ABSTRACT
When minimal functional sequences are used, it is possible to integrate multiple functions on a single peptide chain, like a "single stroke drawing". Here a dual functional peptide was designed by combining in vitro selected catalytic and binding activities. For catalytic activity, we performed in vitro selection for a peptide aptamer binding to hemin by using ribosome display and isolated a peptide that had peroxidase activity in the presence of hemin. By combining the selected catalytic peptide with a peptide antigen, which can be recognized by an antibody, an enzyme-antibody conjugate-like peptide was obtained. This study demonstrates a successful strategy to create dual functionalized peptide chains for use in immunoassays.
Subject(s)
Antibodies/metabolism , Aptamers, Peptide/metabolism , Hemin/metabolism , Oligonucleotides/metabolism , Peroxidase/metabolism , Ribosomes/metabolism , Antibodies/chemistry , Aptamers, Peptide/chemistry , Binding Sites , Catalysis , Hemin/chemistry , Humans , In Vitro Techniques , Kinetics , Oligonucleotides/chemistry , Oxidation-Reduction , Peptide Library , Peroxidase/chemistry , Ribosomes/chemistryABSTRACT
Ribosome display is a very effective and powerful technology for screening functional peptides or polypeptides in vitro. In ribosome display, each peptide or polypeptide (phenotype) links with its corresponding mRNA (genotype) through a ribosome. This link can be achieved by the absence of a stop codon in the mRNA, therefore stalling the ribosome at the end of translation with the nascent random sequence peptide extended by a spacer outside of the ribosome tunnel. In this chapter, we describe a method for the use of a further stabilized peptide-ribosome-mRNA complex for ribosome display.
Subject(s)
Directed Molecular Evolution , Peptides/metabolism , Ribosomes/metabolism , Kinetics , Peptides/genetics , RNA, Messenger/genetics , Ribosomes/chemistry , Ribosomes/genetics , ThermodynamicsABSTRACT
Antibodies were covalently conjugated with poly(ethylene glycol) (PEG) and the properties of the PEGylated antibodies in organic media were investigated. Two types of monoclonal antibody were used in this study. One was a monoclonal antibody (abzyme) that was prepared against a hapten mimicking a transition state of hydrolysis. Another was a monoclonal antibody against estrogen, which is not soluble in water. By electrophoresis and mass spectral analysis, the covalent conjugation with PEG chains was confirmed. The PEGylated antibodies bound to antigens and the PEGylated abzyme catalyzed a hydrolysis reaction in an aqueous solution. The PEGylated antibodies were soluble in dichloromethane and acetone and interacted with antigen either in dichloromethane or in acetone. In conclusion, PEGylated antibodies can be employed as analytical tools for water-insoluble analytes.
