Identification and characterization of recombinant murine interleukin-6 with a C-terminal pentapeptide extension using capillary reversed phase HPLC-MS and edman degradation.
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.
Hammacher
A
A
Joint Protein Structure Laboratory, Ludwig Institute for Cancer Research/Walter and Eliza Hall Institute of Medical Research, Victoria, Australia.
Reid
G E
GE
Moritz
R L
RL
Simpson
R J
RJ
D000818:Animals; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D015850:Interleukin-6 / Q000737:chemistry* / Q000494:pharmacology; D013058:Mass Spectrometry / Q000379:methods*; D051379:Mice; D008934:Mitogens / Q000494:pharmacology; D009842:Oligopeptides / Q000737:chemistry*; D010449:Peptide Mapping / Q000379:methods; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000494:pharmacology
D015850:Interleukin-6; D008934:Mitogens; D009842:Oligopeptides; D011994:Recombinant Proteins