Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins.
István Vida, Zsolt Fazekas, Gergő Gyulai, Dóra Nagy-Fazekas, Gyula Pálfy, Pál Stráner, Éva Kiss, András Perczel
Author Information
István Vida: Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány P. stny. 1/A, Budapest, H-1117, Hungary. ORCID
Zsolt Fazekas: Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány P. stny. 1/A, Budapest, H-1117, Hungary.
Gergő Gyulai: Laboratory of Interfaces and Nanostructures, Institute of Chemistry, Eötvös Loránd University, Pázmány P. stny. 1/A, Budapest, H-1117, Hungary. ORCID
Dóra Nagy-Fazekas: Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány P. stny. 1/A, Budapest, H-1117, Hungary.
Gyula Pálfy: Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány P. stny. 1/A, Budapest, H-1117, Hungary. ORCID
Pál Stráner: MTA-ELTE Protein Modeling Research Group, Eötvös Loránd Research Network (ELKH), Institute of Chemistry, Eötvös Loránd University, Pázmány P. stny. 1/A, Budapest, H-1117, Hungary. ORCID
Éva Kiss: Laboratory of Interfaces and Nanostructures, Institute of Chemistry, Eötvös Loránd University, Pázmány P. stny. 1/A, Budapest, H-1117, Hungary. ORCID
András Perczel: Laboratory of Structural Chemistry and Biology, Institute of Chemistry, Eötvös Loránd University, Pázmány P. stny. 1/A, Budapest, H-1117, Hungary. ORCID
We developed a cost sensitive isotope labelling procedure using a fed-batch fermentation method and tested its efficiency producing the N-, C- and N/ C-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 N-NH Cl and/or C-D-Glc. The consumption rates of NH Cl and D-Glc were quantitatively monitored during fermentation and their ratio was established to be 1:5 (for NH Cl: 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.
References
Curr Protein Pept Sci. 2016;17(2):147-55
[PMID: 26521952]
Biochemistry. 2014 Jun 10;53(22):3540-52
[PMID: 24828921]
