| A recombinant, human elastin polypeptide (EP20-24-24) has been conjugated to polyethylene glycol (PEG). Two bifunctional PEGs were investigated for covalent attachment to the EP20-24-24 polypeptide: PEG-divinyl sulfone (PEG(VS) 2) and vinyl sulfone-PEG-N-hydroxy succinmide ester (VS-PEG-NHS). PEG conjugation to the elastin polypeptide was conducted by addition of one of the functional end-groups of polyethylene glycol with an amine group on the peptide, of which there are nine. The second functional group remains unreacted as a group for use later in attachment, crosslinking, or functionalisation. Preferential reaction of PEG(VS)2 at the N-terminal amine group was achieved by reacting at pH between 7.0–7.5 for 1 hour in a 25x molar excess of PEG(VS)2. In addition, the extent of PEGylation was controlled by varying pH, reaction time and amount of PEG(VS)2. Verification of degree of PEGylation was obtained using MALDI-TOF spectroscopy as well as standard SDS-PAGE followed by immunoblotting. |
