TRAIL和EGFR配体寡肽与力达霉素辅基蛋白融合蛋白制备过程的优化

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。

Optimization of preparation process for the fusion protein consisting of TRAIL,epidermal growth factor receptor/EGFR peptide ligand and apoprotein of lidamycin

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.