Human neurotrophin-3: a one-step peptide mapping method and complete disulfide characterization of the recombinant protein.
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 initial denaturation of NT-3 in 6 M guanidinium chloride (pH6) for 2 hr at 37 degrees 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.
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Protein Structure, Amgen Inc., Thousand Oaks, California 91320, USA.
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Katta
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Rosenfeld
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Rohde
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Haniu
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D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D002918:Chymotrypsin / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D004220:Disulfides / Q000737:chemistry*; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D019791:Guanidine; D006146:Guanidines / Q000494:pharmacology; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors / Q000737:chemistry* / Q000235:genetics; D020933:Neurotrophin 3; D010446:Peptide Fragments / Q000737:chemistry; D010449:Peptide Mapping; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013820:Thermolysin / Q000378:metabolism
D004220:Disulfides; D006146:Guanidines; D009414:Nerve Growth Factors; D020933:Neurotrophin 3; D010446:Peptide Fragments; D011994:Recombinant Proteins; D002918:Chymotrypsin; D013820:Thermolysin; D019791:Guanidine; D003545:Cysteine; D004229:Dithiothreitol