Ligand Binding Domains of human Vascular Endothelial Growth Factor Receptor.

Four cDNA clones, encoding truncated Flt-1 mutants consisting of loop 2, 1-2, 2-3 and 1-3, were amplified by PCR from human placenta cDNA library and the corresponding gene fragments were named Flt-1(2) Flt-1(1-2) Flt-1(2-3) and Flt-1(1-3). In order to detect which part of the extracellular domain was involved in ligand binding, the interaction between Flt-1 mutants and hVEGF(165) was studied with yeast two-hybrid system. The data presented here suggest that both Flt-1(2-3) and Flt-1(1-3) were able to bind hVEGF(165). Two recombinant expression plasmids, pPIC9K/Flt-1(1-3) and pPIC9K/Flt-1(2-3), were constructed and transformed into Pichia pastoris strain GS115, respectively. After 4 days of methanol induction, the amount of the expressed Flt-1s reached 60% of total proteins in supernatant by SDS-PAGE. Recombinant proteins were purified with CM-Sepharose Fast Flow and Sephacryl S-100 chromatography. Biological activities analysis proved that 1-3 loop had slightly better biological activity than 2-3 loop in VEGF(165) binding and in inhibition of HUVEC proliferation stimulated by hVEGF(165).