Biologically active vascular endothelial growth factor as a bacterial recombinant glutathione S‐transferase fusion protein

Abstract

Human VEGF121 (vascular endothelial growth factor isoform 121) was produced as a recombinant fusion protein with GST (glutathione S-transferase) in Escherichia coli. After affinity purification with glutathione, the GST-VEGF121 fusion protein preparation was used to obtain antibodies in mice against commercial hrVEGF (human recombinant VEGF) through immunization. It was also employed successfully to select specific antihuman VEGF antibody fragments of human origin employing phage-display technology. The fusion protein preparation was separated in monomeric, dimeric and oligomeric forms using size-exclusion chromatography. The dimers were recognized by a soluble VEGF receptor 2-Fc chimaera, and stimulated the growth of human umbilical-vein endothelial cells in vitro in a similar fashion to a commercial hrVEGF. The presence of GST in the fusion protein apparently did not affect the correct assembly of dimers and display of residues critical for receptor recognition. The two-step purification method reported in the present paper involves no laborious renaturalization methods, yields 10 mg/l of the mixture of different aggregation states after affinity chromatography, and 5 mg/l of the biologically active dimer after gel filtration, thus providing a source of material for the development of new anti-angiogenic therapeutic molecules.

Enhanced Mitogenic Activity of Recombinant Human Vascular Endothelial Growth Factor VEGF121 Expressed in E. coli Origami B (DE3) with Molecular Chaperones

“…Some studies do not present any yield [ 49 , 54 ] or the data are unclear [ 38 ]. In other works, the yield of purified active VEGF 121 or VEGF 165 is between 1 mg and 5 mg per L of bacterial culture [ 50 , 52 , 53 ], which is similar to the yield of mutant VEGF 121 purified in this work. The yield of recombinantly expressed α 2 -PI 1-8 -VEGF 121 by Zisch et al was 11 mg/L [ 30 ].…”

“…Studies comparing the biological activities of various VEGF preparations have not found any significant differences [ 53 , 63 ]. The biological activity was also comparable to that of the commercial standard in the case of VEGF proteins joined to uncleaved fusion partners, such as thioredoxin or GST [ 49 , 50 ].…”

Characteristics of the specific humoral response in patients with advanced solid tumors after active immunotherapy with a VEGF vaccine, at different antigen doses and using two distinct adjuvants

“…GST-fused human VEGF isoform 121 (GST-hVEGF) was produced in E. coli as previously described [14]. Human VEGF isoform 121 (rhVEGF) was produced in CHO cells [15].…”

Magnetic nanoparticles: a strategy to target the choroidal layer in the posterior segment of the eye

“…rVEGF was produced as a recombinant protein, with VEGF 165 fused to the carboxy-terminus of glutathione-S-transferase (GST)33. It has been previously shown that the presence of GST in the fusion protein does not affect the biofunctionality of VEGF33. Actually, the presence of a GST tag facilitates the purification steps and works as a “spacer” for linking the VEGF to the MNP.…”

“…We used GST alone produced from bacteria transformed with the empty pGEX6p-1 plasmid as control. For protein purification we followed the protocol published by Morera and colleagues33 with minor modifications. In particular, we transformed BL21 strain of E. coli with pGEX6P-VEGF and a single colony was inoculated in 10 ml LB medium.…”