Native using isolation tags

The advantage of such co-purification protocols is that the fully processed protein serving as the bait can allow interactions in a native environment and cellular location to allow isolation of multicomponent complexes. One limitation with this approach is the necessity for an antibody with specific immunoreactivity and immunoprecipitative capability for the bait protein. This drawback can be addressed by expression of the protein with an epitope tag. Excellent antibodies to a variety of epitope tags are available and can be utilized for immunoaffinity purification. Tags such as 6-histidine and GST allow purification using affinity characteristics to nickel and GSH beads, respectively. 

The first evidence of in vitro turnover was reported in a study from the Gronan and Marietta laboratories using both native LipA and a form containing a G-terminal hexahistidine tag. " Purification of the hexahis-tidine-tagged protein from the soluble fraction of crude lysate was performed under anaerobic conditions, and analysis of the resulting as-isolated protein, which was dark brown in color, revealed the presence of 3.4 0.4 atoms of iron and 4.8 0.8 atoms of sulfide per monomer. Subsequent EPR analysis of the dithionite-reduced protein again showed the presence of [4Fe S] clusters, although spin quantification indicated less than 5% of the reduced form. 

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