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Published on: December 18, 2008
Identification and characterization of recombinant murine interleukin-6 with a C-terminal pentapeptide extension
A Hammacher1, G E Reid, R L Moritz
1Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research/Walter and Eliza Hall Institute of Medical Research, Victoria, Australia.
Abstract:
We have identified a preparation of recombinant murine interleukin-6 (mIL-6) that, in addition to the anticipated product, also contained approximately equal amounts of mIL-6 with a C-terminal pentapeptide extension. The extension mutant was generated by readthrough of the stopcodon, and termination at a second in-frame stopcodon 12 base pairs 3' in the expression vector. Aliquots of the preparation were subjected to proteolytic digestion with Asp-N and Lys-C-endopeptidase. The resultant peptides were separated by reversed-phase capillary HPLC, and analysed using a combination of mass spectrometry and N-terminal sequence analysis. These data revealed a C-terminal pentapeptide (Gln-Gly-Ser-Val-Asp) extension, with the authentic stopcodon being translated as glutamine. The extension mutant was isolated by reversed-phase HPLC and shown to have similar mitogenic activity to mIL-6 on murine hybridoma 7TD1 cells.
Insights
A novel recombinant murine interleukin-6 (mIL-6) variant with a C-terminal pentapeptide extension was identified. This extension mutant exhibits similar mitogenic activity to standard mIL-6, suggesting potential implications for cytokine research.
Area of Science:
- Biochemistry
- Molecular Biology
- Immunology
Background:
- Recombinant protein production can yield unexpected variants.
- Post-translational modifications and expression artifacts influence protein function.
Purpose of the Study:
- To characterize a novel C-terminally extended variant of recombinant murine interleukin-6 (mIL-6).
- To assess the biological activity of the identified mIL-6 extension mutant.
Main Methods:
- Proteolytic digestion using Asp-N and Lys-C endopeptidases.
- Separation of peptides via reversed-phase capillary HPLC.
- Analysis using mass spectrometry and N-terminal sequence analysis.
Main Results:
- A recombinant mIL-6 preparation contained equal amounts of the intended product and a C-terminal pentapeptide (Gln-Gly-Ser-Val-Asp) extension mutant.
- The extension was caused by stop codon readthrough and termination at a downstream in-frame stop codon.
- The extension mutant demonstrated comparable mitogenic activity to wild-type mIL-6 on murine hybridoma 7TD1 cells.
Conclusions:
- Stop codon readthrough during recombinant protein expression can generate functional variants.
- The C-terminal pentapeptide extension does not abolish the mitogenic activity of mIL-6.
- This finding highlights the importance of thorough characterization of recombinant proteins.
