Optimized <i>Legionella</i> expression strain for affinity purification of His-tagged membrane proteins eliminates major multimeric contaminant
Author(s)
Vijayrajratnam, Sukhithasri
Patkowski, Jonasz B
Khorsandi, Joshua
Beatty, Wandy L
Kannaiah, Shanmugapriya
Type
Journal Article
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, 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.
Editor(s)
Faucher, Sébastien P
Date Issued
2025-07-01
Date Acceptance
2025-04-10
Citation
Microbiology Spectrum, 2025, 13 (7)
ISSN
2165-0497
Publisher
American Society for Microbiology
Journal / Book Title
Microbiology Spectrum
Volume
13
Issue
7
Copyright Statement
© 2025 Vijayrajratnam et al. This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.
License URL
Identifier
10.1128/spectrum.03222-24
Publication Status
Published
Article Number
e03222-24
Date Publish Online
2025-05-19