Optimized Legionella expression strain for affinity purification of His-tagged membrane proteins eliminates major...

Polyhistidine tags are frequently used for isolating proteins through nickel-nitrilotriacetic acid (Ni-NTA) affinity purification. However, proteins rich in histidine can also bind to the Ni-NTA resin, leading to contamination of the purification with undesired proteins. While attempting to purify the Legionella pneumophila Dot/Icm type IVB secretion system complex for single particle analysis, we encountered an unknown contaminant protein that bound to the Ni-NTA resin and formed uniform particles visible in negative stain electron microscopy (EM). Mass spectrometry identified this protein, which is encoded by the Legionella gene lpg1596 as a homolog of enoyl-CoA hydratase. Modeling of Lpg1596 revealed surface-exposed histidine clusters, which likely explains its ability to bind to the Ni-NTA resin. Moreover, since enoyl-CoA hydratase homologs are known to multimerize, multimers of Lpg1596 would be large enough to be visible by negative stain EM. To address the problematic issue of Lpg1596 binding to the Ni-NTA resin, we constructed and analyzed a L. pneumophila ∆lpg1596 mutant strain. Notably, Ni-NTA affinity purification of lysates from the ∆lpg1596 strain did not contain the contaminant protein or generate observable particles. Since the ∆lpg1596 mutant strain exhibited replication capabilities similar to the wild-type L. pneumophila in macrophages, its deletion will likely not affect pathogenesis studies. To facilitate the deletion of lpg1596 in other Legionella strains, we developed a set of natural transformation vectors with various antibiotic resistance markers. In summary, we present a strategy for removing a common Ni-NTA resin binding protein contaminant in L. pneumophila, which improves single particle analysis outcomes.IMPORTANCENi-NTA purifications are a common method for isolating proteins with a His-tag, but they have a drawback: they often enrich unwanted proteins that are rich in histidines, which can contaminate the sample. We identified one such contaminant in Legionella pneumophila, a protein with homology to enoyl-CoA hydratases (Lpg1596). This protein binds to the Ni-NTA resin and forms particles that are observable in electron microscope (EM) images, interfering with the analysis. By removing the gene responsible for making this protein (lpg1596), the problem was solved, and no unwanted particles appeared in the EM images. The ∆lpg1596 mutant strain is the first optimized strain for purifying His-tagged membrane proteins from Legionella, which can be used for further studies like single particle analysis.