The Effects of Lactose Induction on a Plasmid-Free E. coli T7 Expression System

Abstract

Recombinant production of pharmaceutical proteins like antigen binding fragments (Fabs) in the commonly-used production host Escherichia coli presents several challenges. The predominantly-used plasmid-based expression systems exhibit the drawback of either excessive plasmid amplification or plasmid loss over prolonged cultivations. To improve production, efforts are made to establish plasmid-free expression, ensuring more stable process conditions. Another strategy to stabilize production processes is lactose induction, leading to increased soluble product formation and cell fitness, as shown in several studies performed with plasmid-based expression systems. Within this study we wanted to investigate lactose induction for a strain with a genome-integrated gene of interest for the first time. We found unusually high specific lactose uptake rates, which we could attribute to the low levels of lac-repressor protein that is usually encoded not only on the genome but additionally on pET plasmids. We further show that these unusually high lactose uptake rates are toxic to the cells, leading to increased cell leakiness and lysis. Finally, we demonstrate that in contrast to plasmid-based T7 expression systems, IPTG induction is beneficial for genome-integrated T7 expression systems concerning cell fitness and productivity.

Repetitive Fed-Batch: A Promising Process Mode for Biomanufacturing With E. coli

“…Even though viability did not decrease in cycle two, leaky product (7.7% of total product) could be detected in the supernatant. This behavior has been observed for periplasmic proteins in literature before (Chen et al, 2014;Hausjell et al, 2020).…”

“…Furthermore, lactose has shown to boost productivity in soluble recombinant protein production when compared to IPTG ( Wurm et al, 2016 , 2017a ). For periplasmic recombinant protein production soft induction by lactose is especially important as translocation to the periplasm is the rate limiting step ( Gupta and Shukla, 2017 ; Karyolaimos et al, 2019 ; Hausjell et al, 2020 ).…”

Evolution of Escherichia coli Expression System in Producing Antibody Recombinant Fragments

“…In general, the plasmid-based expression systems exhibit some drawbacks, including the continuous amplification of the plasmid copy number in prolonged cultivations and loss of plasmids over time, propagating the generation of a plasmid-free subpopulation during induction [ 73 ]. In this context, the combination of plasmid-free [ 74 ] and cell-free approaches [ 71 , 75 , 76 ] represent new potential strategies for optimizing E. coli expression platforms (see the next section: New Platforms and new technologies for E. coli -based cell-free expression systems).…”

“…To overcome the variations deriving from different plasmid vectors a very recent benchmark study has introduced the use of the gene integration (GI) approach to improve the production of Fabs expressed in the E. coli periplasmic space in fed-batch fermentations [ 104 ]. Recently, Hausjell and coworkers [ 74 ] have reported the use of a plasmid-free expression technique for Fab antibody fragments using the BL21 (DE3) expression system. According to the “Recombineering approach”, the genes codifying for the heavy and light chains of a Fab fragment were encoded under the control of the T7 promoter and integrated into the genome at the attTN7 site [ 105 ].…”

“…When the same Fab was coexpressed with DsbC in fermentation scale using the ad hoc genetically modified E. coli strain deficient in the Tail specific protease (Tsp) and SRP, an optimal recovery of 2.4 g/L was reached [ 56 ]. Recently, the OmpA-leader sequence (MKKTAIAIAVALAGFATVAQA) was also selected as the best in plasmid-free expression systems (GI) for the production of a functional Fab fragment [ 74 , 104 ].…”