Shi Xianzong, Karkut Tammy, Chamankhah Mahmood, Alting-Mees Michelle, Hemmingsen Sean M, Hegedus Dwayne
Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, Canada S7N 0X2.
Protein Expr Purif. 2003 Apr;28(2):321-30. doi: 10.1016/s1046-5928(02)00706-4.
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.
使用毕赤酵母系统表达一种针对甜菜夜蛾丝氨酸蛋白酶抑制剂的单链抗体（scFv）。为了提高scFv的产量，我们研究了蛋白酶活性、温度、细胞密度、渗透胁迫、培养基成分、pH值和反复诱导等参数。发现毕赤酵母表达几种蛋白酶；然而，调整培养基pH值以限制其活性与提高scFv回收率并无关联。尽管细胞生长速率存在显著差异，但诱导培养基pH值为6.5 - 8.0最有利于scFv的产生。增加接种密度限制了生长潜力，但能产生更高水平的scFv。培养基成分、诱导前的渗透胁迫和温度这三个因素对蛋白质产量影响最大。在诱导培养基中添加精氨酸、酪蛋白氨基酸或乙二胺四乙酸（EDTA）可使scFv产量提高数倍，诱导前生物量积累期间在渗透胁迫条件下培养也有同样效果。在15℃而非30℃孵育可将细胞能够产生scFv的时间从1天延长至7天。在最佳条件下，酵母培养物可产生25 mg/L的功能性scFv，并且可以进行五次反复诱导。
