title: Expression and purification of the p75 neurotrophin receptor transmembrane domain using a ketosteroid isomerase tag.
abstract: BACKGROUND
  Receptors with a single transmembrane (TM) domain are essential for the signal transduction across the cell membrane. NMR spectroscopy is a powerful tool to study structure of the single TM domain. The expression and purification of a TM domain in Escherichia coli (E.coli) is challenging due to its small molecular weight. Although ketosteroid isomerase (KSI) is a commonly used affinity tag for expression and purification of short peptides, KSI tag needs to be removed with the toxic reagent cyanogen bromide (CNBr).
  
  
  RESULTS
  The purification of the TM domain of p75 neurotrophin receptor using a KSI tag with the introduction of a thrombin cleavage site is described herein. The recombinant fusion protein was refolded into micelles and was cleaved with thrombin. Studies showed that purified protein could be used for structural study using NMR spectroscopy.
  
  
  CONCLUSIONS
  These results provide another strategy for obtaining a single TM domain for structural studies without using toxic chemical digestion or acid to remove the fusion tag. The purified TM domain of p75 neurotrophin receptor will be useful for structural studies.
authors.affiliation: Agency for Science, Technology and Research, Nanos, Singapore.
authors.forename: Qingxin
authors.initials: Q
authors.lastname: Li
authors.forename: Angela Shuyi
authors.initials: AS
authors.lastname: Chen
authors.forename: Shovanlal
authors.initials: S
authors.lastname: Gayen
authors.forename: CongBao
authors.initials: C
authors.lastname: Kang
mesh_terms: D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D007535:Isomerases / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007664:Ketosteroids / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D020800:Receptor, Nerve Growth Factor / Q000737:chemistry / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D007664:Ketosteroids; D020800:Receptor, Nerve Growth Factor; D011993:Recombinant Fusion Proteins; D007535:Isomerases