重组人β-淀粉样蛋白1-42大规模发酵及纯化工艺

Large scale fermentation and purification technology of recombinant human β-amyloid peptide 1-42

目的：在毕赤酵母（Pichia pastoris）中高效表达人β-淀粉样蛋白1-42（hAβ1-42），用80L的发酵罐大量制备hAβ1-42。方法：在成功构建表达载体pPICZα—hAβ1-42并电转化至毕赤酵母X-33的基础上，筛选高表达hAβ1-42工程菌，对发酵液的pH值、溶解氧、甲醇流加速度以及诱导表达的时间等发酵条件进行系统优化，通过阳离子交换层析和反向疏水层析对其进行纯化，实现其大规模制备。结果：筛选出的高表达工程菌采用80L发酵罐甲醇诱导补料批式发酵，在pH4．0、溶解氧浓度20％～30％、罐内压力为10psi、甲醇诱导48h时产量最高可达150mg·L^-1；纯化的hAβ1-42经SDS-PAGE分析显示单一区带，相对分子质量约为4200。结论：构建、筛选出高效表达hAβ1-42的毕赤酵母工程菌，并建立稳定的发酵和纯化制备工艺。

Objective To study the efficient expression of the human recombinant β-amyloid protein 1-42 （hAβ1-42）, and produce hAβ1-42 by optimized fermentation parameters on large scale using Pichia pastoris as host system. Methods On the base of construction of the recombinant expression vector pPICZα-Aβ1-42, and the obtained recombinant vector was transformed into the Pichia pastoris, hAβ1-42 was expressed in 80 L fermentor with optimized parameters and the broth was harvested and purified with SP Sepharose and Source TM 30 RPC. Results hAβ1-42 in the broth could reach maximal yield of 150 mg · L^-1 after induced by methanol for 48 h. The expressed product was purified from the fermentating culture. Analytic results revealed that the relative molecular mass of obtained product was about 4, 200. Conclusion hAβ1-42 can be expressed in Pichia pastoris on large scale. A stable fermentation and purification technology is successfully set up.