15N labeling method of peptides using a thioredoxin gene fusion expression system: an application to ACTH‐(1–24)

[摘要]

For structure analysis of peptides by multinuclear NMR, stable isotope-labeled samples are required. A direct over-expression system by E. coli cells does not work for that purpose because of rapid degradation of the peptides and/or the mRNA in host cells. We here developed an over-expression system by means of thioredoxin gene fusion system. The fused protein composed of thioredoxin and the objective peptide was expressed in E. coli and then the peptide part was released by enterokinase. This system was successfully applied for the production of ¹⁵N-labeled human adrenocorticotropic hormone fragment (ACTH-(1-24)) as needed for multinuclear NMR analysis.

[学科分类] 生物化学/生物物理

[关键词] Human adrenocorticotropic hormone;Thioredoxin;Enterokinase;Nuclear magnetic resonance;NMR;nuclear magnetic resonance;E coli;Escherichia coli;HSQC;heteronuclear single quantum coherence spectroscopy;SDS-PAGE;sodium dodecyl sulfate polyacryl amide gel electrophoresis;RP-HPLC;reverse phase high pressure liquid chromatography;TFA;trifluoroacetic acid;NOE;nuclear Overhauser effect;NOESY;nuclear Overhauser effect spectroscopy;TPPI;time proportional phase increment;IPTG;isopropyl-β-d-(−)-thiogalactopyranoside