摘要
类泛素特异性蛋白酶1(Ulp1)是从融合蛋白中移除小分子泛素相关修饰物蛋白(SUMO)标签工艺中的重要工具蛋白酶,在重组多肽和蛋白生产制备中具有广泛应用。Ulp1蛋白易聚集的特性给下游分离纯化工艺带来了诸多挑战。为克服这些问题,本研究设计并重组表达制备了一种S13-Ulp1融合蛋白,以提高Ulp1酶的溶解度,减少聚集,同时降低蛋白等电点,便于采用阴离子交换色谱纯化。通过对发酵、澄清等步骤的工艺参数进行优化,建立了一条发酵表达产量高、纯化工艺简便的S13-Ulp1融合蛋白制备工艺。优化后发酵产量达到了2.34g/L,下游分离纯化总收率为64%,制得的S13-Ulp1蛋白的SDS-PAGE纯度为95%。经酶切试验验证,S13-Ulp1融合蛋白具有与Ulp1相同的特异性,可高效地移除SUMO标签,获得目的多肽。
Ubiquitin-like protein-specific protease 1(Ulp1) is a crucial tool for protease in removing small ubiquitin-related modifier protein(SUMO) tags from the fusion protein. It has been widely used in the production of recombinant peptides and proteins. The easy aggregation of Ulp1 protein brings many challenges to the downstream separation and purification process. An S13-Ulp1 fusion protein was designed and recombinantly expressed to improve the solubility of Ulp1, reduce its aggregation, and decrease its isoelectric point for the convenient purification by anion exchange chromatography. After optimizing the parameters of fermentation and clarification, a preparation process of S13-Ulp1 fusion protein with high yield and simple purification was established. Finally, for S13-Ulp1, the fermentation yield reached 2.34 g/L, the total yield of downstream separation and purification was 64%, and the purity shown in SDS-PAGE analysis was about 95%. The enzymatic reaction assay showed that S13-Ulp1 fusion had the same specificity as Ulp1, which could efficiently remove the SUMO tag to obtain the targeted peptide.
作者
徐海悦
吴勇
李乐乐
黄宗庆
化浩举
冯军
XU Haiyue;WU Yong;LI Lele;HUANG Zongqing;HUA Haoju;FENG Jun(China Institute of Pharmaceutical Industry,Shanghai 201203;Shanghai Duomirui Bio-Technology Co.,Ltd.,Shanghai 201203)
出处
《中国医药工业杂志》
CAS
CSCD
北大核心
2022年第10期1432-1438,共7页
Chinese Journal of Pharmaceuticals
