Biologically active vascular endothelial growth factor as a bacterial recombinant glutathione S-transferase fusion protein.
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.
Morera
Yanelys
Y
Recombinant Antibodies Laboratory, Cancer Research Department, Pharmaceuticals Division, Center for Genetic Engineering and Biotechnology, P.O. Box 6162, Havana 10600, Cuba.
Lamdan
Humberto
H
Bequet
Mónica
M
Ayala
Marta
M
Rojas
Gertrudis
G
Muñoz
Yasmiana
Y
Gavilondo
Jorge V
JV
D000818:Animals; D015153:Blotting, Western; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics*; D005260:Female; D005982:Glutathione Transferase / Q000235:genetics*; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000302:isolation & purification; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D042461:Vascular Endothelial Growth Factor A; D005982:Glutathione Transferase