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Isolation and characterization of modified species of a mutated (Cys125 -Ala) recombinant human interleukin-2
Galina Moya1, Luis Javier González, Vivian Huerta
1Center for Genetic Engineering and Biotechnology, Havana, Cuba. galina.moya@cigb.edu.cu
Abstract:
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.
Insights
This study identified unexpected modifications in recombinant human interleukin-2 (rhIL-2A125) during purification. Researchers discovered N-terminal processing, C-terminal extensions, acetylation, oxidation, and disulfide-linked dimers, revealing complex post-translational variations.
Area of Science:
- Biochemistry
- Protein Chemistry
- Analytical Chemistry
Background:
- Recombinant human interleukin-2 (rhIL-2A125) purification presents challenges due to heterogeneous forms.
- Understanding these variants is crucial for rhIL-2A125 characterization and therapeutic applications.
Purpose of the Study:
- To identify and characterize unknown forms of rhIL-2A125 present in hydrophobic fractions during RP-HPLC purification.
- To elucidate the nature of post-translational modifications and structural variations in rhIL-2A125.
Main Methods:
- Reversed-phase high-performance liquid chromatography (RP-HPLC) for purification.
- Automatic Edman degradation and electrospray ionization mass spectrometry (ESI-MS) for sequence and mass analysis.
- Trypsin digestion, selective peptide blocking, and cation-exchange chromatography for isolating modified peptides.
Main Results:
- Observed partial N-terminal methionine processing and oxidation of Met104 in less hydrophobic fractions (LHF).
- Identified two unexpected C-terminal peptides: one extended and one truncated.
- Characterized N-terminal and lysine acetylation, free cysteine, and covalent linkage to other molecules in more hydrophobic fractions (MHF).
- Detected disulfide-linked covalent dimers of rhIL-2A125.
Conclusions:
- Recombinant rhIL-2A125 exhibits significant heterogeneity due to N-terminal processing, C-terminal modifications, acetylation, oxidation, and dimerization.
- These findings highlight the complexity of rhIL-2A125 post-translational modifications and the importance of advanced analytical techniques for comprehensive characterization.
