ISSN:
1476-5535
Schlagwort(e):
Keywords: baculovirus–insect cell expression vector system (BEVS); Sf-9; HSV protease; glutathione-S-transferase
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Biologie
,
Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
Notizen:
A gene expression system using recombinant Autographa californica nuclear polyhedrosis virus (baculovirus) and Sf-9 cells has been scaled up to the 10-L tank level and shown to be capable of producing herpes simplex virus (HSV) protease in serum-free media. High densities of Spodoptera frugiperda (Sf-9) cells were achieved by modifying two 10-L Biolafitte fermenters specifically for insect cell growth. The existing Rushton impellers were replaced by marine impellers to reduce shear and the aeration system was modified to allow external addition of air/O2 mixtures at low flow rates through either the sparge line or into the head space of the fermenter. To inoculate the tanks, Sf-9 cells were adapted to grow to high cell densities (6–10 × 106 cells ml−1) in shake flasks in serum-free media. With these procedures, cell densities of 5 × 106 cells ml−1 were routinely achieved in the 10-L tanks. These cells were readily infected with recombinant baculovirus expressing the 247-amino acid catalytic domain of the HSV-1 strain 17 protease UL26 gene as a glutathione-S-transferase (GST) fusion protein (GST-247). Three days after infection at a multiplicity of infection (MOI) of 3 pfu cell−1, the GST-247 fusion protein was purified from a cytoplasmic lysate by Glutathione Sepharose 4-B affinity chromatography with reproducible yields of 11–38 mg L−1 of recombinant protein and ≥ 90% purity. Maximum production of this protein was observed at a cell density of 5.0 × 106 cells ml−1.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1038/sj.jim.2900446
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