Improved Expression of Immunotoxin DT_(386)-GMCSF in E.coli
- VernacularTitle:提高免疫毒素DT_(386)-GMCSF在E.coli中表达量的研究
- Author:
Jian-Wei WANG
;
Jing OUYANG
;
Ting CUI
;
Jian-Guo QU
;
Tao HUNG
;
- Publication Type:Journal Article
- Keywords:
Immunotoxin Diphtheria toxin Granulocyte-macrophage colony-stimulating factor Prokaryotic expression Mutant
- From:
China Biotechnology
2006;0(06):-
- CountryChina
- Language:Chinese
-
Abstract:
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.
