Incorporation of Norleucine at Methionine Positions in Recombinant Human Macrophage Colony Stimulating Factor (M-CSF, 4-153) Expressed in Escherichia coli: Structural Analysis

• Published in: Biochemistry (Easton) Vol. 33; no. 14; pp. 4352 - 4362
• Main Authors: Randhawa, Zafar I, Witkowska, H. Ewa, Cone, James, Wilkins, James A, Hughes, Patricia, Yamanishi, Kazuya, Yasuda, Setsuko, Masui, Yoshihiro, Arthur, Patrick
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 12-04-1994
• Subjects: Amino Acid Sequence; Biological and medical sciences; Chromatography, High Pressure Liquid; Cloning, Molecular; Diverse techniques; Escherichia coli; Fundamental and applied biological sciences. Psychology; Humans; Hydrolysis; Macrophage Colony-Stimulating Factor - chemistry; Macrophage Colony-Stimulating Factor - genetics; Macrophage Colony-Stimulating Factor - pharmacology; Mass Spectrometry; Methionine - chemistry; Molecular and cellular biology; Molecular Sequence Data; Norleucine - analysis; Norleucine - chemistry; Peptide Fragments - chemistry; Peptide Fragments - genetics; Peptide Fragments - pharmacology; Protein Conformation; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Recombinant Proteins - pharmacology; Performance evaluation; Human; Norleucine; Edman degradation; Escherichia coli; Chemical method; Electrospray; Analysis method; Bacteria; Macrophage colony stimulating factor; Recombinant protein; Mass spectrometry; Enterobacteriaceae; Quantitative analysis
• ISSN: 0006-2960; 1520-4995
• DOI: 10.1021/bi00180a032

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.