Selection and characterization of an HIV-1 gp120-binding affibody ligand
2006 (English)In: Biotechnology and applied biochemistry, ISSN 0885-4513, E-ISSN 1470-8744, Vol. 45, 93-105 p.Article in journal (Refereed) Published
To evaluate the possibility of generating novel proteins binding to highly glycosylated viral proteins, affibody ligands were selected by bactericiphage display technology to the HIV-1 envelope glycoprotein gp120 (glycoprotein 120), from a combinatorial protein library based on the 58-amino-acid-residue staphylococcal Protein A domain. The predominant variant from the bacteriophage selection was produced in Escherichia coli and characterized by biosenscir analyses. Both univalent and bivalent affibody molecules were shown to bind selectively to the gp120 target molecule in a biosensor analysis. The dissociation equilibrium constants (K.) were determined to be approx. 100 nM for the univalent affibody and 10 nM for the bivalent affibody, confirming the stronger gp120 binding of the bivalent affibody ligand. The affibody constructs were further introduced into the AdS (adenovirus type 5) fibre gene, and the recombinant fibres were shown to bind selectively to gp 120 in a biosensor analysis and to gp160 transiently expressed in African-green-monkey (Cercopithecus aethiops) kidney cells. Neither the affibody ligand nor the AdS fibres showed any virus neutralization activity, suggesting that the affibody bound to a non-neutralizing site on gp 120. To investigate the binding site for the affibody ligand on gp120, CD4 (cluster of differentiation 4) and a panel of mAbs (monoclonal antibodies) known to bind to gp 120 were allowed to compete with the affibody ligand in a biosensor study. Two mAbs, 670-30D and 697-30D, were found to compete with gp120 for overlapping binding sites. Although neutralization effects were not achieved in this initial investigation, the successful selection of a gp120-binding affibody ligand indicates that future affibody-based strategies might evolve to complement antibody-based efforts for HIV-I therapy. Strategies for directed selection of affibody ligands binding to neutralizing epitopes and the potential of using adenovirus for gene-therapy-mediated efforts are discussed.
Place, publisher, year, edition, pages
2006. Vol. 45, 93-105 p.
adenovirus fibre, affibody, bacteriophage display, glycoprotein 120 (gp120), HIV-1, protein engineering, human-immunodeficiency-virus, human monoclonal-antibody, bacterial receptor domain, staphylococcal protein-a, taq dna-polymerase, n-linked glycans, single-chain fv, combinatorial libraries, escherichia-coli, binding-proteins
IdentifiersURN: urn:nbn:se:kth:diva-15970DOI: 10.1042/ba20060016ISI: 000240241500007ScopusID: 2-s2.0-33748525178OAI: oai:DiVA.org:kth-15970DiVA: diva2:334012
QC 201005252010-08-052010-08-05Bibliographically approved