Online analysis of protein inclusion bodies produced in E. coli by monitoring alterations in scattered and reflected light
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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.
KeywordsOnline scattered-light sensor Inclusion bodies Flow cytometry Reflective interference Fourier transform spectra Silicon photonic arrays Optical biosensor
We would like to thank all members of our research groups for providing feedback and suggestions. This work was partially supported by the Russell Berrie Nanotechnology Institute (RBNI) and the Lorry I. Lokey Interdisciplinary Center for Life Sciences and Engineering (LS&E). SiPA samples were prepared at the Micro-Nano Fabrication Unit (MNFU), Technion.
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Conflict of interest
The authors declare that they have no conflict of interest.
Human and animal rights
No research involving human participants and/or animals was carried out.
This study was supported by the Bundesministerium für Wirtschaft und Technologie via Arbeitsgemeinschaft industrieller Forschungsvereinigungen “Otto von Guericke” e. V. within the Zentrale Innovationsoffensive Mittelstand-initiative. Additional funding for this study was provided by the Deutsche Technion-Gesellschaft.
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The authors declare that they agree each with submission of this manuscript. The authors declare that the manuscript has not been submitted to more than one journal for simultaneous consideration.
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