Controllable Autolytic Leaky E. coli Platform for the Recovery of Intracellular Proteins

细胞内 化学 细胞生物学 生物物理学 生物化学 生物
作者
Xia Wu,Jin Ren,Zhiqiang Liu,Su Zheng,Juan Ren,Jian Zha
出处
期刊:Journal of Agricultural and Food Chemistry [American Chemical Society]
标识
DOI:10.1021/acs.jafc.4c06174
摘要

Escherichia coli is a commonly used platform for the production of heterologous proteins. Extraction and purification of intracellularly expressed recombinant proteins rely on efficient cell disruption. To facilitate downstream processing, controlled autolytic cells have been designed that lyse automatically to release intracellular proteins when triggered with an internal or external signal. In the cases when a weak promoter has to be adopted to control autolysis, cell lysis and product release progress slowly even in the presence of surfactants or other adjuvants. In this study, we report an improved autolytic E. coli strain controlled by a weak promoter with higher efficiency without the use of any facilitating chemical. Cell lysis was initiated upon arabinose-induced expression of T4 lysozyme with N-terminal fusion of amphipathic cell-penetrating peptides via a flexible peptide linker. Furthermore, genes involved in membrane permeability were individually deleted and screened for leaky phenotypes. Deletion of lpp (encoding Braun's lipoprotein) combined with the autolytic system caused 96% cell lysis in 4 h of induction and released 84% or 67% of mCherry or a super large Cas13a fusion protein (160.8 kDa), respectively, in 10 h of induction. This autolytic leaky strain shows great promise for protein recovery and library screening.
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