The Study Of Variants Of Human Interleukin-3

Human interleukin-3 (hIL-3) also named Multicolony stimulating factor (Multi-CSF), a multi-lineage cytokine produced principally by activated T-cells, has been shown to stimulate the proliferation, differentiation and survival of haematopoietic stem cells and progenitor cells, which is prospective used in the clinic. However, its clinical use is limited by its systemic toxicity to human. So it is necessary to seeking for new hIL-3 mutants with higher activity. In this paper hIL-3 mutants hIL-3K116V and hIL-3K116W with substitutions at residue 116 lysine respectively by valine and tryptophan were generated by site-directed mutagenesis. The laboratory-scale expression conditions in E.coli BL21 CodonPlus (DE3)-RIL had been optimized and mutants were expressed higher than 40% of total protein in the cells mainly in the form of inclusion body. The inclusion body was denatured, refolded by dialysis and purified by affinity chromatogram with purity higher than 90%. In the study of stimulating TF-1 cells proliferation, hIL-3K116V was 4-fold more potent than hIL-3 and the activity of hIL-3K116W somewhat increased especially at a low concentration. These results show that the Lys116 residue is critical for hIL-3 activity and hIL-3K116V makes a useful focus for the development of more potent hIL-3 substitution.