Membrane Protein Expression in Cell-Free Systems

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Screening of new compounds directed against key protein targets must continually keep pace with emerging antibiotic resistances. Although periplasmic enzymes of bacterial cell wall biosynthesis have been among the first drug targets, compounds directed against the membrane-integrated catalysts are hardly available. A promising future target is the integral membrane protein MraY catalyzing the first membrane associated step within the cytoplasmic pathway of bacterial peptidoglycan biosynthesis. However, the expression of most MraY homologues in cellular expression systems is challenging and limits biochemical analysis. We report the efficient production of MraY homologues from various human pathogens by synthetic cellfree expression approaches and their subsequent characterization. MraY homologues originating from Bordetella pertussis, Helicobacter pylori, Chlamydia pneumoniae, Borrelia burgdorferi, and Escherichia coli as well as Bacillus subtilis were cotranslationally solubilized using either detergent micelles or preformed nanodiscs assembled with defined membranes. All MraY enzymes originating from Gram-negative bacteria were sensitive to detergents and required nanodiscs containing negatively charged lipids for obtaining a stable and functionally folded conformation. In contrast, the Gram-positive B. subtilis MraY not only tolerates detergent but is also less specific for its lipid environment. The MraY⅐nanodisc complexes were able to reconstitute a complete in vitro lipid I and lipid II forming pipeline in combination with the cell-free expressed soluble enzymes MurA-F and with the membrane-associated protein MurG. As a proof of principle for future screening platforms, we demonstrate the inhibition of the in vitro lipid II biosynthesis with the specific inhibitors fosfomycin, feglymycin, and tunicamycin.Proteins involved in bacterial cell wall biosynthesis are highly conserved and often essential. In stage I, the cytoplasmic proteins MurA-F are responsible for the formation of the soluble peptidoglycan precursor (Park's nucleotide). In stage II, the integral membrane protein MraY catalyzes the ligation of Park's nucleotide with the membrane-bound undecaprenylphosphate (C 55 -P) 4 resulting into lipid I. The membrane-associated globular protein MurG catalyzes subsequent glycosyl transfer forming lipid II (Fig. 1). As the final monomeric building block in bacterial cell wall biosynthesis, lipid II will be transported through the membrane into the periplasmic space for further polymerization in stage III of cell wall biosynthesis (1).The bacterial cell wall biosynthetic pathway is an important and traditional target for antibiotics and numerous drugs directed against key catalysts for clinical applications have been developed. However, most known drugs such as the penicillin derivatives affect the periplasmic or extracellular steps of peptidoglycan synthesis catalyzed by soluble enzymes. In contrast, almost no clinical drugs have been developed that are targeted against the integral membrane proteins involve...

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Lipid Requirements for the Enzymatic Activity of MraY Translocases and in Vitro Reconstitution of the Lipid II Synthesis Pathway

Henrich

Engels

et al. 2016

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“…S30 extracts, T7 RNA polymerase, and basic reaction compounds were prepared as previously described (15,16). Reactions were performed in the continuous exchange cell-free (CECF) configuration and incubated for ϳ16 h at 30°C with gentle shaking, if not otherwise stated.…”

Section: Methodsmentioning

confidence: 99%

Preparative Scale Cell-free Production and Quality Optimization of MraY Homologues in Different Expression Modes

Münch

Schneider

et al. 2011

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Background: Functional MraY translocases can be cell-free expressed in high levels. Results: Bacillus subtilis MraY activity is stable and robust, whereas Escherichia coli MraY depends on lipids. Conclusion: Activity of MraY can be modulated by cell-free expression modes. Artificial hydrophobic environments have a strong impact on the MraY sample quality. Significance: New strategy for the efficient production and analysis of drug targets.

“…Cell-free Production of AqpB, Proteoliposome Reconstitution, and Stopped-flow Light Scattering-AqpB was produced according to prior protocols (22, 23) using 2-ml reactors (24). The reaction mix was supplied with 0.1% of Brij-35 for membrane protein solubilization during incubation at 30°C for approx.…”

Section: Methodsmentioning

confidence: 99%

Functional Characterization of a Novel Aquaporin from Dictyostelium discoideum Amoebae Implies a Unique Gating Mechanism

Bülow

Müller-Lucks

Lou

et al. 2012

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Background: Aquaporins are involved in osmoregulation and cell motility. Functional D. discoideum aquaporins are missing. Results: We characterized a putatively gated aquaporin from amoebae that localizes to vacuolar structures, the plasma membrane, and protrusions. Conclusion: Localization and gating hint at functions in osmoregulation and motility. Significance: We identified a novel player in basic physiology of the model organism D. discoideum.