Synthesis of full length PB1‐F2 influenza A virus proteins from ‘Spanish flu’ and ‘bird flu’

The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian isolates. Previous synthetic protocols have been improved to provide a synthetic full length H1N1 type PB1-F2 protein that is encoded by the 'Spanish flu' isolate and an equivalent protein from an avian host that is representative of a highly pathogenic H5N1 'bird flu' isolate, termed SF2 and BF2, respectively. Full length SF2, different mutants of BF2 and a number of fragments of these peptides have been synthesized by either the standard solid-phase peptide synthesis method or by native chemical ligation of unprotected N- and C-terminal peptide fragments. For SF2 chemical ligation made use of the histidine and the cysteine residues located in positions 41 and 42 of the native sequence, respectively, to afford a highly efficient synthesis of SF2 compared to the standard SPPS elongation method. By-product formation at the aspartic acid residue in position 23 was prevented by specific modifications of the SPPS protocol. As the native sequence of BF2 does not contain a cysteine residue two different mutants of BF2 (Y42C) and BF2 (S47C) with appropriate cysteine exchanges were produced. In addition to the full length molecules, fragments of the native sequences were synthesized for comparison of their physical characteristics with those from the H1N1 human isolate A/Puerto Rico/8/34 (H1N1). All peptides were analyzed by mass spectrometry, (1)H NMR spectroscopy, and SDS-PAGE. The protocols allow the synthesis of significant amounts of PB1-F2 and its related peptides.