Study of the Adsorption Behavior of Recombinant Human Growth Hormone onto Peptide-Coated Hydrophobic Surface

Binding of a protein to a molecule involves specific interaction domains that require substrate specificity. Isolating such interaction domains and incorporating them into large polypeptide chains can provide protein-binding epitopes on the surface. These exposed epitopes can recognize their partners on the protein surface and might bind with significant strength as evidenced by a low dissociation constant. This concept allows the opportunity to design relatively small molecules to mimic the macromolecules by exhibiting the same affinity for these binding sites. Thus, developing a peptide-coated polymer surface may offer a viable system for controlled delivery of protein drugs.;The current research explores the adsorption of recombinant human growth hormone (r-hGH) to a peptide-coated colloidal system. The hypothesis is that adsorption of r-hGH to hydrophobic surfaces that have been coated with ligands that demonstrate specific binding to r-hGH will allow high loading of r-hGH without significant denaturation of the bound r-hGH. A novel rhGH-binding peptide named growth hormone binding peptide (GHBpep) allowed binding characteristics similar to that of the soluble form of growth hormone receptor known as growth hormone binding protein (GHBP). This small polypeptide encompasses most of the residues in the hot spot of GHBP that provides most of the binding energy with human growth hormone (HGH).;The project involves preparation and characterization of peptide-modified PLGA nanoparticles by covalently attaching GHBpep to the surface of these nanoparticles using various types of crosslinkers, monovalent and multivalent crosslinkers. Dynamic Light Scattering (DLS) and Laser Doppler Electrophoresis (LDE) techniques were used to characterize the size and charge of nanoparticles, respectively. The extent of surface coverage by the immobilized peptide was estimated using fluorescence spectroscopy. After characterization of the conjugated GHBpep, the interaction of r-hGH with the conjugates was studied under various microenvironmental conditions using traditional methods, such as equilibrium microdialysis and ultrafiltration. The adsorption process was found to be affected by changes in the ionic strength and the pH of the medium. A high degree of association was found at a low concentration of ions, especially at a pH of 5.3 (at the isoelectric point) and at pH of 7.2 when multivalent linkers were utilized.