Expression, purification and biological activity analysis of human vascular endothelial growth factor receptor Flt-1 2-3loop within extracellular domain in Pichia.pastoris

Objective:To study the expression of human Flt 1 2 3loop cDNA in Pichia pastoris and to obtain high level expressed recombinant human Flt 1(2 3) with good biological activity Methods:Amplifying Flt 1(2 3) cDNA by PCR, after confirmed by DNA sequence analysis, the gene was inserted into the Pichia pastoris expression vector pPIC9K containing AOX1 promoter and α secreting signal peptides, the recombinant expression plasmids pPIC9K/Flt 1(2 3) was constructed and transformed into GS115 The His +Mut s phenotype transformants were screened,fermented in flasks and induced by 1% methanol Results:After 4 days of methanol induction, the expressed Flt 1(2 3) comes up to 60% of total proteins in supernatant by SDS PAGE ELISA and Western blot assay proved it having good antigenicity and high specificity The recombinant protein was further purified with CM Sepharose Fast Flow and Sephacryl S 100 chromatography, and was proved having good biological activity to bind hVEGF 165 and to inhibit hUVEC proliferation stimulated by hVEGF 165 Conclusion:High level expression of secreted Flt 1(2 3) with good biological activity were successfully achieved in Pichia pastoris expression system and can be applied to further animal and clinical test