Protein synthesis by solid-phase chemical ligation using a safety catch linker.
The native chemical ligation reaction has been used extensively for the synthesis of the large polypeptides that correspond to folded proteins and domains. The efficiency of the synthesis of the target protein is highly dependent on the number of peptide segments in the synthesis. Assembly of proteins from multiple components requires repeated purification and lyophilization steps that give rise to considerable handling losses. In principle, performing the ligation reactions on a solid support would eliminate these inefficient steps and increase the yield of the protein assembly. A new strategy is described for the assembly of large polypeptides on a solid support that utilizes a highly stable safety catch acid-labile linker. This amide generating linker is compatible with a wide range of N-terminal protecting groups and ligation chemistries. The utility of the methodology is demonstrated 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 product was recovered quantitatively from the solid support and purified in 20%-recovered yield. This strategy should facilitate the synthesis of large polypeptides and should find useful applications in the assembly of protein libraries.
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Departments of Cell Biology and Chemistry, Deparment of Molecular Biology, and The Skaggs Institute of Chemical Biology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Keinan
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Dawson
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D000595:Amino Acid Sequence; D002625:Chemistry, Organic / Q000379:methods; D004220:Disulfides; D007202:Indicators and Reagents; D019402:Macrophage Inflammatory Proteins / Q000138:chemical synthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry; D011506:Proteins / Q000138:chemical synthesis*; D012685:Sepharose; D014764:Viral Proteins
D004220:Disulfides; D007202:Indicators and Reagents; D019402:Macrophage Inflammatory Proteins; D010446:Peptide Fragments; D011506:Proteins; D014764:Viral Proteins; C108459:vMIP-1 protein, Human herpesvirus 8; D012685:Sepharose