Expression, purification, and characterization of a diabody against the most important angiogenesis cell receptor: Vascular endothelial growth factor receptor 2

Abstract

Antibodies and their derivative fragments have long been used as tools in a variety of applications, in fundamental research work, biotechnology, diagnosis, and therapy. Camels produce single heavy-chain antibodies (VHH) in addition to usual antibodies. These minimal-sized binders are very robust and bind the antigen with high affinity in a monomeric state. Vascular endothelial growth factor recepror-2 (VEGFR2) is an important tumor-associated receptor that blockade of its signaling can lead to the inhibition of neovascularization and tumor metastasis. Here, we describe the construction, expression, and purification VEGFR2-specific Diabody. Two variable fragments of a same camel anti-VEGFR2 antibody were linked together by the upper hinge segment of antibody to make a diabody. We showed the ability of diabody to recognition of VEGFR2 on the cell surface by FACS. Diabodies can be produced in the low-cost prokaryotic expression system, so they are suitable molecules for diagnostic and therapeutic issues.

Keywords: Diabody; Nanobody; vascular endothelial growth factor recepror-2.

Figure 1

Construction of pHEN6C-3VGR19-Dia bacterial expression plasmid. The pHEN6C-3VGR19 is a plasmid with one copy of VEGFR2- specific Nanobody cloned between the BstEII and PstI sites. A PCR reaction was performed with the BiNb-Sense and BiNb-Llama-IgG2C-hinge primers and subcloned in the pHEN6C-3VGR19 vector, restricted with PstI and NcoI and ligated to each other. The final construct was named pHEN6c-3VGR19-Dia

Figure 2

Size-exclusion chromatography profile of the IMAC-purified VEGFR2-Nanobody and VEGFR2-Diabody (as indicated), loaded onto a superdex S75 column in PBS buffer

Figure 3

SDS-PAGE (a) and western-blotting (b) analysis of purified VEGFR2-Diabody (Lane 1) and 3VGR19 Nanobody (Lane 2). The MW of the marker lanes was from top to bottom 160,110, 90, 70, 55, 40, 35, 25, 15 and 10 kDa

Figure 4

Flowcytometry analysis of VEGFR2-specific and control Diabody. a, b show the staining of 293KDR (a) and HEK293 (b) with VEGFR2- specific Diabody. c, d show the staining of 293KDR (c) and HEK293 (d) with control diabody (Anti-scorpion diabody)