INCORPORATION OF NORLEUCINE AT METHIONINE POSITIONS IN RECOMBINANT HUMAN MACROPHAGE-COLONY-STIMULATING FACTOR (M-CSF, 4-153) EXPRESSED IN ESCHERICHIA-COLI - STRUCTURAL-ANALYSIS

Expression of the 17.5-kDa truncated form of human recombinant macroph age colony stimulating factor (rM-CSF, 4-153) in Escherichia coli is c omplicated by the replacement of methionine residues by norleucine. In order to detect and quantitate this mistranslational event, the intac t and the S-carboxyamidomethylated proteins were analyzed by amino aci d analysis, automated Edman amino acid sequencing, and electrospray ma ss spectrometry. In addition, the endoproteinase Glu-C generated pepti des were subjected to amino acid sequencing, high-performance liquid c hromatography, and electrospray ionization mass spectrometry. The exte nt of norleucine substitution in different batches of rM-CSF varied be tween 0% and 20%. The relative instability of methionine residues need s to be considered when calculating the extent of norleucine substitut ion at methionine positions. The mass spectrometry of the intact rM-CS F allowed for examination of the distribution of multiply substituted methionine to norleucine species, and it enabled detection and quantit ation of the norleucine incorporation down to the approximately 3% lev el. 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 relia ble and fast method of detection and quantitation of norleucine-contai ning peptides. Norleucine residues were uniformly distributed among al l four methionine positions (10, 27, 61, and 65). A substitution of me thionine by its structural norleucine analog does not have any effect on the activity of the refolded rM-CSF dimers.