利用双启动子质粒在毕赤酵母中高效表达人FGF1  

High-level production of active human fibroblast growth factor 1 using a dual-promoter vector in Pichia pastoris

作　　者：高源[1] 史静静[1] 范代娣[1] GAO Yuan SHI Jingjing FAN Daidi(College of Chemical Engineering, Northwest University, Xi＇an 710069, China)

机构地区：[1]西北大学化工学院,陕西西安710069

出　　处：《西北大学学报（自然科学版）》2017年第5期693-698,共6页Journal of Northwest University（Natural Science Edition）

基　　金：国家自然科学基金资助项目(21576223)

摘　　要：基于p PIC9K构建了同时含有AOX1和FLD1的双启动子质粒,并成功转入毕赤酵母GS115中,建立了一个新型的全长人活性FGF1表达系统。测序结果和SDS-PAGE显示质粒构建成功,两种启动子可同时被甲醇诱导。液质联用分析表明,目的蛋白的氨基酸组成与人源性FGF1基本一致,且在生物活性测定中表现出良好的生物相容性。经过大规模发酵与凝胶过滤层析,重组FGF1蛋白的产量为197 mg/L,约为当前报道的最高表达水平的两倍,且纯度达到97%。Fibroblast growth factor 1（ FGF1）,a considerable member of structurally related polypeptides,has shown therapeutic potential in rebuilding blood vessels,wound healing,and so on. In this paper,a novel system was established for large-scale expression of full-length active human FGF1. A dual-promoter vector,containing both AOX1 and FLD1,was constructed based on p PIC9 K and transformed into P. pastoris GS115 successfully. Notably,the results of SDS-PAGE demonstrated that the two promoters worked simultaneously with methanol induction. LC-MS/MS analysis suggested that the amino acid composition of the protein was almost consistent with human-derived FGF1. Combined with gel filtration chromatography,197 mg pure protein was obtained from per liter culture medium,which was a double yield compared with the highest expression level reported previously.

关 键 词：双启动子质粒 FGF1 毕赤酵母 高效表达 纯化 

分 类 号：Q-3[生物学]