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Differential stability of proteolytically active and inactive recombinant metalloproteinase in Chinese hamster ovary cells
Author(s) -
Morrison Charlotte J.,
McMaster W. Robert,
Piret James M.
Publication year - 1997
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/(sici)1097-0290(19970320)53:6<594::aid-bit7>3.0.co;2-h
Subject(s) - chinese hamster ovary cell , biology , metalloproteinase , flow cytometry , heterologous , mutant , population , microbiology and biotechnology , hamster , cell culture , biochemistry , genetics , enzyme , gene , demography , sociology
Stability of heterologous protein expression during production is critical for regulatory approval of vaccine and therapeutic products. Leishmania GP63, a zinc metalloproteinase that is a potential vaccine candidate, has been expressed on the surface of Chinese hamster ovary (CHO) cells. Flow cytometry was used to follow the stability of GP63 expression. Expression of proteolytically active GP63 (GP63WT) was unstable whether or not methotrexate (MTX) selection was maintained. In contrast, expression of an active site mutant (GP63E265D) was stable under MTX selection. In the absence of selection, the decline in GP63E265D expression was more gradual than the loss of GP63WT expression. Different molecular mechanisms accounted for these losses and resulted in higher growth rate nonproducer populations. A dynamic population model was used to calculate the conversion rates of GP63WT producers to nonproducers. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 594–600, 1997.