TAT-Cytoglobin融合蛋白的原核表达、纯化及生物活性分析

Expression, purification, and characterization of fusion protein TAT-Cytoglobin

摘要:

旨在利用细胞穿膜肽TAT的穿膜作用和细胞珠蛋白 (Cytoglobin, Cygb) 的抗衰老、抗纤维化的功能，将二者通过基因工程的手段融合在一起，以期获得能够穿透细胞屏障的Cygb。通过两次重叠PCR技术获得了TAT-Cygb DNA，将其插入原核表达载体pET22b质粒中，转化至大肠杆菌BL-21, 筛选出可表达TAT-Cygb融合蛋白的大肠杆菌工程菌株。经乳糖诱导表达TAT-Cygb，CM阳离子交换层析 (CM Sepharose Fast Flow Protocol) 获得纯度高达95%的TAT-Cygb融合蛋白，分子量约23 kDa。生物活性实验显示，TAT-Cygb过氧化物酶比活力达到(422.30±0.36) U/mg。TAT-Cygb预处理的Hacat细胞可免受H2O2氧化应激的损伤 (RGR=100%)，同时TAT-Cygb可治疗已被H2O2氧化损伤的细胞 (RGR=98%)，与Cygb处理组相比具有显著差异 (RGR=79%)。该研究首次成功利用大肠杆菌表达系统表达了可穿透细胞膜的、有生物活性的TAT-Cygb融合蛋白，为继续开展Cygb在抗衰老、抗纤维化和抗癌领域的研究奠定了基础。

Abstract:

The aim of this study was to obtain a cell-penetrating cytoglobin (Cygb), which combines the transmenbrane function of cell-penetrating peptides TAT with the anti-aging and anti-fibrotic role of cytoglobin. The Cygb gene was complexed with TAT gene by overlapping PCR, inserted into the vector pET22b to construct the recombinant expression plasmid (pET22b-TAT-Cygb) and then transformed into Escherichia coli BL21 (DE3). The fusion protein TAT-Cygb, whose expression was induced by lactose, was purified by CM Sepharose Fast Flow Protocol and verified by Western blotting. The final TAT-Cygb had a molecular weight of 23 kDa with 95% purity, as shown by SDS-PAGE. As demonstrated by bioactivity experiments, TAT-Cygb exhibited a high specific peroxidase activity up to (422.30±0.36) U/mg. Both TAT-Cygb and Cygb pretreatment group could protect Hacat cells against oxidation of H2O2, but only TAT-Cygb treatment group could remedy cells injuried by H2O2 (RGR=98%), which was significantly different from Cygb treatment group (RGR=79%). We successfully obtained the bioactive and cell-penetrating fusion protein TAT-Cygb that has the potential application in anti-aging, anti-fibrotic and anti-cancer.