TY - JOUR
T1 - Large scale production of the copper enzyme peptidylglycine monooxygenase using an automated bioreactor
AU - Bauman, Andrew T.
AU - Ralle, Martina
AU - Blackburn, Ninian J.
N1 - Funding Information:
The work was supported by a grant from the National Institutes of Health NS27583 to N.J.B. We thank Drs. Betty A. Eipper and Henry T. Keutman for assistance with protein sequencing. We thank Joel Burchfiel for assistance with media preparation and protein purification.
PY - 2007/1
Y1 - 2007/1
N2 - Rat PHM (peptidylglycine α-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: 5 mg/day, B2: 12-15 mg/day), activity (B1: 12-20 μmol O2/min/mg, B2: 24-36 μmol O2/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.
AB - Rat PHM (peptidylglycine α-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: 5 mg/day, B2: 12-15 mg/day), activity (B1: 12-20 μmol O2/min/mg, B2: 24-36 μmol O2/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.
KW - Bioreactor
KW - Copper
KW - Mass spectrometry
KW - Monooxygenase
KW - PHM
KW - Peptidylglycine
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U2 - 10.1016/j.pep.2006.06.016
DO - 10.1016/j.pep.2006.06.016
M3 - Article
C2 - 16931045
AN - SCOPUS:33751422749
SN - 1046-5928
VL - 51
SP - 34
EP - 38
JO - Protein Expression and Purification
JF - Protein Expression and Purification
IS - 1
ER -