提高免疫毒素DT386-GMCSF在E.coli中表达量的研究

以人粒细胞-巨噬细胞集落刺激因子（GM-CSF）受体（GM-CSFR）为靶向的白喉毒素（DT）与GM-CSF免疫毒素DT386-GMCSF为急性髓系白血病提供了一种新的替代治疗途径，但该蛋白在E.coli中的表达量很低，难以进行工业化生产。为探索造成其低表达的关键影响因素，对DT386-GMCSF中的GM-CSF进行了C端的截短表达，发现GM-CSF中L114编码序列可明显影响融合蛋白的表达量。在此基础上，构建了一系列突变体，发现保留1-123位氨基酸且将L114L115V116突变为G114V115T116的突变体DF123GVT的表达量高于DT386-GMCSF，且对来源于高表达GM-CSF受体的HL60细胞的肿瘤单细胞具有相似的细胞毒作用。DF123GVT突变体的获得为GM-CSFR靶向的免疫毒素的开发应用打下了基础。

Improved expression of immunotoxin DT386-GMCSF in E.coli

The development of immunotoxin DT386-GMCSF, a fusion protein which bears the N-terminal 386 amino acids of diphtheria toxin and human granulocyte-macrophage colony-stimulating factor (GM-CSF) and targets the GM-CSF receptor (GM-CSFR), has provided a promising alternative therapy to the acute myeloid leukemia (AML). However, the poor expression of the protein in E.coli is still a bottleneck which limits the industrial production. To identify the critical down-regulating factors on the expression of DT386-GMCSF, a series of truncated mutants of DT386-GMCSF at the C-terminal of GM-CSF were generated and expressed in E.coli. The results showed that the encoding sequences for the L114 of the GM-CSF dramatically impact the expression of DT386-GMCSF. On this basis, a serial of mutants integrating amino acid substitutes were generated. The results revealed that the expression level of the mutant DF123GVT, which harbors the amino acids 1-123 of GM-CSF whose L114L115V116 was substituted with G114V115T116, was evidently higher than that of the DT386-GMCSF, whereas the specific cytotoxicity to blast recovered from mice injected with HL60, a cell line highly expresses the GM-CSFR, was similar. These results have provided an important basis for the future development of the immunotoxins targeting the GM-CSFR.