Selective Release of Recombinant Periplasmic Protein From E. coli Using Continuous Pulsed Electric Field Treatment

Abstract

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.

Potential of Flow Cytometric Approaches for Rapid Microbial Detection and Characterization in the Food Industry—A Review

“…Permanent pores can result in membrane integrity loss and uncontrolled molecule transfer through microbial cell membranes [ 210 ]. Currently, many researchers are focusing on the use of PEF to permeabilize microbial cell membranes of, for instance, microalgae [ 211 ], yeast [ 212 ], and bacteria cells [ 213 ] to extract valuable substances. A recent review by Martínez, Delso, Álvarez, and Raso [ 214 ] summarized the advantages of PEF in comparison to chemical and physical cell permeabilization or disruption methods.…”