Title: Expression tuning in E.coli: discussion of state-of-the-art technologies and multivariate investigation of the pBAD mixed feed system
Language: English
Authors: Marschall, Lukas 
Qualification level: Diploma
Advisor: Herwig, Christoph 
Issue Date: 2016
Number of Pages: 141
Qualification level: Diploma
Background: Tuning of transcription is a powerful process technological tool for efficient recombinant protein production in E. coli. Many challenges such as product toxicity, formation of inclusion bodies, cell death and metabolic burden are associated with non-suitable (too high or too low) levels of recombinant protein expression. Tunable expression systems allow adjusting the recombinant protein expression using process technological means. This enables to exploit the cell's metabolic capacities to a maximum. Aims: In this thesis, tunable recombinant protein expression in E. coli is reviewed thoroughly from a biological and process technological point of view. In a consequent step expression tuning is applied to produce vascular endothelial growth factor-A165 (VEGF-A165), a pharmacologically relevant key player in angiogenesis, in correctly folded and active form in E. coli periplasm. Therefore the well-established system for expression tuning, the E. coli pBAD mixed feed platform, is investigated for the development of an upstream production process. Material and Methods: An E. coli C41 strain with intact L-arabinose metabolism was used in a mixed feed environment with D-glucose as main substrate and L-arabinose as inducing substrate. Following Quality by Design (QbD) principles, the three most promising critical process parameters (CPPs) namely the specific growth rate, specific inducer uptake rate and temperature were investigated in a design of experiments. A 23 factorial design (3 factors at 2 levels) for GIII and 22 factorial design in the case of DsbA signal sequence were conducted, each with a set of 3 center points. Results: So far expression tuning was only addressed in a few studies. For the first time these studies were reviewed with respect to latest findings on induction kinetics and mechanistics. According to the current level of knowledge some promoter system were successfully for expression tuning, in some cases analytical evidence on single cell level is still pending and some attempts did only influence protein expression on population level. For the first time, a promising mixed feed system was applied for tunable protein expression in the periplasm of E. coli. Beside of the observation of quality and quantity dependencies on the investigated CPPs it was demonstrated that the product transcription rate could indirectly be included in an experimental design by the successful use of a tunable promoter system in E. coli. Conclusion: In summary, the use of a tunable expression system was successfully applied in the development of an upstream process for the production of VEGF-A165 in the periplasm of E. coli. We anticipate that expression tuning is able to tackle further issues caused by inappropriate transcription levels and therefore is not only a major benefit for process development, but can pave the way for continuous production of biopharmaceuticals by the issues of constant product quality and culture long term stability
Keywords: all-or- none-induction; E.coli; promoter; mixed feed; tunable; VEGF-A165
URI: https://resolver.obvsg.at/urn:nbn:at:at-ubtuw:1-8950
Library ID: AC13376905
Organisation: E166 - Institut für Verfahrenstechnik, Umwelttechnik und Technische Biowissenschaften 
Publication Type: Thesis
Appears in Collections:Thesis

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