Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins
| Autor: | Gyula Pálfy, István Vida, Zsolt Fazekas, Éva Kiss, András Perczel, Dóra Nagy-Fazekas, Pál Stráner, Gergő Gyulai |
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| Rok vydání: | 2021 |
| Předmět: |
Amyloidogenic Proteins
Bioengineering Applied Microbiology and Biotechnology Biochemistry law.invention 03 medical and health sciences law Labelling Escherichia coli Research Articles 030304 developmental biology 0303 health sciences Isotope 030306 microbiology Chemistry Atomic force microscopy Cost sensitive Active protein Culture Media Isotope Labeling Fermentation Recombinant DNA TP248.13-248.65 Research Article Biotechnology |
| Zdroj: | Microbial Biotechnology Microbial Biotechnology, Vol 14, Iss 3, Pp 1107-1119 (2021) |
| ISSN: | 1751-7915 |
| DOI: | 10.1111/1751-7915.13778 |
| Popis: | Summary We developed a cost sensitive isotope labelling procedure using a fed‐batch fermentation method and tested its efficiency producing the 15N‐, 13C‐ and 15N/13C‐labelled variants of an amyloidogenic miniprotein (E5: EEEAVRLYIQWLKEGGPSSGRPPPS). E5 is a surface active protein, which forms amyloids in solution. Here, we confirm, using both PM‐IRRAS and AFM measurements, that the air–water interface triggers structural rearrangement and promotes the amyloid formation of E5, and thus it is a suitable test protein to work out efficient isotope labelling schemes even for such difficult sequences. E. coli cells expressing the recombinant, ubiquitin‐fused miniprotein were grown in minimal media containing either unlabelled nutrients, or 15N‐NH4Cl and/or 13C‐D‐Glc. The consumption rates of NH4Cl and D‐Glc were quantitatively monitored during fermentation and their ratio was established to be 1:5 (for NH4Cl: D‐Glc). One‐ and two‐step feeding schemes were custom‐optimized to enhance isotope incorporation expressing five different E5 miniprotein variants. With the currently optimized protocols we could achieve a 1.5‐ to 5‐fold increase of yields of several miniproteins coupled to a similar magnitude of cost reduction as compared to flask labelling protocols. We developed a cost sensitive isotope labelling procedure using a fed‐batch fermentation method and tested its efficiency producing the 15N‐, 13C‐ and 15N/13C‐labelled variants of an amyloidogenic miniprotein E5, which is a surface active and forms amyloids in solution. With the currently optimized protocols we could achieve a 1.5‐ to 5‐fold increase of yields of several miniproteins coupled to a similar magnitude of cost reduction as compared to flask labelling protocols. |
| Databáze: | OpenAIRE |
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