Isolation and characterization of modified species of a mutated (Cys125 -Ala) recombinant human interleukin-2.
During purification of recombinant and mutated interleukin-2 (rhIL-2A125) by reversed-phase-high-performance liquid chromatography, more and less hydrophobic fractions named MHF and LHF, respectively are discarded due to the presence of some unidentified forms of rhIL-2Ala125. Using slow and linear gradients of acetonitrile, these fractions were further purified by RP-HPLC, analyzed by automatic Edman degradation, digested with trypsin and analyzed by electrospray ionization mass spectrometry. In all fractions, partial processing of the N-terminal Met residue was observed. In the LHF the Met104 was partially oxidized as sulfoxide. Combining the selective and reversible blocking of tryptic peptides and cation-exchange chromatography, two unexpected C-terminal peptides were selectively isolated. Automatic N-terminal sequencing showed that one of these corresponded to the C-terminal peptide of rhIL-2Ala125 linked to another 11 amino acids (AANDENYALAA) and the other corresponded to the C-terminal peptide of a truncated rhIL-2Ala125 without the C-terminal threonine residue and the extension of the 11 amino acids previously mentioned. MHF contained a mixture of four species of rhIL-2A125 monoacetylated at the N-terminus and at the epsilon-amino groups of internal Lys residues: 8, 32 and 48. Cys58 was found as free cysteine and also covalently linked to Mr 69 and 77 molecules. Covalent dimers of rhIL-2A125 linked through disulfide bridges between Cys58 and Cys105 of different monomers were also found.
D000409:Alanine / Q000737:chemistry*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003545:Cysteine / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007376:Interleukin-2 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D021241:Spectrometry, Mass, Electrospray Ionization
D007376:Interleukin-2; D011994:Recombinant Proteins; D003545:Cysteine; D000409:Alanine