title: Effect of methanol feeding strategies on production and yield of recombinant mouse endostatin from Pichia pastoris.
abstract: Pichia pastoris, a methylotrophic yeast, is an efficient producer of recombinant proteins in which the heterologous gene is under the control of the methanol-induced AOX1 promoter. Hence, the accepted production procedure has two phases: In the first phase, the yeast utilizes glycerol and biomass is accumulated; in the second phase, the yeast utilizes methanol which is used both as an inducer for the expression of the recombinant protein and as a carbon source. Since the yeast is sensitive to methanol concentration, the methanol is supplied gradually to the growing culture. Three methanol addition strategies were evaluated for the purpose of optimizing recombinant endostatin production. Two strategies were based on the yeast metabolism; one responding to the methanol consumption using a methanol sensor, and the other responding to the oxygen consumption. In these two strategies, the methanol supply is unlimited. The third strategy was based on a predetermined exponential feeding rate, controling the growth rate at 0.02 h(-1), in this strategy the methanol supply is limited. Throughout the induction phase glycerol, in addition to methanol, was continuously added at a rate of 1 g L h(-1). Total endostatin production was similar in all three strategies, (400 mg was obtained from 3 L initial volume), but the amount of methanol added and the biomass produced were lower in the predetermined rate method. This caused the specific production of endostatin per biomass and per methanol to be 2 times higher in the predetermined rate than in the other two methods, making the growth control strategy not only more efficient but also more convenient for downstream processing.
authors.affiliation: Biotechnology Unit, National Institutes of Health, NIDDK Building 6, Room B1-33, Bethesda, Maryland 20892-2715, USA.
authors.forename: L B
authors.initials: LB
authors.lastname: Trinh
authors.forename: J N
authors.initials: JN
authors.lastname: Phue
authors.forename: Joseph
authors.initials: J
authors.lastname: Shiloach
mesh_terms: D000818:Animals; D019149:Bioreactors / Q000382:microbiology*; D018929:Cell Culture Techniques / Q000379:methods; D002455:Cell Division; D003094:Collagen / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D043169:Endostatins; D000432:Methanol / Q000378:metabolism*; D051379:Mice; D010446:Peptide Fragments / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification*; D010843:Pichia / Q000145:classification / Q000166:cytology / Q000378:metabolism*; D011786:Quality Control; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013045:Species Specificity
chemical_list: D043169:Endostatins; D010446:Peptide Fragments; D011994:Recombinant Proteins; D003094:Collagen; D000432:Methanol