Producing Peptide Arrays for Epitope Mapping by Intein-Mediated Protein Ligation

Abstract

Peptide arrays are increasingly used to define antibody epitopes and substrate specificities of protein kinases. Their use is hampered, however, by ineffective and variable binding efficiency of peptides, which often results in low sensitivity and inconsistent results. To overcome these limitations, we have developed a novel method for making arrays of synthetic peptides on various membranes after ligating the peptide substrates to an intein-generated carrier protein. We have conducted screening for optimal carrier proteins by immunoreactivity and direct assessment of binding using a peptide derivatized at a lysine sidechain with fluorescein, CDPEK(fluorescein)DS. Ligation of a synthetic peptide antigen to a carrier protein, HhaI methylase, resulted in an improved retention of peptides and an increased sensitivity of up to 10(4)-fold in immunoassay- and epitope-scanning experiments. Denaturing the ligation products with 2% sodium dodecyl sulfate (SDS) or an organic solvent (20% methanol) prior to arraying did not significantly affect the immunoreactivity of the HhaI methylase-peptide product. Because the carrier protein dominates the binding of ligation products and contains one peptide reactive site, the amount of peptide arrayed onto the membranes can be effectively normalized. This technique was utilized in the alanine scanning of hemagglutinin (HA) antigen using two monoclonal antibodies, resulting in distinguishing the different antigen epitope profiles. Furthermore, we show that this method can be used to characterize the antibodies that recognize phosphorylated peptides. This novel approach allows for synthetic peptides to be uniformly arrayed onto membranes, compatible with a variety of applications.

Use of Intein-Mediated Phosphoprotein Arrays to Study Substrate Specificity of Protein Phosphatases

“…The phosphopeptides based on the phosphorylation sites of various PKs were described previously (10). The peptides were dissolved in 5 mM Tris-HCl, pH 7.5, 50 M NaCl at a final concentration of 5 mM.…”

“…We have previously demonstrated production of peptide arrays on nitrocellulose by the ligation of synthetic peptide substrates to an inteingenerated carrier protein (CP) (10). This technique, termed intein-mediated peptide array (IPA), takes advantage of the catalytic activity of an engineered intein to generate a reactive thioester at the C terminus of a CP.…”

“…A peptide possessing an N-terminal cysteine residue is linked to the C terminus of the CP via a native peptide bond using intein-mediated protein ligation (IPL) (11). This method circumvents the problem associated with variable binding by small peptide substrates to various matrices, leading to successful applications for antibody characterization and epitope scanning, with an increase in sensitivity of up to 10 4 - fold compared with the use of peptide antigens (10). …”

Microarray Technology as a Universal Tool for High-Throughput Analysis of Biological Systems

“…al. describe a method where peptides were firstly ligated to an intein-generated carrier protein, which is then bound to a solid support [146]. Because the carrier protein dominates the binding of ligation products and contains one peptide reactive site, the amount of peptides arrayed to the membranes can be effectively normalized.…”

Protein Splicing Mechanisms and Applications

“…This obstacle was overcome using EPL to link peptides to a carrier protein that has good binding characteristics and low antibody reactivity (30 -32). The technique was used to make peptide microarrays (30) and to perform phosphorylation assays followed by Western blot readout on substrates containing phosphorylation sites for c-Src protein tyrosine kinase or Abl protein tyrosine kinase (31,32).…”