"Peptabody": a new type of high avidity binding protein.
A new type of high avidity binding molecule, termed "peptabody" was created by harnessing the effect of multivalent interaction. A short peptide ligand was fused via a semi-rigid hinge region with the coiled-coil assembly domain of the cartilage oligomeric matrix protein, resulting in a pentameric multivalent binding molecule. In the first peptabody (Pab-S) described here, a peptide (S) specific for the mouse B-cell lymphoma BCL1 surface Ig idiotype, was selected from a phage display library. A fusion gene was constructed encoding peptide S, followed by the 24 aa hinge region from camel IgG and a modified 55 aa cartilage oligomeric matrix protein pentamerization domain. The Pab-S fusion protein was expressed in Escherichia coli in a soluble form at high levels and purified in a single step by metal-affinity chromatography. Pab-S specifically bound the BCL1 surface idiotype with an avidity of about 1 nM, which corresponds to a 2 x 10(5)-fold increase compared with the affinity of the synthetic peptide S itself. Biochemical characterization showed that Pab-S is a stable homopentamer of about 85 kDa, with interchain disulfide bonds. Pab-S can be dissociated under denaturing and reducing conditions and reassociated as a pentamer with full-binding activity. This intrinsic feature provides an easy way to combine Pab molecules with two different peptide specificities, thus producing heteropentamers with bispecific and/or chelating properties.
Terskikh
A V
AV
Institute of Biochemistry, University of Lausanne, Epalinges, Switzerland.
Le Doussal
J M
JM
Crameri
R
R
Fisch
I
I
Mach
J P
JP
Kajava
A V
AV
D000818:Animals; D001667:Binding, Competitive; D015153:Blotting, Western; D002850:Chromatography, Gel; D003198:Computer Simulation; D004926:Escherichia coli / Q000235:genetics; D016326:Extracellular Matrix Proteins; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D016393:Lymphoma, B-Cell; D064235:Matrilin Proteins; D051379:Mice; D008807:Mice, Inbred BALB C; D008958:Models, Molecular; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010957:Plasmids; D011485:Protein Binding; D011487:Protein Conformation; D011956:Receptors, Cell Surface / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D017421:Sequence Analysis; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
D016326:Extracellular Matrix Proteins; D006023:Glycoproteins; D008024:Ligands; C577540:Matn1 protein, mouse; D064235:Matrilin Proteins; D010455:Peptides; D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins