title: Optimization of the pilot-scale production of an ice-binding protein by fed-batch culture of Pichia pastoris.
abstract: Ice-binding proteins (IBPs) can bind to the ice crystal and inhibit its growth. Because this property of IBPs can increase the freeze-thaw survival of cells, IBPs have attracted the attention from industries for their potential use in biotechnological applications. However, their use was largely hampered by the lack of the large-scale recombinant production system. In this study, the codon-optimized IBP from Leucosporidium sp. (LeIBP) was constructed and subjected to high-level expression in methylotrophic Pichia pastoris system. In a laboratory-scale fermentation (7 L), the optimal induction temperature and pH were determined to be 25 °C and 6.0, respectively. Further, employing glycerol fed-batch phase prior to methanol induction phase enhanced the production of recombinant LelBP (rLeIBP) by ∼100 mg/l. The total amount of secreted proteins at these conditions (25 °C, pH 6.0, and glycerol fed-batch phase) was ∼443 mg/l, 60 % of which was rLeIBP, yielding ∼272 mg/l. In the pilot-scale fermentation (700 L) under the same conditions, the yield of rLeIBP was 300 mg/l. To our best knowledge, this result reports the highest production yield of the recombinant IBP. More importantly, the rLeIBP secreted into culture media was stable and active for 6 days of fermentation. The thermal hysteresis (TH) activity of rLeIBP was about 0.42 °C, which is almost the same to those reported previously. The availability of large quantities of rLeIBP may accelerate further application studies.
authors.affiliation: Division of Polar Life Sciences, Korea Polar Research Institute, Incheon 406-840, South Korea.
authors.forename: Jun Hyuck
authors.initials: JH
authors.lastname: Lee
authors.forename: Sung Gu
authors.initials: SG
authors.lastname: Lee
authors.forename: Hackwon
authors.initials: H
authors.lastname: Do
authors.forename: Jong Chan
authors.initials: JC
authors.lastname: Park
authors.forename: Eunjung
authors.initials: E
authors.lastname: Kim
authors.forename: Yong-Hoe
authors.initials: YH
authors.lastname: Choe
authors.forename: Se Jong
authors.initials: SJ
authors.lastname: Han
authors.forename: Hak Jun
authors.initials: HJ
authors.lastname: Kim
mesh_terms: D001487:Basidiomycota / Q000235:genetics; D061249:Batch Cell Culture Techniques; D001709:Biotechnology / Q000379:methods*; D002352:Carrier Proteins / Q000235:genetics* / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D005656:Fungal Proteins / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007053:Ice; D060847:Metabolic Engineering; D010843:Pichia / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013696:Temperature
chemical_list: D002352:Carrier Proteins; D003470:Culture Media; D005656:Fungal Proteins; D007053:Ice; D011994:Recombinant Proteins