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Strategy To Approach Stable Production of Recombinant Nattokinase in Bacillus subtilis

机译：枯草芽孢杆菌稳定生产重组纳豆激酶的策略

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Bacillus subtilis (B.subtilis) is widely accepted as an excellent host cell for the secretory production of recombinant proteins.In this study,a shuttle vector was constructed by fusion of Staphylococcus aureus (S.aureus) plasmid pUBHO with Escherichia coli (E.coli) plasmid pUC 18 and used for the expression of nattokinase in B.subtilis.The pUB 110/pUC-based plasmid was found to exhibit high structural instability with the identification of a DNA deletion between two repeated regions.An initial attempt was made to eliminate the homologous site in the plasmid,whereas the stability of the resulting plasmid was not improved.In an alternative way,the pUC18-derived region in this hybrid vector was replaced by the suicidal R6K plasmid origin of E.coli.As a consequence,the pUB110/R6K-based plasmid displayed full structural stability,leading to a high-level production of recombinant nattokinase in the culture broth.This was mirrored by the detection of a very low level of high molecular weight DNAs generated by the plasmid.Moreover,2-fold higher nattokinase production was obtained by B.subtilis strain carrying the pUB110/R6K-based plasmid as compared to the cell with the pAM beta 1-derived vector,a plasmid known to have high structural stability.Overall,it indicates the feasibility of the approach by fusing two compatible plasmid origins for stable and efficient production of recombinant nattokinase in B.subtilis.
机译：枯草芽孢杆菌（B.subtilis）被广泛认为是分泌重组蛋白的优良宿主细胞。在这项研究中，通过金黄色葡萄球菌（S.aureus）质粒pUBHO与大肠杆菌（E.大肠杆菌pUC 18质粒，用于在枯草芽孢杆菌中表达纳豆激酶。基于pUB 110 / pUC的质粒具有很高的结构不稳定性，并鉴定了两个重复区域之间的DNA缺失。消除质粒中的同源位点，而所得质粒的稳定性没有得到改善。以另一种方式，该杂交载体中来自pUC18的区域被大肠杆菌的自杀性R6K质粒来源所替代。基于pUB110 / R6K的质粒显示出完整的结构稳定性，导致在培养液中高水平生产重组纳豆激酶，这通过检测到极低水平的高分子量得以反映质粒产生的DNAs。此外，枯草芽孢杆菌菌株携带基于pUB110 / R6K的质粒的纳豆激酶产量比带有pAM beta 1的载体的细胞高2倍，该质粒具有已知的高结构总体上，它表明了通过融合两个相容的质粒来源以稳定和高效生产枯草芽孢杆菌重组纳豆激酶的方法的可行性。

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