Dataset Information

Proteomics-Based Mechanistic Investigation of Escherichia coli Inactivation by Pulsed Electric Field.

ABSTRACT: The pulsed electric field (PEF) technology has been widely applied to inactivate pathogenic bacteria in food products. Though irreversible pore formation and membrane disruption is considered to be the main contributing factor to PEF's sterilizing effects, the exact molecular mechanisms remain poorly understood. In this study, by using mass spectrometry (MS)-based label-free quantitative proteomic analysis, we compared the protein profiles of PEF-treated and untreated Escherichia coli. We identified a total of 175 differentially expressed proteins, including 52 candidates that were only detected in at least two of the three samples in one experiment group but not in the other group. Functional analysis revealed that the differential proteins were primarily involved in the regulation of cell membrane composition and integrity, stress response, as well as various metabolic processes. Quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis was conducted on the genes of selected differential proteins at varying PEF intensities, which were known to result in different cell killing levels. The qRT-PCR data confirmed that the proteomic results could be reliably used for further data interpretation, and that the changes in the expression levels of the differential candidates were, to a large extent, caused directly by the PEF treatment. The findings of the current study offered valuable insight into PEF-induced cell inactivation.

SUBMITTER: Liu Z

PROVIDER: S-EPMC6857472 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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Proteomics-Based Mechanistic Investigation of <i>Escherichia coli</i> Inactivation by Pulsed Electric Field.

Liu Zhenyu Z Zhao Lingying L Zhang Qin Q Huo Nan N Shi Xiaojing X Li Linwei L Jia Liyan L Lu Yuanyuan Y Peng Yong Y Song Yanbo Y

Frontiers in microbiology 20191108

The pulsed electric field (PEF) technology has been widely applied to inactivate pathogenic bacteria in food products. Though irreversible pore formation and membrane disruption is considered to be the main contributing factor to PEF's sterilizing effects, the exact molecular mechanisms remain poorly understood. In this study, by using mass spectrometry (MS)-based label-free quantitative proteomic analysis, we compared the protein profiles of PEF-treated and untreated <i>Escherichia coli</i>. We ...[more]

PMID: 31781086

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Project description:To date, high-pressure homogenization is the standard method for cell disintegration before the extraction of cytosolic and periplasmic protein from E. coli. Its main drawback, however, is low selectivity and a resulting high load of host cell impurities. Pulsed electric field (PEF) treatment may be used for selective permeabilization of the outer membrane. PEF is a process which is able to generate pores within cell membranes, the so-called electroporation. It can be readily applied to the culture broth in continuous mode, no additional chemicals are needed, heat generation is relatively low, and it is already implemented at industrial scale in the food sector. Yet, studies about PEF-assisted extraction of recombinant protein from bacteria are scarce. In the present study, continuous electroporation was employed to selectively extract recombinant Protein A from the periplasm of E. coli. For this purpose, a specifically designed flow-through PEF treatment chamber was deployed, operated at 1.5 kg/h, using rectangular pulses of 3 μs at specific energy input levels between 10.3 and 241.9 kJ/kg. Energy input was controlled by variation of the electric field strength (28.4-44.8 kV/cm) and pulse repetition frequency (50-1,000 Hz). The effects of the process parameters on cell viability, product release, and host cell protein (HCP), DNA, as well as endotoxin (ET) loads were investigated. It was found that a maximum product release of 89% was achieved with increasing energy input levels. Cell death also gradually increased, with a maximum inactivation of -0.9 log at 241.9 kJ/kg. The conditions resulting in high release efficiencies while keeping impurities low were electric field strengths ≤ 30 kV/cm and frequencies ≥ 825 Hz. In comparison with high-pressure homogenization, PEF treatment resulted in 40% less HCP load, 96% less DNA load, and 43% less ET load. Therefore, PEF treatment can be an efficient alternative to the cell disintegration processes commonly used in downstream processing.

Dataset Information

Proteomics-Based Mechanistic Investigation of Escherichia coli Inactivation by Pulsed Electric Field.

Publications

Proteomics-Based Mechanistic Investigation of <i>Escherichia coli</i> Inactivation by Pulsed Electric Field.

Similar Datasets

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