William Neuberger, “Protein Purification of His-Tagged Cytochrome c nitrite reductase Variants”
Mentor: Arsenio Pacheco, Chemistry & Biochemistry
Poster #134

Cytochrome c nitrite reductase (ccNiR) is a multi-heme enzyme that enables certain microorganisms to interconvert ammonia and nitrite, and the Pacheco group seeks to understand it’s reaction mechanism. This presentation will summarize recent efforts to purify a variant of ccNiR using immobilized metal affinity chromatography (IMAC). The IMAC technique utilizes an agarose-gel column with chelating groups to immobilize divalent metal ions to which the ccNIR variant has an affinity. In this way, the immobilized ions capture and bind the ccNiR to the column. A loading buffer is used to elute anything that is non-specifically bound to the column, leaving only the desired protein on the column. The immediate goal of the project was to optimize the method for stripping the ccNiR from the column. In the past, ccNiR had been stripped from the column using 500 mM imidazole. However, if imidazole is not promptly removed by dialysis after elution, ccNiR loses activity. As an alternative, 50 mM EDTA was tested as a stripping agent. EDTA strongly binds divalent metals and is routinely used to remove these from IMAC columns during column cleaning. However, bound ccNiR was not removed from the column by EDTA, perhaps because the protein prevents the EDTA from readily accessing the metal. At this point, it is unclear why imidazole can successfully strip ccNiR from the column whereas EDTA cannot; we speculate that the smaller imidazole molecule can more readily access the binding site. Future studies are planned that will include loading the IMAC column with different divalent metals. For the present study, the column was charged with Ni²⁺, which is most common. However, other metals such as Co²⁺, Cu²⁺, or Zn²⁺, can also be used. Co²⁺ and Zn²⁺ typically bind proteins more weakly than Ni²⁺, and so may make it easier to recover the bound ccNiR.