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

FEBS Letters

Jane Dyson, H.¹  Prytulla, Stefan¹  Wright, Peter E.¹ 

[1] Department of Molecular Biology and Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, CA 92037, USA

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;   

DOI  :  10.1016/S0014-5793(96)01278-1

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 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.

【 授权许可】

Unknown