Preparing recombinant “Split AEP” for protein labeling

Muge Ma, Simon Tang, Matthew T. Dickerson, Louis Luk

Research output: Chapter or section in a book/report/conference proceedingChapter or section

1 Citation (SciVal)

Abstract

A variant originated from Oldenlandia affinis asparaginyl ligase, OaAEP1-C247A, has emerged as an ideal tool for protein labeling. However, its preparation was laborious and time-consuming. It is recombinantly produced as a zymogen, requiring acid activation and four chromatographic steps; despite these extensive steps, the catalytically active enzyme exhibited only moderate purity. Here, we report a novel preparation protocol, in which the cap and catalytically active core domains are produced as separate entities. The active enzyme can be obtained in two chromatographic steps, immobilized metal affinity chromatography (IMAC) and size exclusion chromatography (SEC), with no acid activation required, thereby shortening the purification procedure from at least 2 days to less than 6 h. In addition to the original C247A mutation which enhanced reaction with various amino nucleophiles, an extra D29E mutation was introduced to prevent self-cleavage, which led to noticeable improvements in homogeneity and activity of the enzyme. Indeed, the resulting “split AEP” (i.e., core domain of OaAEP1-D29E/C247A) exhibited improved catalytic efficiency constant (kcat/KM) that was found to be ∼3-fold higher than that of the original acid-activated counterpart (OaAEP1-C247A). Furthermore, we described a protein labeling protocol that couples the enzymatic reaction with an irreversible chemical transformation, thereby enabling high conversion of labeled protein with a lowered amount of reagent. Precisely, an alternative Asn-Cys-Leu (NCL) recognition sequence was used for substrate recognition. As the byproduct contains an N-terminal cysteine, it can be transformed into an inert 1,2 aminothiol motif by reacting with formylphenyl boronic acid (FPBA). Finally, the opportunities and challenges associated with the use of asparaginyl ligase are discussed.
Original languageEnglish
Title of host publicationModern Methods of Drug Design and Development
EditorsMatthew Lloyd
Pages501-540
Number of pages40
DOIs
Publication statusPublished - 5 Aug 2023

Publication series

NameMethods in Enzymology
Volume690
ISSN (Print)0076-6879
ISSN (Electronic)1557-7988

Bibliographical note

Funding Information:
We would like to thank for the financial support provided by BBSRC ( BB/T015799/1 ) for Louis Y. P. Luk as well as the UKRI PhD studentship for T. M. Simon Tang (1928909).

Publisher Copyright:
© 2023

Keywords

  • Asparaginyl endopeptidase
  • Asparaginyl ligase
  • Byproduct quenching
  • OaAEP
  • Peptide ligation

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry

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