Improved large scale culture of Methylophilus methylotrophus for 13C/15N labeling and random fractional deuteration of ribonucleotides.
Isotopic labeling of RNA with 13C and 15N has become a routine procedure in structural studies by NMR spectroscopy. The methodology in this paper describes the random fractional deuteration of RNA using the obligate methylotropic bacterium, Methylophilus methylotrophus. This bacterium was grown using a non-deuterated carbon source in 52:48 D20/H20 and we have shown that all protons in the ribonucleotides except for the ribose H1 become 52% randomly fractionally deuterated. Improved growth conditions for this organism are also described that yield higher cell densities in liquid culture, which is applicable for all labeling procedures.
Batey
R T
RT
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139, USA.
Cloutier
N
N
Mao
H
H
Williamson
J R
JR
D002247:Carbon Isotopes; D003903:Deuterium; D006088:Gram-Negative Aerobic Bacteria / Q000254:growth & development* / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007553:Isotope Labeling / Q000379:methods*; D009682:Magnetic Resonance Spectroscopy; D009587:Nitrogen Isotopes; D012265:Ribonucleotides; D013696:Temperature
D002247:Carbon Isotopes; D009587:Nitrogen Isotopes; D012265:Ribonucleotides; D003903:Deuterium