毕赤酵母表达肽抗生素hPAB-β的纯化

Purification of peptide antibiotic hPAB-β from recombinant Pichia pastoris

目的在成功构建肽抗生素hPAB-β重组毕赤酵母的基础上,深入探讨酵母表达hPAB-β的规模化纯化方法。方法采用高密度发酵法在3.7L发酵罐中对pPIC9K-hPAB-β/GS115优5重组菌株进行发酵,收集发酵液,依次采用W-UF-Ⅱ过滤系统超滤、反相层析、阳离子交换、分子筛层析等纯化技术对目标表达产物逐级进行纯化,目的蛋白用15%的Tricine-SDS-PAGE电泳进行跟踪分析。最终纯化产物的生物学活性用平板琼脂扩散法进行测定。结果在培养至工程菌菌体湿重约200～250g/L时,以甲醇诱导培养基诱导目的蛋白表达,48h后菌体湿重达280g/L,目标产物表达量约75mg/L。发酵液经Mr10000膜超滤,达到去除大部分酵母色素和浓缩样品的目的(体积浓缩约2/3)。再经反相层析、离子交换、分子筛层析纯化,最终目的产物的纯度达98%以上,得率达32.5%。且纯化的目的蛋白具有良好的杀灭金黄色葡萄球菌和铜绿假单胞菌的活性。结论酵母表达肽抗生素hPAB-β经膜超滤、反相层析、离子交换和分子筛层析四步纯化,实现了去除非目的蛋白、酵母色素及脱盐的目的,为规模化制备hPAB-β创造了前提。

Objective To probe the purification of human peptide antibiotics （hPAB-β） derived from high-density fermentation of Pichia pastoris. Methods The recombinant Pichia pastoris strain pPIC9K-hPAB-β/GS115 you5 was cultured in a fermentor （3.7 L） and induced with methanol to express hPAB-β. The fermentative supematant was collected and subjected to purification with membrane ultraffiltration, reverse phase chromatography, cation exchange, and molecular sieve chromatography by turns. The existence of hPAB-β during the processes was determined by Tricine-SDS-PAGE analysis. The biological activity of the purified hPAB-β was identified by agar-well diffusion assay. Resuits The expression of recombinant hPAB-β was induced by methanol when the wet weight of Pichia pastoris achieved 200 - 250 g/L. The wet weight of Pichia pastoris reached around 280 g/L after 48 h of continuous induction and the yield of hPAB-β was about 75 mg/L. The purification of hPAB-β from fermentative supematants with the Mr 10 000 membrane ultrafiltration was almost removed the saccharemycetic pigments and the volume containing the target protein was also concentrated around 2/3. The final purity of hPAB-β was above 98% after purification by reverse phase chromatography, cation exchange, and molecular sieve chromatography. The purified recombinant peptide hPAB-β retained its bacteriocidal activity against Staphylococcus aureus and Pseduomonas aeruginosa. Conclusion The purification strategy of peptide antibiotics hPAB-β from the fermentative supematants of recombinant Pichia pastoris was succesfully constructed, which will facilitate the large-scale preparation of hPAB-β from recombinant Pichia pastoris.