Expression and characterization of functional human vascular endothelial growth factor (VEGF165) in Kluyveromyces lactis

Abstract

Among the vascular endothelial growth factor (VEGF) family variants, the 165-amino acid isoform (VEGF165) is the best characterized and most potent endothelial cell mitogenic factor. It is known that VEGF165 mediates angiogenesis and has the potential for the therapeutic applications. In this study, the expression system of Kluyveromyces lactis that produces the recombinant human VEGF165 has been evaluated. The gene encoding human VEGF165 was successfully cloned in the pKLAC2 expression vector containing a strong LAC4 promoter, after which a pKLAC2-VEGF165 plasmid was constructed. After the transformation, the recombinant human vascular endothelial growth factor 165 (rhVEGF165) was expressed in K. lactis GG799 cells (~ 5.7 mg/L) confirmed by SDS-PAGE and Western blotting and downstream purification processed comprising ammonium sulphate precipitation and affinity chromatography. The biological activity of the purified rhVEGF165 was confirmed by the proliferation of the human umbilical vein-derived endothelial cells (HUVEC) in a dose- and time-dependent manner. The K. lactis-derived rhVEGF165 exhibited a higher proliferative activity compared with a commercially available rhVEGF165 with a half-maximal effective concentration of 3.0. Cell migration analysis was conducted to evaluate the in vitro wound healing effect of the produced rhVEGF165 via a scratch assay. These findings indicate that K. lactis could be a suitable host for secreting bioactive human VEGF165 for therapeutic use.