Large scale production of the copper enzyme peptidylglycine monooxygenase using an automated bioreactor.
Rat PHM (peptidylglycine alpha-hydroxylating monooxygenase; EC 1.14.17.3) expressed in CHO DG44 cells as a recombinant protein (rat PHMcc, residues 42-356 cloned in the pCIS vector, A.S. Kolhekar, H.T. Keutman, R. E. Mains, A.S.W. Quon, B.A. Eipper, Biochemistry 36 (1997) 10901-10909), was produced in two different bioreactors, a Cellmax 100 (B1) and an Accusyst-MiniMax (B2). B2 contains features not present in B1, which contribute to environmental control, and ease of operation, and was more successful at producing high quality PHM than B1 in both yield (B1: 5mg/day, B2: 12-15 mg/day), activity (B1: 12-20 micromol O(2)/min/mg, B2: 24-36 micromol O(2)/min/mg), and viability (B1: <6 months, B2: indefinite). Additionally, B1 exhibited clipping at Ser 61, and a decline in quality late in the run. PHM from B2 was of consistent quality and homogeneity throughout the run. The increased yield and purity made possible collection of visible spectra of the Cu(II) sites, and mass spectrometric data not previously available.
Bauman
Andrew T
AT
Department of Environmental and Biomolecular Systems, OGI School of Science and Engineering, Oregon Health and Sciences University, Beaverton, OR 97006, USA.
Ralle
Martina
M
Blackburn
Ninian J
NJ
D000818:Animals; D001331:Automation; D019149:Bioreactors; D001709:Biotechnology / Q000295:instrumentation; D016466:CHO Cells; D006224:Cricetinae; D003470:Culture Media; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism; D008647:Mesocricetus; D006899:Mixed Function Oxygenases / Q000096:biosynthesis*; D009097:Multienzyme Complexes / Q000096:biosynthesis*; D051381:Rats
D003470:Culture Media; D009097:Multienzyme Complexes; D006899:Mixed Function Oxygenases; C035189:peptidylglycine monooxygenase; D005947:Glucose