William Neuberger, “Purification of His-Tagged Cytochrome C Nitrite Reductase Variants”
Mentor: Andy Pacheco, Chemistry & Biochemistry, Letters & Science (College of)
Poster #161
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 its 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, varying concentrations of EDTA were tested as a stripping agent. EDTA strongly binds divalent metals and is routinely used to remove these from IMAC columns during column cleaning. However, 50 mM of EDTA failed to elute ccNiR, and EDTA was found to be insoluble at concentrations much above 200 mM. We were also concerned that very high EDTA concentrations might rip an essential calcium ion from ccNiR, and a future activity assay will confirm or deny that. In a separate study, the IMAC column was loaded with Co2+ instead of the more common Ni2+, but the Co2+ didn’t hold the protein.