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

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.