Asparaginyl Ligases: New Enzymes for the Protein Engineer's Toolbox

Abstract

Enzyme‐catalysed site‐specific protein modifications enable the precision manufacture of conjugates for the study of protein function and/or for therapeutic or diagnostic applications. Asparaginyl ligases are a class of highly efficient transpeptidases with the capacity to modify proteins bearing only a tripeptide recognition motif. Herein, we review the types of protein modification that are accessible using these enzymes, including N‐ and C‐terminal protein labelling, head‐to‐tail cyclisation, and protein‐protein conjugation. We describe the progress that has been made to engineer highly efficient ligases as well as efforts to chemically manipulate the enzyme reaction to favour product formation. These enzymes are powerful additions to the protein engineer‘s toolbox.

One-step asparaginyl endopeptidase (OaAEP1)-based protein immobilization for single-molecule force spectroscopy

“…Finally, the protein with an NGL sequence can be immobilized to the GL-coated surface via the Oa AEP1-mediate enzymatic ligation for half an hour. 18,53,54…”

“…Finally, the protein with an NGL sequence can be immobilized to the GL-coated surface via the OaAEP1-mediate enzymatic ligation for half an hour. 18,53,54 To demonstrate the applicability of this method for the AFM-SMFS study, a characterized protein, enhanced Green Fig. 1 Scheme of surface protein immobilization using different methods.…”

Rational domestication of a plant-based recombinant expression system expands its biosynthetic range

“…Although some vaccines, enzymes, and antibodies produced in plant-based systems are on the market or in late-stage trials, there remains a gap in terms of peptide production. There is an opportunity to develop plant systems for producing stabilised therapeutic peptides, by capitalising on recent advances in the application of plant peptide ligation machinery (Jackson et al, 2020;Rehm et al, 2021). Some plants natively produce stable head-to-tail cyclic disulfide-rich peptides (cycDRPs), with the best characterised being the three-disulfide containing cyclotide peptide family (Craik et al, 1999) and the single-disulfide containing sunflower trypsin inhibitor (SFTI) peptide (Luckett et al, 1999).…”

“…Although some vaccines, enzymes, and antibodies produced in plant-based systems are on the market or in late-stage trials, there remains a gap in terms of peptide production. There is an opportunity to develop plant systems for producing stabilised therapeutic peptides, by capitalising on recent advances in the application of plant peptide ligation machinery (Jackson et al, 2020; Rehm et al, 2021).…”

Enzymatic Protein–Protein Conjugation through Internal Site Verified at the Single-Molecule Level

“…Here, we used two enzymes, Sortase A and Oa AEP1, for protein immobilization and conjugation. Thus, it can be easily applied to high-precision SMFS measurement for similar protein studies. − …”