Optimized <i>Legionella</i> expression strain for affinity purification of His-tagged membrane proteins eliminates major multimeric contaminant.

ABSTRACT: 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, wh

SUBMITTER: Vijayrajratnam S 

PROVIDER: S-EPMC12211046 | biostudies-literature | 2025 Jul

REPOSITORIES: biostudies-literature