Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins

Abstract

We developed a cost sensitive isotope labelling procedure using a fed-batch fermentation method and tested its efficiency producing the ¹⁵ N-, ¹³ C- and ¹⁵ N/¹³ 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 ¹⁵ N-NH₄ Cl and/or ¹³ C-D-Glc. The consumption rates of NH₄ Cl and D-Glc were quantitatively monitored during fermentation and their ratio was established to be 1:5 (for NH₄ 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.