重组毕氏酵母中降解hIFN-βHSA蛋白酶的鉴定及其活性控制

Definition and characterization of protease of fusion human IFNβ-HSA expressed in pichia pastoris

研究了重组毕赤酵母KM71/pPIC9K-IFNβ-HSA表达融合蛋白hIFNβ-HSA过程中产物降解的问题。十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和Western-Blotting分析结果表明,发酵液中除了9.0×104的目的融合蛋白hIFNβ-HSA外,同时还出现了6.5×104的降解带,进一步结合HPLC分析证实该降解发生在融合蛋白hIFNβ-HSA的HSA区域。在此基础上,采用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)活性电泳的方法,确定在融合蛋白hIFNβ-HSA表达的过程中,引起融合蛋白hIFNβ-HSA降解的蛋白酶为蛋白酶B(Proteinase B,PrB)。最后,确定了可以抑制融合蛋白hIFNβ-HSA降解的最佳的蛋白酶抑制剂EDTA,通过在诱导表达阶段添加不同浓度的蛋白酶抑制剂EDTA,使融合蛋白的表达量达到22.18 mg/mL,与空白对照组相比增加了61%。

The problem for the expression of recombinant human IFNβ-HSA fusion protein in Pichia pastoris KM71/IH was the degradation of the target proteins during fermentation. The analysis by SDS-PAGE and Western-blot showed that 9.0 × 10^4 fusion protein and 6.5 × 10^4 degradation band appeared in the fermentation broth. The degradation appeared in the HSA region of recombinant human IFNβ-HSA fusion protein was confirmed by HPLC analysis. The protease caused the degradation of fusion protein IFNβ- HSA was proteinase B and EDTA was the best protease inhibitor. By adding of EDTA into the fermentation medium, the quantity of expression of the fusion protein is up to 22. 18 mg/mL, and creased by 61% compared with the blank reference.