The E. coli pET expression system revisited—mechanistic correlation between glucose and lactose uptake

Wurm, David Johannes; Veiter, Lukas; Ulonska, Sophia; Eggenreich, Britta; Herwig, Christoph; Spadiut, Oliver

doi:10.1007/s00253-016-7620-7

Cited by 54 publications

(86 citation statements)

References 51 publications

(59 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A batch and fed-batch phase for biomass generation were followed by an induction phase using a mixed feed medium with glucose as primary carbon source and lactose as carbon source as well as inducer. Detailed information about the bioreactor setup and media composition can be found elsewhere [32]. …”

Section: Methodsmentioning

confidence: 99%

A Novel Application for Low Frequency Electrochemical Impedance Spectroscopy as an Online Process Monitoring Tool for Viable Cell Concentrations

Slouka

Wurm

Brunauer

et al. 2016

Sensors

Self Cite

View full text Add to dashboard Cite

New approaches in process monitoring during industrial fermentations are not only limited to classical pH, dO2 and offgas analysis, but use different in situ and online sensors based on different physical principles to determine biomass, product quality, lysis and far more. One of the very important approaches is the in situ accessibility of viable cell concentration (VCC). This knowledge provides increased efficiency in monitoring and controlling strategies during cultivations. Electrochemical impedance spectroscopy—EIS—is used to monitor biomass in a fermentation of E. coli BL21(DE3), producing a recombinant protein using a fed batch-based approach. Increases in the double layer capacitance (Cdl), determined at frequencies below 1 kHz, are proportional to the increase of biomass in the batch and fed batch phase, monitored in offline and online modes for different cultivations. A good correlation of Cdl with cell density is found and in order to get an appropriate verification of this method, different state-of-the-art biomass measurements are performed and compared. Since measurements in this frequency range are largely determined by the double layer region between the electrode and media, rather minor interferences with process parameters (aeration, stirring) are to be expected. It is shown that impedance spectroscopy at low frequencies is a powerful tool for cultivation monitoring.

show abstract

Section: Methodsmentioning

confidence: 99%

A Novel Application for Low Frequency Electrochemical Impedance Spectroscopy as an Online Process Monitoring Tool for Viable Cell Concentrations

Slouka

Wurm

Brunauer

et al. 2016

Sensors

Self Cite

View full text Add to dashboard Cite

show abstract

“…Instead, we hypothesized that the reason for this strong divergence could originate from the plasmids. All employed pET plasmids from our previous studies [20,23,24,34] were high copy number plasmids, which also carried a lacI gene, thereby introducing much more repressor protein into the cells. The GI strain however, only expressed LacI from the copies in the genome [35].…”

Section: Correlation Between Q Sglu and Q Slacmaxmentioning

confidence: 99%

“…Recent studies from our group showed that there is a correlation between the maximum specific lactose uptake rate (q s,lac,max ) and the specific glucose uptake rate (q s,glu ) in E. coli BL21 (DE3) strains carrying pET plasmids. For several different pET plasmids for expression of various products, a mechanistic model for this correlation has been established, which can serve as a basis for steering product titers, product properties and/or product location [20,23,24].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

Self Cite

View full text Add to dashboard Cite

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.

show abstract

“…These systems are applied in the means that the first carbon source is linked to cell growth and cell energy, whereas, the second carbon source is used to regulate recombinant protein production (Sagmeister et al 2013a). Mixed feed systems were shown to increase productivity of heterologous products (Anjou and Daugulis 2000; Jungo et al 2007; Wurm et al 2016). …”

Section: Introductionmentioning

confidence: 99%

A novel method to recover inclusion body protein from recombinant E. coli fed-batch processes based on phage ΦX174-derived lysis protein E

Ehgartner

Sagmeister

Langemann

et al. 2017

Appl Microbiol Biotechnol

Self Cite

View full text Add to dashboard Cite

Production of recombinant proteins as inclusion bodies is an important strategy in the production of technical enzymes and biopharmaceutical products. So far, protein from inclusion bodies has been recovered from the cell factory through mechanical or chemical disruption methods, requiring additional cost-intensive unit operations. We describe a novel method that is using a bacteriophage-derived lysis protein to directly recover inclusion body protein from Escherichia coli from high cell density fermentation process: The recombinant inclusion body product is expressed by using a mixed feed fed-batch process which allows expression tuning via adjusting the specific uptake rate of the inducing substrate. Then, bacteriophage ΦX174-derived lysis protein E is expressed to induce cell lysis. Inclusion bodies in empty cell envelopes are harvested via centrifugation of the fermentation broth. A subsequent solubilization step reveals the recombinant protein. The process was investigated by analyzing the impact of fermentation conditions on protein E-mediated cell lysis as well as cell lysis kinetics. Optimal cell lysis efficiencies of 99% were obtained with inclusion body titers of >2.0 g/l at specific growth rates higher 0.12 h−1 and inducer uptake rates below 0.125 g/(g × h). Protein E-mediated cell disruption showed a first-order kinetics with a kinetic constant of −0.8 ± 0.3 h−1. This alternative inclusion body protein isolation technique was compared to the one via high-pressure homogenization. SDS gel analysis showed 10% less protein impurities when cells had been disrupted via high-pressure homogenization, than when empty cell envelopes including inclusion bodies were investigated. Within this contribution, an innovative technology, tuning recombinant protein production and substituting cost-intensive mechanical cell disruption, is presented. We anticipate that the presented method will simplify and reduce the production costs of inclusion body processes to produce technical enzymes and biopharmaceutical products.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-017-8281-x) contains supplementary material, which is available to authorized users.

show abstract

The E. coli pET expression system revisited—mechanistic correlation between glucose and lactose uptake

Cited by 54 publications

References 51 publications

A Novel Application for Low Frequency Electrochemical Impedance Spectroscopy as an Online Process Monitoring Tool for Viable Cell Concentrations

A Novel Application for Low Frequency Electrochemical Impedance Spectroscopy as an Online Process Monitoring Tool for Viable Cell Concentrations

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

A novel method to recover inclusion body protein from recombinant E. coli fed-batch processes based on phage ΦX174-derived lysis protein E

Contact Info

Product

Resources

About