BACTERIAL EXPRESSION OF LYMPHOMA RNRNASE IMMUNOTOXIN

本研究的目的是利用细菌高水平表达系统表达蛙源重组核糖核酸酶（rpRNase），无论是单独表达还是与B细胞淋巴瘤特异性人源化单链抗体（rpRNase-hLL 2scFv）融合表达。 由于rpRNase在哺乳动物细胞中的胞质毒性以及其保持酶活性所必需的N-末端焦谷氨酰残基的需要，将rpRNase表达为重组蛋白的尝试没有成功。 建议在E.大肠杆菌中的包涵体含有一个N-末端甲基。 开发了溶解和复性重组蛋白的策略，所述重组蛋白经后修饰以恢复生物活性结构。 将研究重组蛋白在无细胞系统或组织培养中的RNA酶活性、抑制或蛋白质合成方面的生物活性。 对于免疫毒素融合蛋白，掺入可裂解肽接头以促进内化后rpRNase释放。 生物活性重组rpRNase将与hLL 2化学缀合，并且其体外和体内（在荷瘤小鼠中）的抗肿瘤效果将与rpRNase-hLL 2scFv的抗肿瘤效果进行比较。拟定商业应用：开发一种经济有效的策略来生产单独的或作为与B细胞淋巴瘤特异性单链抗体的融合蛋白的生物活性重组rpRNase。该重组rpRNase可用作致敏肿瘤的抗肿瘤剂或用作与不同肿瘤特异性的抗体缀合的免疫毒素。 该融合蛋白可用于治疗B细胞淋巴瘤。

The objective of this project is to employ a bacterial high-level expression system to express recombinant ribonuclease of frog (Rana Pipiens) origin (rpRNase), either alone, or as a fusion protein with a humanized single chain antibody specific for B-cell lymphoma (rpRNase-hLL2scFv). Attempts to express rpRNase as recombinant protein were not successful because of its cytosolic toxicity in mammalian cells and its necessary requirement for an N-terminal pyroglutamyl residue to remain enzymatically active. The proposal suggests the production of rpRNase or rpRNase-hLL2scFv in E. coli as inclusion bodies containing an N-terminal Methionine. Strategies are developed to solubilize and renature the recombinant proteins which are post-translationally modified to resume a bioactive structure. The biological activities of the recombinant proteins in terms of RNase activity, inhibition or protein synthesis in cell-free system or in tissue culture will be studied. For the immunotoxin fusion protein, a cleavable peptide linker is incorporated to facilitate rpRNase release upon internalization. The bioactive recombinant rpRNase will be chemically conjugated to hLL2, and its anti-tumor effect, both in vitro and in vivo (in tumor-bearing mice), will be compared with that of rpRNase-hLL2scFv. PROPOSED COMMERCIAL APPLICATION: The development of an economic and efficient strategy to produce a bioactive recombinant rpRNase either alone or as fusion protein to a B-cell lymphoma specific single chain antibody. The recombinant rpRNase can be used as an anti-tumor agent for sensitize tumors or as immunotoxin conjugated to antibodies of different tumor specificities. The fusion protein can be used for the treatment of B-cell lymphoma.