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首页> 外文期刊>Biotechnology Progress >Integration of Bacteria Capture via Filtration and in Situ Lysis for Recovery of Plasmid DNA under Industry-Compatible Conditions
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Integration of Bacteria Capture via Filtration and in Situ Lysis for Recovery of Plasmid DNA under Industry-Compatible Conditions

机译：通过过滤和原位裂解整合细菌捕获以在工业兼容条件下回收质粒DNA

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摘要

Combining capture and lysis of the bacteria with partial purification of the plasmid DNA is beneficial for the design of efficient plasmid production processes at larger scale.Such an approach is possible when the bacteria are captured by filtration.Taking industrial requirements into account,however,such a capture requires complex filtration mixtures containing retentive additives such as bentonite and polycations.This makes the straightforward transfer of established lysis protocols to in situ lysis difficult.In this contribution,the different steps of such a protocol are designed for complex filter cakes,including fragilization (by lysozyme),lysis (alkaline pH/ acidic pH,70/37 °C,urea/NaC1/Triton),and specific elution (pH,NaCl,CaCl2,guanidinium hydrochloride).Results are compared in regard to plasmid quality (topoisomeric form) and quantity (compared to the yield obtained by a commercial miniprep of a small aliquot of the bacteria suspension from the bioreactor).Best results in these terms were obtained by the Triton lysis protocol performed at 37 °C (30 min of contact with a lysis buffer composed of 50 mM Tris pH 8,1% Triton,1 g/L lysozyme,and 6 M guanidinium hydrochloride) followed by the specific elution of the plasmid DNA in 50 mM Tris buffer pH 8.
机译：将细菌的捕获和裂解与质粒DNA的部分纯化结合起来有利于大规模设计高效的质粒生产工艺。当通过过滤捕获细菌时，这种方法是可行的。捕集需要复杂的过滤混合物，其中包含膨润土和聚阳离子等保持性添加剂。这使得将既定的裂解方案直接转移至原位裂解变得困难。为此，该方案的不同步骤设计用于复杂的滤饼，包括脆化（通过溶菌酶），裂解（碱性pH /酸性pH，70/37°C，尿素/ NaC1 / Triton）和比洗脱（pH，NaCl，CaCl2，盐酸胍）进行比较。结果与质粒质量（拓扑异构体）进行了比较形式和数量（与从商业化的小量制备的生物反应器中细菌悬浮液的等分试样获得的产量相比）。 e项是通过在37°C（与50 mM Tris pH 8,1％Triton，1 g / L溶菌酶和6 M盐酸胍）组成的裂解缓冲液接触30分钟的条件下进行的Triton裂解方案获得的。在50 mM Tris缓冲液pH 8.中特异性洗脱质粒DNA

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