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Biotechnology and Applied Biochemistry (2001) 34, (199–204) (Printed in Great Britain)

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Identification of the ligand-binding domain of human vascular-endothelial-growth-factor receptor Flt-1

Li Ma*, Xiaoning Wang*¹, Zhiqing Zhang†, Xiaoming Zhou†, Aijun Chen† and Lihong Yao†

*Institute of Molecular Immunology, The First Military Medical University, Guangzhou 510515, People's Republic of China, and †State Key Laboratory of Molecular Virology and Genetic Engineering, Beijing 100052, People's Republic of China

Key words: gene expression, Pichia pastoris, yeast two-hybrid.

Abbreviations used: (h)VEGF, (human) vascular endothelial growth factor; (s)Flt-1, (soluble) VEGF receptor; HUVEC, human umbilical-vein endothelial cells; CM-carboxymethyl; IgSF, immunoglobulin superfamily; X-gal, 5-bromo-4-chloroindol-3-yl b-D-galactopyranoside.

¹To whom correspondence should be addressed (e-mail xnwang@hotmail.com).

The vascular-endothelial-growth-factor (VEGF) receptor Flt-1 has been shown to be involved in vasculogenesis and angiogenesis. The receptor is characterized by seven immunoglobulin-like loops within the extracellular domain and the first three N-terminal immunoglobulin-like loops are involved in high-affinity binding of VEGF. The minimal extracellular domains of Flt-1 to achieve VEGF binding were screened using the yeast two-hybrid system. The result showed that the binding capacity of loop2-3 was close to that of loop1-3. The two truncated mutants consisting of loop2-3 and loop1-3 were expressed in the methylotrophic yeast Pichia pastoris at high levels (0.3 mg/litre). The corresponding proteins, named soluble (s)Flt-1(2-3) and sFlt-1(1-3), were purified. An in vitro biological activity assay showed that the binding capacity of sFlt-1(2-3) to human VEGF₁₆₅ and the inhibiting effect of it on human umbilical-vein endothelial cell proliferation stimulated by human VEGF₁₆₅ were close to those of sFlt-1(1-3). Animal tests showed that sFlt-1(2-3) could significantly inhibit the formation of regenerated blood vessels stimulated by hVEGF₁₆₅.

Received 8 June 2001/14 September 2001; accepted 18 September 2001

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