Efficient Expression Based In Exoli Cell-free System,Quality Optimization And Functional Characterization Of Multiple Drug Target Proteins

Cell-free (CF) expression systems have emerged in recent times as promisingtools in order to accelerate recombinant protein expression approaches. There areseveral advantages over the in vivo protein synthesis in using the E. coli CFexpression system, including the elimination of a living host environment duringprotein overexpression with problems of toxic effects of the recombinant proteins tothe host cell physiology. The open accessibility of the reaction could be easily tooperate the addition of assistant molecules to provide a suitable environment forcorrect folding of recombinant proteins. The ease in which large quantities of extractsmay be prepared and CF expression reactions be carried out in small volumes andincubated in short times are already sufficient for the production of proteins. Theimplement of expression vector or PCR fragment template creates very powerful toolsfor proteomic applications. The coupled protein expression and functional assayscould be developed to high throughput screening approaches for special ligands.MraY translocase catalyzes the first committed membrane bound step ofbacterial peptidoglycan synthesis leading to the formation of lipid I. The essentialmembrane protein therefore has a high potential as target for drug screeningapproaches to develop antibiotics against Gram-positive as well as Gram-negativebacteria. However, the production of large integral membrane proteins inconventional cellular expression systems is still very challenging. Cell-free expressiontechnologies have been optimized in recent times for the production of membraneproteins in the presence of detergents (D-CF), lipids (L-CF), or as precipitates (P-CF).We report the development of preparative scale production protocols for the MraYhomologues of Escherichia coli and Bacillus subtilis in all three cell-free expressionmodes followed by their subsequent quality evaluation. Although both proteins can becell-free produced at comparable high levels, their requirements for optimalexpression conditions differ markedly. B. subtilus MraY was stably folded in all three expression modes and showed highest translocase activities after P-CF productionfollowed by defined treatment with detergents. In contrast, the E. coli MraY appearsto be unstable after post-or cotranslational solubilization in detergent micelles.Expression kinetics and reducing conditions were identified as optimizationparameters for the quality improvement of E. coli MraY. Most remarkably, in contrastto B. subtilis MraY the E. coli MraY has to be stabilized by lipids and only theproduction in the L-CF mode in the presence of preformed liposomes resulted instable and translocase active protein samples.Human glucosamine-6-phosphate N-acetyltransferase (GNA1) is required forthe de novo synthesis of N-acetyl-D-glucosamine-6-phosphate representing anessential precursor in UDP-GlcNAc biosynthesis. Therefore, GNA1could become anattractive drug target for antimicrobials based on structural and kinetic differences inbetween human and microbial GNA1homologues. We report the development ofefficient cell-free expression strategies to rapidly produce preparative scale amountsof human GNA1derivatives suitable for throughput screening purposes. Cell-freeexpression yielded routinely more than five mg of GNA1in one ml of reactionmixture within two hours. We demonstrate that the derivative GNA1-sGFP canfurther be used for fast monitoring and inhibitor screening directly in the reactionmixtures without previous purification. The inhibitor glucose-6-phosphate to humanGNA1was identified by inhibition assay. We suggest that the cell-free production ofhuman GNA1and its derivative GNA1-sGFP provides a novel, fast and efficientapproach in order to support enzymatic assays and to develop throughput screeningapproaches for inhibitors.