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Characterization of the AlkS/P-alkB-expression system as an efficient tool for the production of recombinant proteins in Escherichia coli fed-batch fermentations

机译:AlkS / P-alkB表达系统的表征是在大肠杆菌补料分批发酵中生产重组蛋白的有效工具

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摘要

The availability of suitable, well-characterized, and robust expression systems remains an essential requirement for successful metabolic engineering and recombinant protein production. We investigated the suitability of the Pseudomonas putida GPo1-derived AlkS/P-alkB expression system in strictly aqueous cultures. By applying the apolar inducer dicyclopropylketone (DCPK) to express green fluorescent protein (GFP) from this system in Escherichia coli and analyzing the resulting cultures on single-cell level by flow cytometry, we found that this expression system gives rise to a homogeneous population of cells, even though the overall system is expected to have a positive feed-back element in the expression of the regulatory gene alkS. Overexpressing E. coli's serine hydroxymethyltransferase gene glyA, we showed that the system was already fully turned in at inducer concentrations as low as 0.005% (v/v). This allows efficient mass production of recombinant enzymes even though DCPK concentrations from 0.05% to 0.01% over the course of a fully aerated cultivation in aqueous medium. Therefore, we elaborated the optimum induction procedure for production of the biocatalytically promising serine hydroxymethyltransferase and found volumetric and specific productivity to increase with specific growth rate in glucose-limited fed-batch cultures. Acetate excretion as a result of recombinant protein production could be avoided in an optimized fermentation protocol by switching earlier to a linear feed. This protocol resulted in a production of a final cell dry weight (CDW) concentration of 52 g/L, producing recombinant GlyA with a maximum specific activity of 6.3 U/mg total protein.
机译:合适的,特征明确的和健壮的表达系统的可用性仍然是成功进行代谢工程和重组蛋白生产的基本要求。我们调查了恶臭假单胞菌GPo1衍生的AlkS / P-alkB表达系统在严格水性培养中的适用性。通过应用非极性诱导剂二环丙基酮(DCPK)在大肠杆菌中从该系统表达绿色荧光蛋白(GFP)并通过流式细胞术在单细胞水平上分析所得培养物,我们发现该表达系统产生了一个均一的即使整个系统有望在调节基因alkS的表达中具有正反馈元素。过表达大肠杆菌的丝氨酸羟甲基转移酶基因glyA,我们显示该系统已经以低至0.005%(v / v)的诱导剂浓度完全上交。即使在含水培养基中完全充气培养的过程中,即使DCPK浓度从0.05%到0.01%,也可以有效地大量生产重组酶。因此,我们详细阐述了生产具有生物催化潜力的丝氨酸羟甲基转移酶的最佳诱导程序,并发现在葡萄糖限制的分批补料培养中,体积和比生产率会随特定的生长速率而增加。在优化的发酵方案中,可以通过更早地转换为线性饲料来避免由于重组蛋白生产而导致的乙酸排泄。该方案导致产生的最终细胞干重(CDW)浓度为52 g / L,产生重组GlyA,其最大比活性为6.3 U / mg总蛋白。

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