title: Online analysis of protein inclusion bodies produced in E. coli by monitoring alterations in scattered and reflected light.
abstract: The online monitoring of recombinant protein aggregate inclusion bodies during microbial cultivation is an immense challenge. Measurement of scattered and reflected light offers a versatile and non-invasive measurement technique. Therefore, we investigated two methods to detect the formation of inclusion bodies and monitor their production: (1) online 180° scattered light measurement (λ = 625 nm) using a sensor platform during cultivation in shake flask and (2) online measurement of the light reflective interference using a porous Si-based optical biosensor (SiPA). It could be shown that 180° scattered light measurement allows monitoring of alterations in the optical properties of Escherichia coli BL21 cells, associated with the formation of inclusion bodies during cultivation. A reproducible linear correlation between the inclusion body concentration of the non-fluorescent protein human leukemia inhibitory factor (hLIF) carrying a thioredoxin tag and the shift ("Δamp") in scattered light signal intensity was observed. This was also observed for the glutathione-S-transferase-tagged green fluorescent protein (GFP-GST). Continuous online monitoring of reflective interference spectra reveals a significant increase in the bacterium refractive index during hLIF production in comparison to a non-induced reference that coincide with the formation of inclusion bodies. These online monitoring techniques could be applied for fast and cost-effective screening of different protein expression systems.
authors.affiliation: Institut für Technische Chemie, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5, 30167, Hannover, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Ude
authors.affiliation: Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, 32000, Haifa, Israel.
authors.forename: Nadav
authors.initials: N
authors.lastname: Ben-Dov
authors.affiliation: Institut für Technische Chemie, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5, 30167, Hannover, Germany.
authors.forename: André
authors.initials: A
authors.lastname: Jochums
authors.affiliation: Institut für Technische Chemie, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5, 30167, Hannover, Germany.
authors.forename: Zhaopeng
authors.initials: Z
authors.lastname: Li
authors.affiliation: Department of Biotechnology and Food Engineering, Technion-Israel Institute of Technology, 32000, Haifa, Israel.
authors.forename: Ester
authors.initials: E
authors.lastname: Segal
authors.affiliation: Institut für Technische Chemie, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5, 30167, Hannover, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Scheper
authors.affiliation: Institut für Technische Chemie, Gottfried Wilhelm Leibniz Universität Hannover, Callinstraße 5, 30167, Hannover, Germany. beutel@iftc.uni-hannover.de.
authors.forename: Sascha
authors.initials: S
authors.lastname: Beutel
mesh_terms: D019149:Bioreactors / Q000382:microbiology; D002623:Chemistry Techniques, Analytical; D003584:Cytological Techniques / Q000379:methods*; D004926:Escherichia coli / Q000737:chemistry* / Q000254:growth & development / Q000378:metabolism; D002479:Inclusion Bodies / Q000737:chemistry*; D008027:Light; D011994:Recombinant Proteins / Q000032:analysis*; D015203:Reproducibility of Results
chemical_list: D011994:Recombinant Proteins
keywords: Flow cytometry; Inclusion bodies; Online scattered-light sensor; Optical biosensor; Reflective interference Fourier transform spectra; Silicon photonic arrays