Production of human serum albumin by sugar starvation induced promoter and rice cell culture.
Human serum albumin (HSA) is the most widely used clinical serum protein. Currently, commercial HSA can only be obtained from human plasma, due to lack of commercially feasible recombinant protein expression systems. In this study, inducible expression and secretion of HSA by transformed rice suspension cell culture was established. Mature form of HSA was expressed under the control of the sucrose starvation-inducible rice alpha Amy3 promoter, and secretion of HSA into the culture medium was achieved by using the alpha Amy3 signal sequence. High concentrations of HSA were secreted into culture medium in a short time (2-4 days) by sucrose depletion after cell concentrations had reached a peak density in culture medium containing sucrose. The recombinant HSA had the same electrophoretic mobility as commercial HSA and was stable and free from apparent proteolysis in the culture medium. In a flask scale culture with repeated sucrose provision-depletion cycles, HSA was stably produced with yields up to 11.5% of total medium proteins or 15 mg/L per cycle after each sucrose provision-depletion cycle. A bubble column type bioreactor was designed for production of HSA. In the bioreactor scale culture, HSA was produced with yields up to 76.4 mg/L 4 days after sucrose depletion. HSA was purified from the culture medium to high purity by a simple purification scheme. Enrichment of HSA in culture medium simplifies downstream purification, minimizes protease degradation, and may reduce production cost. The combination of a DNA construct containing the alpha Amy3 promoter and signal sequence, and the use of a rice suspension cell culture can provide an effective system for the production of recombinant pharmaceutical proteins.
Huang
Li-Fen
LF
Institute of Molecular Biology, Academia Sinica, Taipei 115, Nankang, Taiwan, ROC.
Liu
Yu-Kuo
YK
Lu
Chung-An
CA
Hsieh
Shie-Liang
SL
Yu
Su-May
SM
D001483:Base Sequence; D019149:Bioreactors; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D003470:Culture Media; D004274:DNA, Recombinant / Q000235:genetics; D006801:Humans; D066298:In Vitro Techniques; D012275:Oryza / Q000166:cytology / Q000235:genetics* / Q000378:metabolism*; D030821:Plants, Genetically Modified; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D012709:Serum Albumin / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D013395:Sucrose / Q000378:metabolism; D014170:Transformation, Genetic; D000516:alpha-Amylases / Q000235:genetics / Q000378:metabolism
D003470:Culture Media; D004274:DNA, Recombinant; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012709:Serum Albumin; D013395:Sucrose; D000516:alpha-Amylases