Gene synthesis, high-level expression and assignment of backbone 15N and 13C resonances of soybean leghemoglobin.
A synthetic gene for apoleghemoglobin-a from soybean, optimized for expression in Escherichia coli has been designed and synthesized by a recursive polymerase chain reaction technique. The protein has been expressed with high efficiency and a purification protocol has been developed. The holoprotein is readily reconstituted by the addition of heme. 15N- and 15N,13C-labeled samples were produced and backbone 15N and 13C assignments were determined by 2D and 3D NMR spectroscopy. Comparison of the chemical shifts of 13C(alpha) and 13CO with random coil shifts revealed a pattern of secondary structure which correlates well with the one previously derived from homonuclear NMR data and low-resolution X-ray crystallography.
Prytulla
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Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Dyson
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HJ
Wright
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PE
D000595:Amino Acid Sequence; D001483:Base Sequence; D002247:Carbon Isotopes; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007874:Leghemoglobin / Q000737:chemistry / Q000235:genetics*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009587:Nitrogen Isotopes; D013025:Glycine max / Q000737:chemistry*
D002247:Carbon Isotopes; D004274:DNA, Recombinant; D007874:Leghemoglobin; D009587:Nitrogen Isotopes