Human Fl Gene Expression In Escherichia Coli

Hematopoietic growth factors (HGFs) are an important part of cytokines, which show an important role in the mechanism of blood cells building and regulation. HGFs mainly consist of Erythropoietin (EPO), Granulocyte Colony-Stimulating Factor (G-CSF), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Macrophage Colony-Stimulating Factor (M-CSF), Interlukin-3 (IL-3), Stem Cell Factor (SCF), Thrombopoietin (TPO), flt3(fins-like tyrosine kinase3) ligand and so on. FL (flt3 ligand) was identified in 1994 to be a type I transmembrane protein that stimulates the proliferation and differentiation of hematopoietic stem cells and primitive uncommitted progenitor cells . As SCF, FL has low biological activity alone, but it shows strong synergistic roles with other cytokines. One major difference between SCF and FL appears to be their effect on mast cells. SCF stimulates both the proliferation and activation of mast cells, while the flt3 ligand has no effect on mast cells . In addition, the human FL(hFL) also stimulates the proliferation of dendritic cells The hFL gene localizes on the proximal portion of chromosome 19q13.3 . hFL contains 235 amino acids with a signal peptide of first 26 amino acids, 156 amino acids of extracellular domain, 23 amino acids of transmembrane domain, and 30 amino acids of cytoplasmic tail. hFL contains 6 cysteins, which can form three intramolecuar disulfide bonds, and N-linked sugars in two sites.It has been suggested recently that FL, administered either alone or in combination with other cytokines, can inhibit the growth and metastasis of malignancies of the liver, lung and breast. Multiple isoforms of hFL have been identified. The predominant isoform of hFL is the trans-membrane protein that exerts its biological activity at the cell surface. Trans-membrane FL protein can be cleaved proteolytically to produce a soluble form of the protein that is also biologically active. In order to obtain a high yield of rhFL, the out-membrane domain of hFL gene has been synthesized by PCR and expressed in E.coli.There are four parts in the dissertation:1. Secrete expression of hFL.2. Fusion expression of hFL.3. Non-secrete, non-fusion expression of hFL.4. Characterization of rhFL and its effect on dendritic cells(DCs)...