Single-chain antibody fragment production in Pichia pastoris: Benefits of prolonged pre-induction glycerol feeding.
Secretory production of a single-chain antibody fragment (scFv) by recombinant Pichia pastoris using the methanol inducible AOX1 promoter is limited biochemically by retarded secretion, and economically by the high demand for pure oxygen. To address the problem, the adaptation phase with growth-limiting feeding of glycerol before the production phase was optimized. In a standard procedure with a short glycerol-feeding phase before induction, scFv accumulated in the supernatant only after 15 h. Conversely, scFv started to appear immediately in the medium upon methanol induction when the glycerol-feeding phase was extended to 18 h. Interestingly, despite a significantly lower cell density in the cultivation with extended glycerol feeding, the same amount of functional product of 300 mg/L was obtained about 30 h after the start of glycerol feeding with both methods. mRNA analysis revealed that the higher and faster production of the product was related to longer lasting induction of the scFv mRNA. Additional effects of a better adaptation of the secretion machinery may be suggested by higher expression of unfolded protein response-related genes KAR2 and PDI. A clear benefit of the longer glycerol-feeding phase was a 75% reduction of the consumption of both pure oxygen and methanol, and a significantly lower cell density, which would be beneficial for down-stream purification of the product.
Institute for Biotechnology, Martin-Luther-University Halle-Wittenberg, Halle, Germany.
Narendar K
NK
Khatri
Dörte
D
Gocke
Oliver
O
Trentmann
Peter
P
Neubauer
Frank
F
Hoffmann
D019149:Bioreactors / Q000382:microbiology*; D001709:Biotechnology / Q000379:methods*; D005990:Glycerol / Q000378:metabolism*; D010843:Pichia / Q000378:metabolism*; D057127:Single-Chain Antibodies / Q000096:biosynthesis*
D057127:Single-Chain Antibodies; D005990:Glycerol