Ligand Binding Domains of human Vascular Endothelial Growth Factor Receptor

MA Li^*, WANG Xiao-Ning
( Institute of Molecular Immunology, The First Military Medical University, Guangzhou 510515, China )
ZHANG Zhi-Qing, ZHOU Xiao-Ming, ZENG Ge-Fei, CHEN Ai-Jun
( National Laboratory of Molecular Virology and Genetic Engineering, Beijing 100052, China )

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 hVEGF₁₆₅ 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 hVEGF₁₆₅. 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 VEGF₁₆₅ binding and in inhibition of HUVEC proliferation stimulated by hVEGF₁₆₅.
Key words    Flt-1； VEGF； yeast two-hybrid； Pichiapastoris； gene expression

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