Article

Journal of Protein Chemistry

, Volume 15, Issue 4, pp 351-358

Human neurotrophin-3: A one-step peptide mapping method and complete disulfide characterization of the recombinant protein

  • John O. HuiAffiliated withProtein Structure, Amgen Inc.
  • , John LeAffiliated withProtein Structure, Amgen Inc.
  • , Viswanatham KattaAffiliated withProtein Structure, Amgen Inc.
  • , Robert RosenfeldAffiliated withProtein Chemistry, Amgen Inc.
  • , Michael F. RohdeAffiliated withProtein Structure, Amgen Inc.
  • , Mitsuru HaniuAffiliated withProtein Structure, Amgen Inc.

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Abstract

Human neurotrophin-3 (NT-3) is a member of the nerve growth factor (NGF) family of neurotrophic factors, and the recombinant protein is being developed as a therapeutic for neurodegenerative diseases. The final product purity and lot-to-lot variation are monitored routinely by peptide mapping. However, only the N-terminal region of NT-3 was susceptible to proteolysis under native conditions. Complete digestion required that the protein be chemically modified by reduction and S-alkylation prior to proteolysis. Complete proteolytic degradation of the protein was achieved simply by an intial denaturation of NT-3 in 6 M guanidinium chloride (pH 6) for 2 hr at 37°C, followed by a tenfold dilution with the digestion buffer (0.1 M Tris-HCl, 1 mM CaCl2 at pH 7.0) and immediate addition of chymotrypsin at 1% by weight. Direct comparison of the peptide map with an identical aliquot that had been reduced and alkylated also allowed the establishment of the cystine linkages present in NT-3: Cys14 to Cys79, Cys57 to Cys108, and Cys67 to Cys110. This disulfide structure is homologous to the NGF family of neurotrophic factors.

Key words

Neurotrophins peptide mapping disulfide characterization