Design of experiments-based high-throughput strategy for development and optimization of efficient cell disruption protocols.

Abstract

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.