Details
| Original language | English |
|---|---|
| Pages (from-to) | 4147-4159 |
| Number of pages | 13 |
| Journal | Applied Microbiology and Biotechnology |
| Volume | 100 |
| Issue number | 9 |
| Early online date | 4 Mar 2016 |
| Publication status | Published - May 2016 |
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 Escherichiacoli 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.
Keywords
- Flow cytometry, Inclusion bodies, Online scattered-light sensor, Optical biosensor, Reflective interference Fourier transform spectra, Silicon photonic arrays
ASJC Scopus subject areas
- Biochemistry, Genetics and Molecular Biology(all)
- Biotechnology
- Immunology and Microbiology(all)
- Applied Microbiology and Biotechnology
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In: Applied Microbiology and Biotechnology, Vol. 100, No. 9, 05.2016, p. 4147-4159.
Research output: Contribution to journal › Article › Research › peer review
}
TY - JOUR
T1 - Online analysis of protein inclusion bodies produced in E. coli by monitoring alterations in scattered and reflected light
AU - Ude, Christian
AU - Ben-Dov, Nadav
AU - Jochums, André
AU - Li, Zhaopeng
AU - Segal, Ester
AU - Scheper, Thomas
AU - Beutel, Sascha
N1 - Funding Information: 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. 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.
PY - 2016/5
Y1 - 2016/5
N2 - 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 Escherichiacoli 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.
AB - 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 Escherichiacoli 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.
KW - Flow cytometry
KW - Inclusion bodies
KW - Online scattered-light sensor
KW - Optical biosensor
KW - Reflective interference Fourier transform spectra
KW - Silicon photonic arrays
UR - http://www.scopus.com/inward/record.url?scp=84960172557&partnerID=8YFLogxK
U2 - 10.1007/s00253-016-7403-1
DO - 10.1007/s00253-016-7403-1
M3 - Article
C2 - 26940052
AN - SCOPUS:84960172557
VL - 100
SP - 4147
EP - 4159
JO - Applied Microbiology and Biotechnology
JF - Applied Microbiology and Biotechnology
SN - 0175-7598
IS - 9
ER -