title: Enhanced truncated-t-PA (CT-b) expression in high-cell-density fed-batch cultures of Pichia pastoris through optimization of a mixed feeding strategy by response surface methodology.
abstract: Recently, Pichia pastoris has been the focal point of interest as an expression system for production of many recombinant proteins. The study and optimization of feeding strategy are of major importance to achieve maximum volumetric productivity in fed-batch cultivations. Among different feeding strategies used in P. pastoris fed-batch cultures, those trying to maintain a constant specific growth rate have usually resulted in superior productivities. The objective of the present study was to investigate and optimize the co-feeding of glycerol and methanol to attain maximum expression of t-PA in P. pastoris fed-batch cultures with constant specific growth rate. The experiments were designed by response surface methodology, considering the specific feeding rates of methanol and glycerol as independent variables. In each experiment, glycerol and methanol were fed according to a predetermined equation to maintain a constant specific growth rate. It was found that with glycerol feeding for higher specific growth rates, the inhibitory properties of glycerol are more pronounced, while the best expression level was achieved when the ratio of µ set glycerol to that of methanol was around 1.67. In all specific growth rates tested, almost a similar ratio of the specific glycerol feeding rate to that of methanol led to the maximum protein production and activity. The statistical model predicted the optimal operating conditions for µ set glycerol and that of methanol to be 0.05 and 0.03 h(-1), respectively. Applying the optimum strategy, maximum of 52 g/L biomass, 300 mg/L t-PA and 340,000 IU/mL enzyme activity were obtained.
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Mohammad Reza
authors.initials: MR
authors.lastname: Kazemali
authors.affiliation: Cancer Genetics Research Group, Breast Cancer Research Center, Academic Center for Education Culture and Research, Tehran, Iran.
authors.forename: Keivan
authors.initials: K
authors.lastname: Majidzadeh
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Soroush
authors.initials: S
authors.lastname: Sardari
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Amir Hossein
authors.initials: AH
authors.lastname: Saadati
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Farzaneh
authors.initials: F
authors.lastname: Barkhordari
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Ahmad
authors.initials: A
authors.lastname: Adeli
authors.affiliation: Biotechnology Research Center, Pasteur Institute of Iran, Tehran, Iran.
authors.forename: Fereidoun
authors.initials: F
authors.lastname: Mahboudi
authors.affiliation: Department of Industrial Biotechnology, National Institute of Genetic Engineering and Biotechnology, PO Box 14965-161, Tehran, Iran. maghsoudi@nigeb.ac.ir.
authors.forename: Amir
authors.initials: A
authors.lastname: Maghsoudi
mesh_terms: D015870:Gene Expression; D006801:Humans; D010843:Pichia / Q000235:genetics / Q000254:growth & development*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000096:biosynthesis* / Q000235:genetics
chemical_list: D011994:Recombinant Proteins; D010959:Tissue Plasminogen Activator
keywords: Mixed feeding strategy; Pichia pastoris; Plasminogen activator; Response surface methodology; Specific growth rate