Glauche Florian, Pilarek Maciej, Bournazou Mariano Nicolas Cruz, Grunzel Petra, Neubauer Peter
Chair of Bioprocess Engineering, Institute of Biotechnology Technische Universität Berlin Berlin Germany.
Faculty of Chemical and Process Engineering Warsaw University of Technology Warsaw Poland.
Eng Life Sci. 2016 Oct 5;17(11):1166-1172. doi: 10.1002/elsc.201600030. eCollection 2017 Nov.
Efficient and reproducible cell lysis is a crucial step during downstream processing of intracellular products. The composition of an optimal lysis buffer should be chosen depending on the organism, its growth status, the applied detection methods, and even the target molecule. Especially for high-throughput applications, where sample volumes are limited, the adaptation of a lysis buffer to the specific campaign is an urgent need. Here, we present a general design of experiments-based strategy suitable for eight constituents and demonstrate the strength of this approach by the development of an efficient lysis buffer for Gram-negative bacteria, which is applicable in a high-throughput format in a short time. The concentrations of four lysis-inducing chemical agents EDTA, lysozyme, Triton X-100, and polymyxin B were optimized for maximal soluble protein concentration and ß-galactosidase activity in a 96-well format on a Microlab Star liquid handling platform under design of experiments methodology. The resulting lysis buffer showed the same performance as a commercially available lysis buffer. The developed protocol resulted in an optimized buffer within only three runs. The established procedure can be easily applied to adapt the lysis buffer to other strains and target molecules.
高效且可重复的细胞裂解是细胞内产物下游处理过程中的关键步骤。最佳裂解缓冲液的组成应根据生物体、其生长状态、所应用的检测方法，甚至目标分子来选择。特别是对于高通量应用，由于样品体积有限，使裂解缓冲液适应特定实验的需求迫在眉睫。在此，我们提出一种基于实验设计的通用策略，该策略适用于八种成分，并通过开发一种适用于革兰氏阴性菌的高效裂解缓冲液来证明这种方法的优势，该缓冲液可在短时间内以高通量形式应用。在实验设计方法下，在Microlab Star液体处理平台上，以96孔板形式对四种裂解诱导化学试剂乙二胺四乙酸（EDTA）溶菌酶、曲拉通X-100和多粘菌素B的浓度进行了优化，以实现最大可溶性蛋白浓度和β-半乳糖苷酶活性。所得的裂解缓冲液表现出与市售裂解缓冲液相同的性能。所开发的方案仅通过三次运行就得到了优化的缓冲液。所建立的程序可以很容易地应用于使裂解缓冲液适应其他菌株和目标分子。
