多个药物靶标蛋白在大肠杆菌无细胞系统高效表达及功能表征
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摘要
无细胞蛋白质表达体系作为一种新兴的重组蛋白合成技术，相对于传统的体内蛋白表达系统具有如下明显的优势：没有细胞结构的限制，应用于生产对宿主细胞有毒害作用的外源蛋白质；反应操作简易，耗时短；反应体系开放，易于调节反应条件；亦可偶联其它的工艺步骤，加快后续的重组蛋白质纯化、功能表征和结构解析；反应体系小，能够直接以质粒或者PCR产物作为模板在微孔板上同时平行进行合成反应，有利于建立高通量的蛋白质合成和配体筛选平台。
细菌肽聚糖生物合成途径的MraY转位酶将底物胞壁酸锚定于细胞膜磷脂双分子层内侧。作为细菌合成细胞壁的必需因子，MraY转位酶是新型广谱高效抗生素的重要靶标。受制于膜蛋白在体内蛋白质合成体系表达量过低等因素限制，MraY转位酶的生化功能和结构解析的研究进展极为缓慢。本工作采用无细胞蛋白质合成体系成功表达和纯化了制备级的革兰氏阳性枯草芽孢杆菌的和革兰氏阴性大肠杆菌MraY转位酶，并完成蛋白质功能表征和特异性抑制剂筛选。虽然两个MraY转位酶在无细胞合成体系的三种表达模式中(P-CF，D-CF和L-CF)均可以获得较高的产量，但是对最佳条件的选择却有明显差异。枯草芽孢杆菌MraY转位酶在所有的表达模式中均可正确折叠， P-CF模式表达的MraY转位酶在经过表面活性剂DDM重悬处理后，具有最高的酶活性。相比之下，P-CF和D-CF模式表达的大肠杆菌MraY转位酶与表面活性剂形成蛋白胶束之后，蛋白结构的稳定性受到较大影响，酶活力丧失。通过降低无细胞合成体系反应温度和提高体系内还原剂浓度，可以一定程度上提高大肠杆菌MraY转位酶的稳定性，但是酶活性低且重复性不高。L-CF模式添加的脂质体可以显著提高大肠杆菌MraY转位酶的稳定性，制备的蛋白样品能够在较长时间内保持酶活力。
人类葡糖胺6-磷酸N-乙酰转移酶GNA1是尿嘧啶-5′-二磷酸-N-乙酰葡萄糖胺生物合成途径的一个重要酶，将底物乙酰辅酶A的乙酰基转移到葡糖胺-6-磷酸，形成N-乙酰葡糖胺-6-磷酸。作为新型的抗生素和抗癌靶标的GNA1转移酶，人类的GNA1转移酶和微生物同源物在蛋白结构和酶反应动力学具有明显的差异。本工作实现了人类GNA1转移酶以及融合sGFP的GNA1转移酶在大肠杆菌无细胞蛋白合成体系的功能性表达。每毫升反应液2小时内可以合成超过5mg的GNA1转移酶，未经纯化的GNA1-sGFP融合蛋白可以替代纯化的GNA1转移酶用于蛋白质合成快速检测，功能表征和抑制剂筛选。人类GNA1转移酶和GNA1-sGFP融合蛋白的酶活性被葡萄糖-6-磷酸特异性抑制，且抑制趋势相同，说明无细胞蛋白合成体系表达的GNA1转移酶以及GNA1-sGFP融合蛋白可以进行快速的酶功能表征和抑制剂的高通量筛选。
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.
细菌肽聚糖生物合成途径的MraY转位酶将底物胞壁酸锚定于细胞膜磷脂双分子层内侧。作为细菌合成细胞壁的必需因子，MraY转位酶是新型广谱高效抗生素的重要靶标。受制于膜蛋白在体内蛋白质合成体系表达量过低等因素限制，MraY转位酶的生化功能和结构解析的研究进展极为缓慢。本工作采用无细胞蛋白质合成体系成功表达和纯化了制备级的革兰氏阳性枯草芽孢杆菌的和革兰氏阴性大肠杆菌MraY转位酶，并完成蛋白质功能表征和特异性抑制剂筛选。虽然两个MraY转位酶在无细胞合成体系的三种表达模式中(P-CF，D-CF和L-CF)均可以获得较高的产量，但是对最佳条件的选择却有明显差异。枯草芽孢杆菌MraY转位酶在所有的表达模式中均可正确折叠， P-CF模式表达的MraY转位酶在经过表面活性剂DDM重悬处理后，具有最高的酶活性。相比之下，P-CF和D-CF模式表达的大肠杆菌MraY转位酶与表面活性剂形成蛋白胶束之后，蛋白结构的稳定性受到较大影响，酶活力丧失。通过降低无细胞合成体系反应温度和提高体系内还原剂浓度，可以一定程度上提高大肠杆菌MraY转位酶的稳定性，但是酶活性低且重复性不高。L-CF模式添加的脂质体可以显著提高大肠杆菌MraY转位酶的稳定性，制备的蛋白样品能够在较长时间内保持酶活力。
人类葡糖胺6-磷酸N-乙酰转移酶GNA1是尿嘧啶-5′-二磷酸-N-乙酰葡萄糖胺生物合成途径的一个重要酶，将底物乙酰辅酶A的乙酰基转移到葡糖胺-6-磷酸，形成N-乙酰葡糖胺-6-磷酸。作为新型的抗生素和抗癌靶标的GNA1转移酶，人类的GNA1转移酶和微生物同源物在蛋白结构和酶反应动力学具有明显的差异。本工作实现了人类GNA1转移酶以及融合sGFP的GNA1转移酶在大肠杆菌无细胞蛋白合成体系的功能性表达。每毫升反应液2小时内可以合成超过5mg的GNA1转移酶，未经纯化的GNA1-sGFP融合蛋白可以替代纯化的GNA1转移酶用于蛋白质合成快速检测，功能表征和抑制剂筛选。人类GNA1转移酶和GNA1-sGFP融合蛋白的酶活性被葡萄糖-6-磷酸特异性抑制，且抑制趋势相同，说明无细胞蛋白合成体系表达的GNA1转移酶以及GNA1-sGFP融合蛋白可以进行快速的酶功能表征和抑制剂的高通量筛选。
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.
引文
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