title: Incorporation of norleucine at methionine positions in recombinant human macrophage colony stimulating factor (M-CSF, 4-153) expressed in Escherichia coli: structural analysis.
abstract: Expression of the 17.5-kDa truncated form of human recombinant macrophage colony stimulating factor (rM-CSF, 4-153) in Escherichia coli is complicated by the replacement of methionine residues by norleucine. In order to detect and quantitate this mistranslational event, the intact and the S-carboxyamidomethylated proteins were analyzed by amino acid analysis, automated Edman amino acid sequencing, and electrospray mass spectrometry. In addition, the endoproteinase Glu-C generated peptides were subjected to amino acid sequencing, high-performance liquid chromatography, and electrospray ionization mass spectrometry. The extent of norleucine substitution in different batches of rM-CSF varied between 0% and 20%. The relative instability of methionine residues needs to be considered when calculating the extent of norleucine substitution at methionine positions. The mass spectrometry of the intact rM-CSF allowed for examination of the distribution of multiply substituted methionine to norleucine species, and it enabled detection and quantitation of the norleucine incorporation down to the approximately 3% level. Selective ion chromatograms of molecular ions of interest obtained in reversed-phase high-performance liquid chromatography/electrospray ionization mass spectrometry of proteolytic fragments offered a reliable and fast method of detection and quantitation of norleucine-containing peptides. Norleucine residues were uniformly distributed among all four methionine positions (10, 27, 61, and 65). A substitution of methionine by its structural norleucine analog does not have any effect on the activity of the refolded rM-CSF dimers.
authors.affiliation: Otsuka America Pharmaceutical Inc., Rockville, Maryland 20850.
authors.forename: Z I
authors.initials: ZI
authors.lastname: Randhawa
authors.forename: H E
authors.initials: HE
authors.lastname: Witkowska
authors.forename: J
authors.initials: J
authors.lastname: Cone
authors.forename: J A
authors.initials: JA
authors.lastname: Wilkins
authors.forename: P
authors.initials: P
authors.lastname: Hughes
authors.forename: K
authors.initials: K
authors.lastname: Yamanishi
authors.forename: S
authors.initials: S
authors.lastname: Yasuda
authors.forename: Y
authors.initials: Y
authors.lastname: Masui
authors.forename: P
authors.initials: P
authors.lastname: Arthur
authors.forename: C
authors.initials: C
authors.lastname: Kletke
mesh_terms: D000595:Amino Acid Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004926:Escherichia coli; D006801:Humans; D006868:Hydrolysis; D016173:Macrophage Colony-Stimulating Factor / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D013058:Mass Spectrometry; D008715:Methionine / Q000737:chemistry*; D008969:Molecular Sequence Data; D009646:Norleucine / Q000032:analysis / Q000737:chemistry*; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000494:pharmacology
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D016173:Macrophage Colony-Stimulating Factor; D009646:Norleucine; D008715:Methionine