A second Ni affinity step was performed to remove the cleaved tags and the TEV protease.
The protein solution was diluted with 50 mM HEPES, pH 7.5, 500 mM NaCl, and 5% glycerol to dilute the imidazole followed by a second Ni-NTA affinity purification, and the flow through was collected.
A second Ni-NTA affinity step was used in conjunction with 30K Amicon Filters to concentrate the BASS.
The resulting 6His.tag-FPR fusion protein was purified using Ni-NTA chromatography, and, after thrombin treatment, a second affinity Ni-NTA chromatography separates the 6His.tag extension from the XacFPR protein.
The lysate was supplemented with 1 U/ml Benzonase (Sigma-Aldrich, St . Louis MO) and 1 mM PMSF, cleared by centrifugation, and loaded onto a Ni-affinity resin.
Subsequently, cells were broken by sonication and the cleared lysate was applied to a HisTrap Ni-affinity column and proteins eluted with a stepwise imidazol gradient.
The lysate was cleared from debris by centrifugation and then was loaded onto a Ni-affinity resin.
The U11-48K Zn-finger domain was first purified using Ni-affinity chromatography followed by TEV protease cleavage.
The recombinant lipase was purified using a Ni-affinity column according to the manufacturer's protocol.
Lane 5: Flow-through of the second Ni-NTA affinity column.
