Protein synthesis by native chemical ligation: Expanded scope by using straightforward methodology

The total chemical synthesis of proteins has great potential for increasing our understanding of the molecular basis of protein function. The introduc tion of native chemical ligation techniques to join unprotected peptides ne xt to a cysteine residue has greatly facilitated the synthesis of proteins of moderate size. Were, we describe a straightforward methodology that has enabled us to rapidly analyze the compatibility of the native chemical liga tion strategy for X-Cys ligation sites, where X is any of the 20 naturally occurring amino acids. The simplified methodology avoids the necessity of s pecific amino acid thioester linkers or alkylation of C-terminal thioacid p eptides. Experiments using matrix-assisted laser-desorption ionization MS a nalysis of combinatorial ligations of LYRAX-C-terminal thioester peptides t o the peptide CRANK show that all 20 amino acids are suitable for ligation, with Val, lie, and Pro representing less favorable choices because of slow ligation rates. To illustrate the method's utility, two 124-aa proteins we re manually synthesized by using a three-step, four-piece ligation to yield a fully active human secretory phospholipase AZ and a catalytically inacti ve analog. The combination of flexibility in design with general access bec ause of simplified methodology broadens the applicability and versatility o f chemical protein synthesis.