Protein synthesis by solid-phase chemical ligation using a safety catch linker

The native chemical ligation reaction has been used extensively for the syn thesis of the large polypeptides that correspond to folded proteins and dom ains. The efficiency of the synthesis of the target protein is highly depen dent on the number of peptide segments in the synthesis. Assembly of protei ns from multiple components requires repeated purification and lyophilizati on steps that give rise to considerable handling losses. In principle, perf orming the ligation reactions on a solid support would eliminate these inef ficient steps and increase the yield of the protein assembly. A new strateg y is described for the assembly of large polypeptides on a solid support th at utilizes a highly stable safety catch acid-labile linker. This amide gen erating linker is compatible with a wide range of N-terminal protecting gro ups and ligation chemistries. The utility of the methodology is demonstrate d by a three-segment synthesis of vMIP I, a chemokine that contains all 20 natural amino acids and has two disulfide bonds. The crude polypeptide prod uct was recovered quantitatively from the solid support and purified in 20% -recovered yield. This strategy should facilitate the synthesis of large po lypeptides and should find useful applications in the assembly of protein l ibraries.