Rapid and Efficient Purification of Functional Collectin-12 and Its Opsonic Activity against Fungal Pathogens

Figure 2

Purification of recombinant CL-12. (a) Illustrative diagram for CL-12 purification. (b) Purification of recombinant CL-12 by Ni-NTA magnetic beads. rCL-12ED-expressed culture supernatants were incubated with Ni-NTA magnetic beads, followed by washing with buffer containing 50 mM imidazole and elution with buffer containing 250 mM imidazole. Purification samples were analyzed by 4~12% SDS-PAGE. Lane 1, loading material; lane 2, flow-through; lane 3, eluate. (c) Determination of impurities in the eluate. Supernatants from the cultivation of CHO/control were conducted with the same procedure of purification. The eluate of beads incubated with supernatants of CHO/control cultivation (lane 1) or rCL-12ED-expressed CHO cultivation (lane 2) was analyzed by 4~12% SDS-PAGE. (d) Impurity removal by an anion exchange spin column. CL-12-containing eluates were run onto an anion exchange spin column after removal of imidazole by a desalting spin column. Bound proteins were fractionated with a NaCl gradient (0~0.5 M) and analyzed by 4~12% SDS-PAGE. (e) Analysis of purified CL-12. CL-12-containing fractions seen in panel (d) were pooled, desalted, and concentrated to the desired concentration. The purity and oligomeric pattern were then determined in SDS-PAGE (e) and western blot (f) analysis. Lane 1, rCL-12R&D; lane 2, purified rCL-12ED. Arrow indicates the major rCL-12ED band.

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