Bioreactor production of human alpha(1)-antitrypsin using metabolically regulated plant cell cultures.
Transgenic rice cell cultures, capable of producing recombinant human alpha(1)-antitrypsin (rAAT), were scaled up from shake flasks to a 5-L bioreactor. The maximum specific growth rates (mu(max)) observed from two bioreactor runs were 0.40 day(-1) (doubling time of 1.7 days) and 0.47 day(-1) (doubling time of 1.5 days), and the maximum specific oxygen uptake rates were 0.78 and 0.84 mmol O(2)/(g dw h). Using a metabolically regulated rice alpha-amylase (RAmy3D) promoter, signal peptide, and terminator, sugar deprivation turned on rAAT expression, and rAAT was secreted into the culture medium. After 1 day of culture in sugar-free medium, there was still continued biomass growth, oxygen consumption, and viability. Extracellular concentrations of 51 and 40 mg active rAAT/L were reached 1.7 and 2.5 days, respectively, after induction in a sugar-free medium. Volumetric productivities for two batch cultures were 7.3 and 4.6 mg rAAT/(L day), and specific productivities were 3.2 and 1.6 mg rAAT/(g dw day). Several different molecular weight bands of immunoreactive rAAT were observed on immunoblots.
Trexler
Melody M
MM
Department of Chemical Engineering and Materials Science, One Shields Avenue, University of California, Davis, California 95616, USA.
McDonald
Karen A
KA
Jackman
Alan P
AP
D019149:Bioreactors; D015153:Blotting, Western; D006801:Humans; D012275:Oryza / Q000166:cytology* / Q000235:genetics; D010100:Oxygen / Q000378:metabolism; D030821:Plants, Genetically Modified; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000096:biosynthesis; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis*; D000516:alpha-Amylases / Q000235:genetics
D011994:Recombinant Proteins; D000515:alpha 1-Antitrypsin; D000516:alpha-Amylases; D010100:Oxygen