Cloning, expression and purification of the cytokine FLT3-ligand for protein interaction studies
thesisposted on 2021-05-23, 16:50 authored by Lauren Alicia Agro
The FLT3-FLT3L receptor complex is responsible for the proliferation and differentiation of hematopoietic progenitor and stem cells, and is involved in several hematological malignancies. A truncated, soluble form of FLT3L was amplified from the full-length sequence by PCR and cloned into a genomic integration and expression vector for Kluyveromyces lactis. Expression and secretion of the 161 amino acid FLT3L was analyzed by SDS-PAGE and Western blot and showed smeared bands around 50kDa consistent with a high level of varying glycosylation as expected. A yield of 100ug/mL of culture was obtained. Purification was performed by Ni-affinity chromatography and the protein was confirmed to be in the elution fraction via mass spectrometry analysis. Purified FLT3L was able to activate RAW264.7 cells after 24hr incubation period causing large vacuoles and a dendritic appearance. FLT3L will be utilized for LARC protein interaction studies to search for interactors that might serve as therapeutic agents in human body fluids and cell culture models of myeloid leukemia.