Site-Specific Sequential Protein Labeling Catalyzed by a Single Recombinant Ligase

J Am Chem Soc. 2019 Oct 30;141(43):17388-17393. doi: 10.1021/jacs.9b09166. Epub 2019 Oct 16.

Abstract

Protein ligases of defined substrate specificity are versatile tools for protein engineering. Upon completion of the reaction, the products of currently reported protein ligases contain the amino acid sequence that is recognized by that same ligase, resulting in repeated cycles of ligation and hydrolysis as competing reactions. Thus, previous efforts to sequentially label proteins at distinct positions required ligases of orthogonal specificity. A recombinant Oldenlandia affinis asparaginyl endopeptidase, OaAEP1, is promiscuous for incoming nucleophiles. This promiscuity enabled us to define a nucleophile composed of natural amino acids that is ligated efficiently to the substrate yet yields a product that is poorly recognized by OaAEP1. Proteins modified with an efficient recognition module could be readily modified to yield a defined product bearing a cleavage-resistant motif, whereas proteins containing this inferior recognition motif remained essentially unmodified. We demonstrate the versatility of the N- or C-terminal protein modifications obtainable with this approach and modify the N- and C-termini of a single substrate protein in a sequential, site-specific manner in excellent yield.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Catalysis
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Green Fluorescent Proteins / chemistry
  • Green Fluorescent Proteins / metabolism
  • Oldenlandia / enzymology
  • Protein Engineering / methods*
  • Proteins / chemistry*
  • Proteins / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Single-Domain Antibodies / chemistry

Substances

  • Proteins
  • Recombinant Proteins
  • Single-Domain Antibodies
  • Green Fluorescent Proteins
  • Cysteine Endopeptidases
  • asparaginylendopeptidase