Enhanced, simplified expression of perdeuterated hemoglobin for NMR structure and dynamics.
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 for NMR characterization of structure and dynamics. In addition, the low sensitivity of NMR demands can be achieved without major sacrifice to yield. We applied the approach to human adult hemoglobin (Hb A), a 64 kDa, tetrameric protein that requires significant post-expression processing. This aspect accentuates the need for high yield. Using specially adapted JM109(DE3) Escherichia coli, we developed a shake-flask approach to express >90% deuterated NMR samples. Typical yields were 2.5-fold higher than obtained from cells adapted by more-traditional methods, while deuteration levels were increased by 17%. Ultimately, a 200 mL culture was sufficient to obtain ((2)H, (15)N)-labeled Hb A sufficient for a 200 microM, 400 microL NMR sample. This avoids need for additional equipment for fermentation, which was used in previous protocols to express Hb A. It also allows a much smaller culture volume than often required by such equipment, for corresponding linear reductions in the cost of labeled starting materials. We tested the adaptation protocol with both JM109 and JM109(DE3) E. coli, and with pre- and post-transformation with the Hb A expression plasmid (pHE7). The (DE3) strain consistently outperformed its parent strain in response to adaptation, with the latter failing to survive adaptation in multiple trials. In addition, pre-transformed cells were consistently more receptive to adaptation. Finally, we also detail updated protocols to isolate Hb A in its functional form.
Department of Chemistry & Chemical Biology, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Cogswell Lab, Troy, NY 12180, USA.
Xinji
X
Guo
James G
JG
Kempf
D003903:Deuterium / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015870:Gene Expression; D006441:Hemoglobin A / Q000737:chemistry* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D019906:Nuclear Magnetic Resonance, Biomolecular; D010957:Plasmids / Q000235:genetics; D011487:Protein Conformation; D014170:Transformation, Genetic
D006441:Hemoglobin A; D003903:Deuterium