Preparing recombinant “Split AEP” for protein labeling

Ma, Muge, Tang, Simon T.M., Dickerson, Matthew T. and Luk, Louis Y.P. ORCID: https://orcid.org/0000-0002-7864-6261 2023. Preparing recombinant “Split AEP” for protein labeling. Lloyd, Matthew, ed. Methods in Enzymology, Vol. 690. Methods in Enzymology, Elsevier, pp. 501-540. (10.1016/bs.mie.2023.07.004)

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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.

Item Type:	Book Section
Date Type:	Publication
Status:	Published
Schools:	Chemistry
Publisher:	Elsevier
ISSN:	0076-6879
Date of Acceptance:	5 August 2023
Last Modified:	06 Mar 2024 15:06
URI:	https://orca.cardiff.ac.uk/id/eprint/162638

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