Optimal conditions for the expression of a single-chain antibody (scFv) gene in Pichia pastoris

A Pichia pastoris system was used to express a single-chain antibody (scFv) targeted against Mamestra configurata (bertha armyworm) serpins. To improve scFv production we examined parameters such as proteinase activity, temperature, cell density, osmotic stress, medium composition, pH, and reiterati...

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Bibliographic Details
Published in:	Protein expression and purification 2003-04, Vol.28 (2), p.321-330
Main Authors:	Shi, Xianzong, Karkut, Tammy, Chamankhah, Mahmood, Alting-Mees, Michelle, Hemmingsen, Sean M, Hegedus, Dwayne
Format:	Article
Language:	eng
Subjects:	Animals Blotting, Western Cloning, Molecular Colony Count, Microbial Culture Media - pharmacology Electrophoresis, Polyacrylamide Gel Endopeptidases - genetics Endopeptidases - metabolism Hydrogen-Ion Concentration Immunoglobulin Fragments - genetics Immunoglobulin Fragments - metabolism Methanol - pharmacology Moths - chemistry Osmotic Pressure Pichia - cytology Pichia - genetics Pichia pastoris Recombinant Proteins - drug effects Recombinant Proteins - genetics Recombinant Proteins - metabolism scFv Serpin Serpins - immunology Single-chain antibody Temperature Time Factors
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Summary:	A Pichia pastoris system was used to express a single-chain antibody (scFv) targeted against Mamestra configurata (bertha armyworm) serpins. To improve scFv production we examined parameters such as proteinase activity, temperature, cell density, osmotic stress, medium composition, pH, and reiterative induction. P. pastoris was found to express several proteases; however, adjustment of medium pH to limit their activity did not correlate with increased scFv recovery. Induction medium pH values of 6.5–8.0 were most conducive to scFv production, despite significant differences in cell growth rates. Increasing inoculum density limited growth potential but gave rise to higher levels of scFv production. Three factors, medium composition, pre-induction osmotic stress, and temperature, had the greatest effects on protein production. Supplementation of the induction medium with arganine, casamino acids, or EDTA increased scFv production several fold, as did cultivation under osmotic stress conditions during pre-induction biomass accumulation. Incubation at 15 versus 30 °C extended the period whereby cells were capable of producing scFv from 1 to 7 days. Under optimal conditions, yeast cultures yielded 25 mg/L of functional scFv and could be subject to five reiterative inductions.
ISSN:	1046-5928 1096-0279