Neuraminidase (NA) is one of the targets for the development of new antivirals against the influenza virus. The recombinant Escherichia coli cells, namely the strains BL21(DE3)pLysS and ArcticExpress(DE3) were used to produce the influenza virus neuraminidase. Although the different conditions of induction were tested, the accumulation of over-expressed NA in insoluble fraction occurred independently of these conditions. The level of over-expressed protein represents 26.15 % of the total cellular proteins. Therefore, the aim of these study was to design the procedure for isolation of recombinant neuraminidase from IBs and subsequently its solubilization and refolding to its native and active form. The highest purity of IBs (86 %) was achieved after repeatedly washing for at least five times with 2 M urea. The best solubilizing agent for releasing NA from IBs was the solution of 8 M urea at pH 8.0 with 94.8 ± 0.4 mg/L released proteins. The most appropriate buffer for refolding of solubilized NA was found to be 50 mM Tris-HCl at pH 7.5 (102 ± 24.2 mg proteins) and the addition of glycerol or arginine had no stimulating effect on protein recovery. The determination of non-glycosylated activity of refolded NA monomer (Km = 0.51 g/L; Vmax = 9.73 U/mg; kcat = 8.76 s-1) using fetuin as a substrate in the coupled enzyme reaction system was the highlight of this work. This procedure provides a way to produce active form of NA monomer by recombinant E. coli cells.

中文翻译：

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从重组大肠杆菌产生的包涵体中纯化病毒神经氨酸酶。

神经氨酸酶（NA）是针对流感病毒的新型抗病毒药物开发的目标之一。重组大肠杆菌细胞，即菌株BL21（DE3）pLysS和ArcticExpress（DE3）被用于生产流感病毒神经氨酸酶。尽管测试了不同的诱导条件，但不溶级分中过度表达的NA的积累与这些条件无关。过表达的蛋白质水平占总细胞蛋白质的26.15％。因此，这些研究的目的是设计从IBs中分离重组神经氨酸酶的程序，然后将其溶解并重折叠为其天然和活性形式。用2 M尿素反复洗涤至少五次后，IB的纯度最高（86％）。从IBs释放NA的最佳增溶剂是pH 8.0的8 M尿素与94.8±0.4 mg / L释放的蛋白质的溶液。发现用于重溶NA的最合适的缓冲液是pH 7.5的50 mM Tris-HCl（102±24.2 mg蛋白质），添加甘油或精氨酸对蛋白质回收率没有刺激作用。本研究的重点是使用胎球蛋白作为底物测定重折叠NA单体（Km = 0.51 g / L; Vmax = 9.73 U / mg; kcat = 8.76 s-1）的非糖基化活性。 。该程序提供了通过重组大肠杆菌细胞产生活性形式的NA单体的方法。5（102±24.2 mg蛋白质）和添加甘油或精氨酸对蛋白质回收率没有刺激作用。本研究的重点是使用胎球蛋白作为底物测定重折叠NA单体（Km = 0.51 g / L; Vmax = 9.73 U / mg; kcat = 8.76 s-1）的非糖基化活性。 。该程序提供了通过重组大肠杆菌细胞产生活性形式的NA单体的方法。5（102±24.2 mg蛋白质）和添加甘油或精氨酸对蛋白质回收率没有刺激作用。本研究的重点是使用胎球蛋白作为底物测定重折叠NA单体（Km = 0.51 g / L; Vmax = 9.73 U / mg; kcat = 8.76 s-1）的非糖基化活性。 。该程序提供了通过重组大肠杆菌细胞产生活性形式的NA单体的方法。