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 ap proximately equal amounts of mIL-6 with a C-terminal pentapeptide exte nsion, The extension mutant was generated by readthrough of the stopco don, and termination at a second in-frame stopcodon 12 base pairs 3' i n the expression vector. Aliquots of the preparation were subjected to proteolytic digestion with Asp-N and Lys-C-endopeptidase, The resulta nt peptides were separated by reversed-phase capillary HPLC, and analy sed using a combination of mass spectrometry and N-terminal sequence a nalysis, These data revealed a C-terminal pentapeptide (Gln-Gly-Ser-Va l-Asp) extension, with the authentic stopcodon being translated as glu tamine, The extension mutant was isolated by reversed-phase HPLC and s hown to have similar mitogenic activity to mIL-6 on murine hybridoma 7 TD1 cells. (C) 1997 John Wiley & Sons, Ltd.