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目的建立长链人胰岛素样生长因子-1(Long chain Arg3 human insulin-like growth factor-1,LR3IGF-1)的分离纯化方法。方法将LR3IGF-1毕赤酵母工程菌接种至30 L发酵罐进行高密度发酵,发酵液经离心、超滤浓缩后,采用SP Sepharose FF阳离子交换层析、Phenyl Sepharose FF疏水层析、S-100凝胶过滤层析和DEAE Sepharose FF层析对发酵上清液中的表达产物进行分离纯化,测定蛋白浓度,计算蛋白收率,并分析纯化样品的浓度。采用MTT法检测各步纯化样品促NIH-3T3细胞增殖的活性。结果纯化LR3IGF-1的蛋白总收率为28%,RP-HPLC纯度可达95%以上。随着纯化过程中样品纯度的增加,LR3IGF-1的活性也相应增强,各纯化步骤基本不影响LR3IGF-1的生物活性。结论初步建立了LR3IGF-1的分离纯化工艺,其简便有效,为LR3IGF-1的规模化生产奠定了基础。
Objective To establish a method for the isolation and purification of long chain human insulin-like growth factor-1 (LR3IGF-1). Methods The Pichia pastoris strain LR3IGF-1 was inoculated into a 30 L fermentor for high-density fermentation. After the fermentation broth was centrifuged and concentrated by ultrafiltration, SP Sepharose FF cation exchange chromatography, Phenyl Sepharose FF hydrophobic chromatography, S-100 Gel filtration chromatography and DEAE Sepharose FF chromatography to separate and purify the expression product in the fermentation supernatant, measure the protein concentration, calculate the protein yield, and analyze the purified sample concentration. The activity of purified NIH-3T3 cells was detected by MTT assay. Results The total protein yield of purified LR3IGF-1 was 28%. The purity of RP-HPLC was over 95%. The LR3IGF-1 activity increased with the increase of the purity of the sample during purification, and the purification steps did not affect the biological activity of LR3IGF-1. Conclusion The separation and purification of LR3IGF-1 was preliminarily established, which was simple and effective and laid the foundation for the large-scale production of LR3IGF-1.