title: Ligand Binding Domains of human Vascular Endothelial Growth Factor Receptor.
abstract: 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).
authors.affiliation: Institute of Molecular Immunology, The First Military Medical University, Guangzhou 510515, China. hmzmary@hotmail.com
authors.forename: Li
authors.initials: L
authors.lastname: Ma
authors.forename: Xiao-Ning
authors.initials: XN
authors.lastname: Wang
authors.forename: Zhi-Qing
authors.initials: ZQ
authors.lastname: Zhang
authors.forename: Xiao-Ming
authors.initials: XM
authors.lastname: Zhou
authors.forename: Ge-Fei
authors.initials: GF
authors.lastname: Zeng
authors.forename: Ai-Jun
authors.initials: AJ
authors.lastname: Chen