dimer interface, suggesting that immobilization per se would not be expected to disrupt this interface. It must be considered, however, that a functional dimer would require two C-termini to be immobilized in close proximity, which is unlikely at the immobilization densities of our columns. Therefore, on balance, we suspect that the dimerization equilibrium is only applicable in a minority of protein units immobilized on the column. We posit that more fully exploiting the dimerization equilibrium in the column format would yield even more robust separations. The surest way to obtain homogeneous populations of dimers on-column would likely be to link two monomers together (e.g., with a polypeptide chain), tuning dimerization affinity through mutagenesis of the residues contributing to inter-monomer interactions, and immobilizing this dimer through a single attachment point. Dimerization could also be exploited in other separation formats. These directions are the subject of current efforts.