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

Objective To study the biological activity of soluble recombinant peptides represent the FLT 1 1 3 and 2 3 loops of human VEGF receptor. Methods Two recombinant expression plasmids, pPIC9K/FLT 1(1 3) and pPIC9K/FLT 1(2 3), were constructed and then separately transformed into Pichia pastoris GS115. Soluble recombinant protein was purified with CM Sepharose fast flow chromatography and Sephacryl S 100 chromatography. Biological activities were analyzed with indirect ELISA, competition binding experiment, cell based binding experiment, HUVEC MTT assay and [ 3H] thymidine incorporation assay. Results After 4 days of 1% methanol induction, the expressed FLT 1(1 3) was up to 60% of total proteins in supernatant. Compared with FLT 1(1 3), the expressed FLT 1(2 3) was proved having almost the same biological activity to bind hVEGF 165 and to inhibit HUVEC proliferation stimulated by hVEGF 165 . Conclusion High level expression of soluble FLT 1(1 3) and FLT 1(2 3) with good biological activity was achieved in Pichia pastoris .