Characterizing Enhanced Fractional 13-C Enrichment in Proteins

When adding a fractional amount of u-13C-glucose to bacterial growth media at the time of induction the amount of incorporated C-13 exceeds the fractional amount. This effect is understood as reserving the enriched nutrient for protein expression versus cell proliferation. To date, typical results for 10%w/w u-13C-glucose sample added at the time of induction leads to an incorporation ca. 17% C-13 in the expressed protein (yeast ubiquitin). The incorporation of C-13 was highly dependent on the time of induction. To achieve optimal incorporation, it is necessary to induce prior to the onset of stationary phase. If the enriched nutrient is added after the onset of stationary phase, an enhancement is observed, but not of the same magnitude (ca. 13%). A number of questions arise from this work. For example, it is natural to wonder if the enhancement can be improved through variations in the protocol. Also prior work observed poor preservation of 13Cα-13Cβ spin pairs in the amino acids ILV when adding 10% u-13C-glucose to expression media at induction, leading to the absence of Cβ signals in HNCACB spectra for ILV, a potentially useful editing effect. Both the enhancement and Cα-Cβ performance have been evaluated under a variety of addition expression conditions, and will be reported. Specifically, we investigated if it is possible to restore the 13Cα-13Cβ ILV spin pairs in fractionally enriched samples