Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins. Issue 3 (19th March 2021) - Vida, István
Summary: 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. 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, 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.