Expression of soluble Flt‐1 gene in Pichia pastoris and the effect of the product on multiple‐myeloma cells in vitro

Abstract

VEGF (vascular endothelial growth factor), a potent angiogenic molecule specific for vascular endothelial cells, is overexpressed in most tumours including MM (multiple myeloma) and closely associated with tumour growth and prognosis. It has been shown that a soluble fragment of the VEGF receptor Flt‐1 (Fms‐like tyrosine kinase‐1) [sFlt‐1 (soluble Flt‐1)] has antiangiogenic properties by way of its antagonist activity against VEGF. VEGF and its receptors have been shown to be targets for treating tumours. In the present study, sFlt‐1 gene was expressed in Pichia pastoris and the product was applied for studying the effect on KM3 MM cells. sFlt‐1 gene was inserted into the pPICZαA vector and the expressed product was analysed by SDS/PAGE, immunoblot and ELISA. The sFlt‐1 protein was expressed by 0.5% (v/v) methanol induction and it accumulated up to 23% of total proteins in the supernatant. The product was further purified with metal‐chelating resin [Ni‐NTA (Ni 2+ ‐nitrilotriacetate)]. The functional analysis of the sFlt‐1 protein was performed with HUVEC (human umbilical‐vein endothelial cells) proliferation assay. We next showed that the sFlt‐1 protein acted directly on MM cells and inhibited the VEGF‐induced proliferation of MM cells with MTT [3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐2 H ‐tetrazolium bromide] and 3 H uptake assay. The sFlt‐1 protein blocked VEGF‐induced ERK (extracellular‐signal‐regulated kinase) phosphorylation and inhibited the MAPK (mitogen‐activated protein kinase) signalling cascades. The present study demonstrated that anti‐MM activity of the sFlt‐1 protein, coupled with its antiangiogenic effects, provides the basis for clinical trials of this agent to improve the outcome in MM.