Bacterial fermentation and isotope labelling optimized for amyloidogenic proteins

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.     

Acetalation of 1,6-anhydro-1(6)-thio-D-glucitol

Acetalation of 1,6-anhydro-1(6)-thio-D-glucitol (1a) with acetone, formaldehyde, or benzaldehyde afforded 2,3:4,5-diacetals (2a, 2b, and 2c) whose structure, after desulfurization, was proved by mass spectrometry. Upon partial hydrolysis of 2a, one of the isopropylidene groups was split off, and the other migrated to O-3,O-4 to give 4b. In 4b, in the stable conformation, OH-2 occupies an equatorial position, whereas OH-5 is axially oriented. Accordingly, OH-2 reacts faster than OH-5 on methylation of 4b, giving 4e. Hydrolysis of the isopropylidene group of the 2,5-di-O-methyl derivative 4d and subsequent mesylation afforded the corresponding 3,4-di-O-mesyl compound 1c, which showed significant ulcerostatic activity.