title: Beyond Antibodies: Development of a Novel Protein Scaffold Based on Human Chaperonin 10.
abstract: Human Chaperonin 10 (hCpn10) was utilised as a novel scaffold for presenting peptides of therapeutic and diagnostic significance. Molecular dynamic simulations and protein sizing analyses identified a peptide linker (P1) optimal for the formation of the quarternary hCpn10 heptamer structure. hCpn10 scaffold displaying peptides targeting Factor VIIa (CE76-P1) and CD44 (CP7) were expressed in E. coli. Functional studies of CE76-P1indicated nanomolar affinity for Factor VIIa (3 nM) similar to the E-76 peptide (6 nM), with undetectable binding to Factor X. CE76-P1was a potent inhibitor of FX activity (via inhibition of Factor VIIa) and prolonged clot formation 4 times longer than achieved by E-76 peptide as determined by prothrombin time (PT) assays. This improvement in clotting function by CE76-P1, highlights the advantages of a heptamer-based scaffold for improving avidity by multiple peptide presentation. In another example of hCPn10 utility as a scaffold, CP7 bound to native CD44 overexpressed on cancer cells and bound rCD44 with high affinity (KD9.6 nM). The ability to present various peptides through substitution of the hCpn10 mobile loop demonstrates its utility as a novel protein scaffold.
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland (UQ), Brisbane, QLD 4072, Australia.
authors.forename: Abdulkarim M
authors.initials: AM
authors.lastname: Alsultan
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland (UQ), Brisbane, QLD 4072, Australia.
authors.forename: David Y
authors.initials: DY
authors.lastname: Chin
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland (UQ), Brisbane, QLD 4072, Australia.|Centre for Advanced Imaging, University of Queensland (UQ), Brisbane, QLD 4072, Australia.|Australian Research Council Training Centre for Biopharmaceutical Innovation, University of Queensland (UQ), Brisbane, QLD 4072, Australia.
authors.forename: Christopher B
authors.initials: CB
authors.lastname: Howard
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland (UQ), Brisbane, QLD 4072, Australia.
authors.forename: Christopher J
authors.initials: CJ
authors.lastname: de Bakker
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland (UQ), Brisbane, QLD 4072, Australia.|Australian Research Council Training Centre for Biopharmaceutical Innovation, University of Queensland (UQ), Brisbane, QLD 4072, Australia.
authors.forename: Martina L
authors.initials: ML
authors.lastname: Jones
authors.affiliation: Australian Institute for Bioengineering and Nanotechnology (AIBN), University of Queensland (UQ), Brisbane, QLD 4072, Australia.|School of Chemical Engineering, University of Queensland (UQ), Brisbane, QLD 4072, Australia.|Australian Research Council Training Centre for Biopharmaceutical Innovation, University of Queensland (UQ), Brisbane, QLD 4072, Australia.
authors.forename: Stephen M
authors.initials: SM
authors.lastname: Mahler
mesh_terms: D001665:Binding Sites; D001777:Blood Coagulation / Q000187:drug effects; D018835:Chaperonin 10 / Q000737:chemistry*; D015942:Factor VIIa / Q000494:pharmacology*; D006801:Humans; D018960:Hyaluronan Receptors / Q000378:metabolism*; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D019151:Peptide Library; D010455:Peptides / Q000737:chemistry* / Q000494:pharmacology*; D011257:Pregnancy Proteins / Q000737:chemistry*; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D013491:Suppressor Factors, Immunologic / Q000737:chemistry*
chemical_list: C497483:CD44 protein, human; D018835:Chaperonin 10; D018960:Hyaluronan Receptors; D019151:Peptide Library; D010455:Peptides; D011257:Pregnancy Proteins; D013491:Suppressor Factors, Immunologic; C025124:early pregnancy factor; D015942:Factor VIIa