Gene synthesis, high‐level expression and assignment of backbone 15N and 13C resonances of soybean leghemoglobin

[摘要]

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^α 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.

[学科分类] 生物化学/生物物理

[关键词] Leghemoglobin;Gene synthesis;NMR assignment;Secondary structure;dNTP;deoxyribonucleotide;DSS;sodium 2;2-dimethyl-2-silapentane-5-sulfonate;IPTG;isopropyl thiogalactoside;Lb;leghemoglobin;PAGE;polyacrylamide gel electrophoresis;PCR;polymerase chain reaction;TFA;trifluoro acetic acid;X-gal;5-bromo-4-chloro-3-indolyl-β-d-galactoside