Figure 2. Purification of Recombinant Mical^redoxCH protein.

(A) Coomassie-stained bands (arrow) corresponding to the Nus-tagged Mical^redoxCH protein were observed in multiple collection tubes following IPTG-induced bacterial expression, lysis, Ni²⁺-NTA affinity chromatography, and elution with 250 mM imidazole. MW in kDa (also for B-D).

(B) Samples from (A) containing Ni²⁺-NTA affinity purified Nus-tagged Mical^redoxCH protein were combined and subjected to thrombin digestion (+) to remove the Nus tag. A Coomassie-stained band corresponding in size to the cleaved Mical^redoxCH protein was observed following thrombin digestion (arrow). Note, that uncleaved Nus-tagged Mical^redoxCH protein is readily seen in the absence of thrombin (arrowhead).

(C) As seen following Coomassie-staining, Cation exchange chromatography was used to separate Mical^redoxCH protein (arrow) from contaminating proteins, including chaperonins and the Nus tag. Proteins were eluted with a gradient of NaCl by increasing the percentage of S-B buffer. Sample tubes 5 through 13 (*) were utilized for (D).

(D) Gel filtration chromatography allowed the further purification of the Mical^redoxCH protein as seen following Coomassie-staining such that the protein collected in tubes 7 and 8 (*) was combined, concentrated to a working volume, and utilized for the results presented in Figures 3 and 4. We find an overall yield of ~2 mg of highly-pure active Mical^redoxCH protein can be routinely generated from 6 L of bacterial culture using this purification strategy and the starting culture volume can be scaled-up in order to purify larger amounts of Mical^redoxCH protein.