Monitoring proteolysis of recombinant human interferon-gamma during batch culture of Chinese hamster ovary cells.
Proteolytic cleavage of recombinant human interferon-gamma (IFN-gamma) expressed in Chinese hamster ovary (CHO) cells during batch fermentation has been monitored by mass spectrometric peptide mapping. IFN-gamma was purified from cell-free culture supernatant by immunoaffinity chromatography and cleaved by endoprotease Asp-N. Peptide fragments were resolved by reverse-phase HPLC and identified by a combination of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) and automated N-terminal peptide sequencing. Using this approach, a peptide was identified as the C-terminal fragment of the IFN-gamma polypeptide. Analysis of this peptide by MS indicated that the recombinant IFN-gamma polypeptide secreted by CHO cells was truncated by at least ten amino acids, initially at Gln133-Met134. No full length (143 amino acids) polypeptide molecules were observed at any stages of the fermentation. Additional proteolytic cleavages at basic amino acids N-terminal of Gln133 occurred during the later stages of the culture resulting in a heterogeneous IFN-gamma polypeptide with "ragged" C-termini.
Goldman
M H
MH
Research School of Biosciences, University of Kent, Canterbury, United Kingdom.
James
D C
DC
Ison
A P
AP
Bull
A T
AT
D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D002452:Cell Count; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D010450:Endopeptidases / Q000378:metabolism; D005069:Evaluation Studies as Topic; D005285:Fermentation; D006801:Humans; D007371:Interferon-gamma / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011994:Recombinant Proteins
D011994:Recombinant Proteins; D007371:Interferon-gamma; D010450:Endopeptidases