Improved Fmoc-solid-phase peptide synthesis of an extracellular loop of CFTR for antibody selection by the phage display technology.
Cystic fibrosis (CF), a life-shortening genetic disease, is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene that codes for the CFTR protein, the major chloride channel expressed at the apical membrane of epithelial cells. The development of an imaging probe capable of non-invasively detect CFTR at the cell surface could be of great advantage for the management of CF. With that purpose, we synthesized the first extracellular loop of CFTR protein (ECL1) through fluorenylmethyloxycarbonyl (Fmoc)-based microwave-assisted solid-phase peptide synthesis (SPPS), according to a reported methodology. However, aspartimide formation, a well-characterized side reaction in Fmoc-SPPS, prompted us to adopt a different side-chain protection strategy for aspartic acid residues present in ECL1 sequence. The peptide was subsequently modified via PEGylation and biotinylation, and cyclized through disulfide bridge formation, mimicking the native loop conformation in CFTR protein. Herein, we report improvements in the synthesis of the first extracellular loop of CFTR, including peptide modifications that can be used to improve antigen presentation in phage display for selection of novel antibodies against plasma membrane CFTR.
['Ferreira|Vera F C|VFC|https://orcid.org/0000-0002-5644-3199', 'Correia|João D G|JDG|https://orcid.org/0000-0002-7847-4906', 'Farinha|Carlos M|CM|https://orcid.org/0000-0002-5467-1710', 'Mendes|Filipa|F|https://orcid.org/0000-0003-0646-1687']
D000906:Antibodies D061505:Cell Surface Display Techniques D019005:Cystic Fibrosis Transmembrane Conductance Regulator D005449:Fluorenes D006801:Humans D010455:Peptides D060327:Solid-Phase Synthesis Techniques
extracellular loop CFTR cystic fibrosis solid-phase peptide synthesis aspartimide PEGylation biotinylation cyclization