Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease from<i>Pseudomonas aeruginosa</i>

Nji, Emmanuel; Li, Dianfan; Doyle, D.A.; Caffrey, Martin

doi:10.1107/s2053230x14017865

Cited by 5 publications

(2 citation statements)

References 23 publications

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“…Several attempts to obtain well-diffracting crystals of LysP failed 40,41 . To obtain stable protein for structure determination, camelid nanobodies were generated and screened against LysP (Extended Data Fig.…”

Section: Cryo-em Structure Determinationmentioning

confidence: 99%

Structural insights intoPseudomonas aeruginosalysine-specific uptake mechanism for extremely low pH regulation

Bicer,

Matsuoka,

Moumbock

et al. 2024

Preprint

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Under conditions of extremely low pH, in addition to transporting lysine, bacterial lysine–specific permease (LysP) interacts with the transcriptional regulator CadC to upregulate cadBA operon expression. cadBA encodes CadA, which decarboxylates lysine to cadaverine, and CadB, which exports cadaverine to the environment to reduce acidity. This process is crucial for survival of pathogenic bacteria in their hosts. Here, we report the inward-occluded (3.2 – 5.3 Å) cryo-EM structure of Pseudomonas aeruginosa LysP bound to L-lysine and in complex with a nanobody. L–lysine is coordinated by hydrophobic stacking, cation-π interactions and hydrogen bonding mostly with polar uncharged LysP residues. LysP reconstituted into liposomes showed robust and specific transport of L-lysine with the transport being inhibited by L–4–thialysine (S–2–aminoethyl–L–cysteine). These findings inform our understanding of the specific recognition, inhibition, and transport mechanism of L–lysine by LysP, which will have important ramifications for the design of antibiotics to target bacterial LysP.

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Section: Cryo-em Structure Determinationmentioning

confidence: 99%

Structural insights intoPseudomonas aeruginosalysine-specific uptake mechanism for extremely low pH regulation

Bicer,

Matsuoka,

Moumbock

et al. 2024

Preprint

Self Cite

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“…This purification workflow has shown promise in the crystallization of other membrane proteins [24]. For instance, stable crystals were obtained from lysine-specific permease [25] and CdsD [26] extracts purified by IMAC and SEC in preliminary X-ray diffraction studies. Nonetheless, in the case of STEAP1, this approach appears to not be yielding enough protein concentration for crystallization studies, prompting the development of new isolation bioprocesses.…”

Section: Introductionmentioning

confidence: 99%

Specific Six-Transmembrane Epithelial Antigen of the Prostate 1 Capture with Gellan Gum Microspheres: Design, Optimization and Integration

Batista-Silva

Gomes

Barroca-Ferreira

et al. 2023

IJMS

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This work demonstrates the potential of calcium- and nickel-crosslinked Gellan Gum (GG) microspheres to capture the Six-Transmembrane Epithelial Antigen of the Prostate 1 (STEAP1) directly from complex Komagataella pastoris mini-bioreactor lysates in a batch method. Calcium-crosslinked microspheres were applied in an ionic exchange strategy, by manipulation of pH and ionic strength, whereas nickel-crosslinked microspheres were applied in an affinity strategy, mirroring a standard immobilized metal affinity chromatography. Both formulations presented small diameters, with appreciable crosslinker content, but calcium-crosslinked microspheres were far smoother. The most promising results were obtained for the ionic strategy, wherein calcium-crosslinked GG microspheres were able to completely bind 0.1% (v/v) DM solubilized STEAP1 in lysate samples (~7 mg/mL). The target protein was eluted in a complexed state at pH 11 with 500 mM NaCl in 10 mM Tris buffer, in a single step with minimal losses. Coupling the batch clarified sample with a co-immunoprecipitation polishing step yields a sample of monomeric STEAP1 with a high degree of purity. For the first time, we demonstrate the potential of a gellan batch method to function as a clarification and primary capture method towards STEAP1, a membrane protein, simplifying and reducing the costs of standard purification workflows.

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Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters

Bird

Nettleship

Järvinen

et al. 2016

Advances in Experimental Medicine and Biology

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The production of recombinant integral membrane proteins for structural and functional studies remains technically challenging due to their relatively low levels of expression. To address this problem, screening strategies have been developed to identify the optimal membrane sequence and expression host for protein production. A common approach is to genetically fuse the membrane protein to a fluorescent reporter, typically Green Fluorescent Protein (GFP) enabling expression levels, localization and detergent solubilisation to be assessed. Initially developed for screening the heterologous expression of bacterial membrane proteins in Escherichia coli, the method has been extended to eukaryotic hosts, including insect and mammalian cells. Overall, GFP-based expression screening has made a major impact on the number of membrane protein structures that have been determined in the last few years.

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Cloning, expression, purification, crystallization and preliminary X-ray diffraction of a lysine-specific permease fromPseudomonas aeruginosa

Cited by 5 publications

References 23 publications

Structural insights intoPseudomonas aeruginosalysine-specific uptake mechanism for extremely low pH regulation

Structural insights intoPseudomonas aeruginosalysine-specific uptake mechanism for extremely low pH regulation

Specific Six-Transmembrane Epithelial Antigen of the Prostate 1 Capture with Gellan Gum Microspheres: Design, Optimization and Integration

Expression Screening of Integral Membrane Proteins by Fusion to Fluorescent Reporters

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