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 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 degrees 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.
Shi
Xianzong
X
Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, Canada S7N 0X2.
Karkut
Tammy
T
Chamankhah
Mahmood
M
Alting-Mees
Michelle
M
Hemmingsen
Sean M
SM
Hegedus
Dwayne
D
D000818:Animals; D015153:Blotting, Western; D003001:Cloning, Molecular; D015169:Colony Count, Microbial; D003470:Culture Media / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000378:metabolism*; D000432:Methanol / Q000494:pharmacology; D009036:Moths / Q000737:chemistry; D009997:Osmotic Pressure; D010843:Pichia / Q000166:cytology / Q000235:genetics*; D011994:Recombinant Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism; D015843:Serpins / Q000276:immunology; D013696:Temperature; D013997:Time Factors
D003470:Culture Media; D007128:Immunoglobulin Fragments; D011994:Recombinant Proteins; D015843:Serpins; C055966:immunoglobulin Fv; D010450:Endopeptidases; D000432:Methanol