Cloning an artificial gene encoding angiostatic anginex: From designed peptide to functional recombinant protein.
Anginex, a designed peptide 33-mer, is a potent angiogenesis inhibitor and anti-tumor agent in vivo. Anginex functions by inhibiting endothelial cell (EC) proliferation and migration leading to detachment and apoptosis of activated EC's. To better understand tumor endothelium targeting properties of anginex and enable its use in gene therapy, we constructed an artificial gene encoding the biologically exogenous peptide and produced the protein recombinantly in Pichia pastoris. Mass spectrometry shows recombinant anginex to be a dimer and circular dichroism shows the recombinant protein folds with beta-strand structure like the synthetic peptide. Moreover, like parent anginex, the recombinant protein is active at inhibiting EC growth and migration, as well as inhibiting angiogenesis in vivo in the chorioallantoic membrane of the chick embryo. This study demonstrated that it is possible to produce a functionally active protein version of a rationally designed peptide, using an artificial gene and the recombinant protein approach.
Angiogenesis Laboratory, Research Institute for Growth and Development, Department of Pathology, Maastricht University and University Hospital, Maastricht, The Netherlands.
Ricardo J M G E
RJ
Brandwijk
Irina
I
Nesmelova
Ruud P M
RP
Dings
Kevin H
KH
Mayo
Victor L J L
VL
Thijssen
Arjan W
AW
Griffioen
D020533:Angiogenesis Inhibitors / Q000032:analysis / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics / Q000494:pharmacology; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D042783:Endothelial Cells / Q000187:drug effects; D018014:Gene Transfer Techniques; D005813:Genes, Synthetic / Q000235:genetics*; D006801:Humans; D018919:Neovascularization, Physiologic / Q000187:drug effects; D010455:Peptides; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D015202:Protein Engineering / Q000379:methods*; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D011994:Recombinant Proteins / Q000032:analysis / Q000096:biosynthesis / Q000737:chemistry
D020533:Angiogenesis Inhibitors; D010455:Peptides; D011506:Proteins; D011994:Recombinant Proteins; C111993:betapep-25 protein, synthetic