Enhanced, simplified expression of perdeuterated hemoglobin for NMR structure and dynamics

摘要: Abstract An advanced protocol is provided to adapt cells for enhanced proliferation in and expression from deuterated minimal media. For large proteins (>20–30 kDa), deuteration levels >90% are essential NMR characterization of structure dynamics. In addition, the low sensitivity demands can be achieved without major sacrifice yield. We applied approach human adult hemoglobin (Hb A), a 64 kDa, tetrameric protein that requires significant post-expression processing. This aspect accentuates need high Using specially adapted JM109(DE3) Escherichia coli , we developed shake-flask express samples. Typical yields were 2.5-fold higher than obtained by more-traditional methods, while increased 17%. Ultimately, 200 mL culture was sufficient obtain ( 2 H, 15 N)-labeled Hb A 200 μM, 400 μL sample. avoids additional equipment fermentation, which used previous protocols A. It also allows much smaller volume often required such equipment, corresponding linear reductions cost labeled starting materials. tested adaptation with both JM109 E. pre- post-transformation plasmid (pHE7). The (DE3) strain consistently outperformed its parent response adaptation, latter failing survive multiple trials. pre-transformed more receptive adaptation. Finally, detail updated isolate functional form.