title: Fluorescence based recognition elements for glucose sensing using the glucose-galactose binding protein from Escherichia coli
creator: Neuner, Lisa
description: Abstract  The biotechnology industry is growing rapidly and is embedded in a highly regulated and dynamic environment. Under the regulations and guidelines of the Food and Drug Association, short FDA, a pharmaceutical company must ensure the quality of biotechnological products. Within the last few years so called in-line technologies revolutionized the batch production procedure in the biopharmaceutical industry towards continuous manufacturing and real-time monitoring of critical vitality parameters in a bioreactor. As a key benefit, it is expected that with this technology, final product deviations can be reduced by up to 50%. Furthermore, studies have been shown that regulating vitality parameters such as glucose can improve overall product quality. Despite the increasing adoption and expected doubling of the market in the next years, there are challenges. Continuous manufacturing methods are not yet well characterized for therapeutic proteins due to a lack of (bio-)analytical in-line technology.  The overall objective of this work was to develop optical glucose biosensor recognition elements that can measure ‘in-line’ physiological glucose concentrations. For that purpose, the well-researched glucose-galactose binding protein in E. coli (GGBP) was genetically and chemically modified to enhance the spectral properties of the biosensing element. Several single-labelled as well as self-calibrated dual-labelled systems were developed. The candidate proteins were designed, synthesized, and labelled with commercially available fluorophores. The best result for the single-labelled glucose sensing system was achieved with the combination of GGBP variant H152C/A213R/L238S labelled with the newly developed commercially available dye Atto 465. A linear dependency between 1-75 mM glucose with a total fluorescence increase of +48% could be demonstrated.  Furthermore, this work presents for the first time, selective labeling of FRET-based biosensor recognition elements based on the recently discovered π-clamp strategy (Nature Chem., 2016). For this purpose, a chemical synthesis of perfluoro arylated fluorophore (XC113) has been developed. It could be shown that dual labeling of a FRET pair was highly accurate and selective. GGBP variant H152C/A213R/L238S_DGTN280-283FCPF labeled with XC113 and DY585 proved the idea of the concept and a successful labeling of the pi-clamp. A fluorescence transfer rate of 18% was obtained and a maximal fluorescence intensity change by 12% upon binding of glucose was observed. In a future experiment, this mutant can be further improved by using Atto 495 and DY585 as a FRET pair and an overly sensitive ratiometric glucose biosensor could be developed for bioreactor environments.
date: 2023
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/32504/1/Dissertation_FINAL_Lisa%20Neuner_01.10.2020_pdfA.pdf
identifier: DOI:10.11588/heidok.00032504
identifier: urn:nbn:de:bsz:16-heidok-325043
identifier:   Neuner, Lisa  (2023) Fluorescence based recognition elements for glucose sensing using the glucose-galactose binding protein from Escherichia coli.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/32504/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng