CAJ | 학술논문

目的对以包涵体形式表达的 TRAIL 和 EGFR 配体寡肽与力达霉素辅基蛋白融合蛋白 Ec-LDP-TRAIL 的制备过程进行优化。 　　方法对大肠杆菌原核表达 Ec-LDP-TRAIL 目的蛋白的温度、诱导物浓度、起始菌体密度及时间等条件进行优化；在Ni2+亲和层析纯化蛋白过程中对样品预处理以及纯化缓冲液成分进行优化；对纯化后蛋白的分步透析复性过程进行一系列优化，并通过基于 ELISA 的结合活性实验分析Ec-LDP-TRAIL 与肿瘤细胞的结合能力。 　　结果经过工艺优化后，纯化后的融合蛋白 Ec-LDP-TRAIL纯度达95%以上，复性后 LB 培养基活性蛋白收率约为2.2 mg/L，与初始复性条件相比提高近2倍。复性后融合蛋白 Ec-LDP-TRAIL 显示出能够与人表皮癌 A431和人大细胞肺癌 H460细胞的结合活性。 　　结论融合蛋白 Ec-LDP-TRAIL 制备过程的优化为后续研发和生产奠定了实验基础，同时也为其他以包涵体形式表达的基于 TRAIL 的抗肿瘤蛋白药物的制备提供借鉴。
목적대이포함체형식표체적 TRAIL 화 EGFR 배체과태여력체매소보기단백융합단백 Ec-LDP-TRAIL 적제비과정진행우화。 　　방법대대장간균원핵표체 Ec-LDP-TRAIL 목적단백적온도、유도물농도、기시균체밀도급시간등조건진행우화；재Ni2+친화층석순화단백과정중대양품예처리이급순화완충액성분진행우화；대순화후단백적분보투석복성과정진행일계렬우화，병통과기우 ELISA 적결합활성실험분석Ec-LDP-TRAIL 여종류세포적결합능력。 　　결과경과공예우화후，순화후적융합단백 Ec-LDP-TRAIL순도체95%이상，복성후 LB 배양기활성단백수솔약위2.2 mg/L，여초시복성조건상비제고근2배。복성후융합단백 Ec-LDP-TRAIL 현시출능구여인표피암 A431화인대세포폐암 H460세포적결합활성。 　　결론융합단백 Ec-LDP-TRAIL 제비과정적우화위후속연발화생산전정료실험기출，동시야위기타이포함체형식표체적기우 TRAIL 적항종류단백약물적제비제공차감。
Objective To optimize the preparation process of the fusion protein Ec-LDP-TRAIL expressed in the form of inclusion body. Methods Based on the selective antitumor activity of TRAIL in tumor cells rather than normal cells, and the target of EGFR ligand peptide, we constructed a bi-specific fusion protein consisting of TRAIL, EGFR ligand peptide and lidamycin. Conditions of fermentation of engineering bacteria, preparation and dissolution of inclusion body, Ni2+affinity chromatography of the target protein and refolding through stepwise dialysis were investigated. Results After optimization, the purity of fusion protein Ec-LDP-TRAIL was more than 95% by immobilized Ni2+ affinity chromatography under denaturing conditions. Besides that, after optimization of the refolding process using orthogonal design, the yield of active fusion protein Ec-LDP-TRAIL was 2.2 mg from 1 L LB medium which was 2-fold higher than that of the initial refolding conditions. The purified fusion protein Ec-LDP-TRAIL could bind to both human epidermoid carcinoma A431 cells and human large cell lung carcinoma H460 cells. Conclusions Optimization of preparation process for Ec-LDP-TRAIL expressed as the form of inclusion body provides the experimental basis for the future development and production of the protein. Furthermore, it might serve as a relatively practical system for the preparation of other TRAIL-based proteins expressed in the form of inclusion body.