Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt.
Department of Biotechnology, Institute of Graduate Studies and Research, Alexandria University, Alexandria, Egypt.
Protein Expr Purif. 2021 May;181:105820. doi: 10.1016/j.pep.2021.105820. Epub 2021 Jan 11.
In previous studies Pseudomonas aeruginosal-ASNase complete coding sequence gene, 984 bp (GenBank accession number KU161101.2) was isolated by PCR, cloned into pET28a(+) vector, expressed in E. coli DE3(BL21) pLysS, purified to apparent homogeneity and biochemically characterized. In the present work we highlight large scale production, affinity purification of the recombinant enzyme, effect of osmolytes on the stability of the l-ASNase and cytotoxicity on different cancer cell lines. Successful overexpression was achieved in E. coli as a 6-His-Tag fusion protein after 18 h of induction with lactose at a concentration of 2 g/L in fermentation medium and at 37 °C. The recombinant enzyme was purified to homogeneity using Ni chelated Fast Flow Sepharose resin with 19758.8 specific activity and 10.28 purification fold. With respect to the effect of osmolytes on the stability of the purified enzyme, the majority of the tested osmolytes namely 5% maltose, 5% mannitol, 30% glycerol and 5% BSA were found to increase the stability of the recombinant l-ASNase as compared to the free enzyme. Triple negative breast cancer cell line, MDA-MB-231 treated with recombinant l-ASNase showed significant morphological changes and the IC of the purified enzyme was found to be 3.1 IU. Human leukemia cell line, THP-1 treated with l-ASNase showed apoptotic bodies and morphological changes with IC of the purified enzyme 1.75 IU. Moreover, the purified recombinant l-ASNase was found to induced cytotoxic effects on colorectal adenocarcinoma cell line, Caco-2 with IC of 68.28 IU. Results of apoptosis assay on THP-1 cells revealed that the purified l-ASNase induced early and late apoptosis at 14.16% and 7.56 respectively as compared to the control untreated cells.
在之前的研究中，通过 PCR 从铜绿假单胞菌中分离出 ASNase 完整编码序列基因，长 984bp（GenBank 登录号 KU161101.2），克隆到 pET28a(+)载体中，在大肠杆菌 DE3(BL21) pLysS 中表达，纯化至明显均一性并进行了生化特性分析。在本工作中，我们强调了大规模生产、重组酶的亲和纯化、渗透剂对 l-ASNase 稳定性的影响以及对不同癌细胞系的细胞毒性。在发酵培养基中乳糖浓度为 2g/L、37°C 下，成功实现了乳糖诱导 18 小时后，大肠杆菌的过表达，得到 6-His-Tag 融合蛋白。通过 Ni 螯合 Fast Flow Sepharose 树脂对重组酶进行了纯化，获得了 19758.8U/mg 的比活性和 10.28 的纯化倍数。就渗透剂对纯化酶稳定性的影响而言，与游离酶相比，大多数测试的渗透剂，即 5%麦芽糖、5%甘露醇、30%甘油和 5%BSA，均能提高重组 l-ASNase 的稳定性。用重组 l-ASNase 处理三阴性乳腺癌细胞系 MDA-MB-231 后，细胞形态发生明显变化，纯化酶的 IC 为 3.1IU。用 l-ASNase 处理人白血病细胞系 THP-1 后，细胞出现凋亡小体和形态变化，纯化酶的 IC 为 1.75IU。此外，纯化的重组 l-ASNase 对结直肠腺癌细胞系 Caco-2 表现出细胞毒性作用，IC 为 68.28IU。THP-1 细胞的凋亡检测结果表明，与未处理的对照细胞相比，纯化的 l-ASNase 诱导早晚期凋亡分别为 14.16%和 7.56%。
