Expression Of Recobinant Human FLT-3 Ligand Extracellular Domain In Pichia Pastoris And Its Biological Characteristics

OBJECTIVE: FLT-3 ligand (fins-like tyrosine kinase receptor-3 ligand, FL) was a recently described growth factor affecting early hematopoietic progenitor cells, that play a key role in the growth and differentiation of primitive hematopoietic cells. FL promotes the survival, proliferation, and differentiation of hematopoietic progenitors in synergy with other growth factors. Preliminary clinical trials demonstrate that FL treatment resulted in an antitumor response against human malignances and development of a variety of bioreactor systems, and results from the efficacy of ex vivo expanded hematopoietic cells for transplantation therapy. To establish a highly efficient expression system of recombinant human FL, and detect expressed product biological activity, a recombinant Pichia Pastoris was constructed. METHODS: An artificial gene for FL extracellular domain cDNA was synthesized by using favored genetic codons of Pichia Pastroris. By inserting human FL extracellular domain cDNA coding 156 amino acid residues into Pichia pastoris expression vector pPIC9K containing AOX1 promoter and the sequences of alpha secreting signal peptides, a recombinant expression plasmid pPIC9K-FL was constructed, and integrated into the alcohol oxidase region of the host genome. Transformation was done by electroporation. Human FL extracellular domain cDNA transformed to yeast host strain KM71, then His+ Mutsphenotype transformant was screened out and cultured in flasks, and rhFL was expressed under the induction of 0.5% methanol. RESULTS: SDS-PAGE showed that after being induced with 0.5% methanol foi 48 hours, the expressed product, a 20 kDa glycoprotein that was secreted into the medium, existed in supernatant in the form of soluble molecule. SDS-PAGE showed efficient expression and secretion of the rhFL. The highest yield (108 mg/L) was obtained when expression was induced with 0.5% methanol for 96 hours. Western-blot analyses showed good antigenicity and specificity of expressed product, recognized specifically by rhFL polyclonal antibody. Compared with its counterpart that expressed by E.coli, the expressed protein was glycosylated by the yeast, migrating at 20 kDa on SDS/PAGE. Deglycosylation with PNGase F resulted in a decrease in apparent molecular mass from 20 kDa to 18kDa. Biological assay proved that the expressed product could stimulate the proliferation of CD34+ hematopoietic cells. CONCLUSION: FLT-3 ligand extracellular domain was successfully expressed. The study lays a foundation for further application of the expressed product in expansion of hematopoietic stem cells and tumor immunotherapy. Last, recobinant human FL can be used to generate large numbers of antigen-presenting dendritic cells from CD34+ peripheral blood progenitor cells that might be ideally used for immunotherapeutic approaches.