On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation

Peptide asparaginyl ligases (PALs) are precision tools for peptide cyclization, cell-surface labelling, protein semisynthesis and protein conjugation. PALs are expressed as inactive proenzymes requiring low pH activation. During activation, a large portion of the cap domain of the proenzyme that cov...

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Main Authors: Chua, Niying, Wong, Yee Hwa, El Sahili, Abbas, Liu, Chuan Fa, Lescar, Julien
Other Authors: School of Biological Sciences
Format: Article
Language:English
Published: 2023
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Online Access:https://hdl.handle.net/10356/169274
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spelling sg-ntu-dr.10356-1692742023-07-17T15:31:49Z On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation Chua, Niying Wong, Yee Hwa El Sahili, Abbas Liu, Chuan Fa Lescar, Julien School of Biological Sciences NTU Institute of Structural Biology Science::Biological sciences Asparaginyl Endopeptidase Peptideasparaginyl Ligase Peptide asparaginyl ligases (PALs) are precision tools for peptide cyclization, cell-surface labelling, protein semisynthesis and protein conjugation. PALs are expressed as inactive proenzymes requiring low pH activation. During activation, a large portion of the cap domain of the proenzyme that covers the substrate binding site is proteolytically removed, exposing the active site to solvent and releasing a population of heterogenous active enzymes. The availability of a readily active ligase not requiring acid activation and subsequent purification of active forms would facilitate manufacturing and streamline applications. Here, we engineered the OaAEP1b-C247A hyperactive ligase via serial truncations along the linker connecting the cap and core domain of the proenzyme. The recombinant expression of the truncated constructs was carried out in Escherichia coli. Following a solubilization/refolding protocol, one truncated construct termed 'OaAEP1b-C247A-∆351' could be overexpressed in the insoluble fraction, purified, and displayed a level of ligase activity comparable to the acid-activated OaAEP1b-C247A enzyme. This constitutively active protein can be stored for up to 2 years at -80 °C and readily used for peptide cyclization and protein conjugation. We were able to express and purify a stable constitutively active asparaginyl ligase that can be stored for months without significant activity loss. The removal of the low pH proenzyme activation step eliminates the heterogeneity introduced by this procedure. The yield of purified recombinant active ligase that can be routinely obtained per 100 mL of E. coli cell culture is about 0.9 mg. This recombinant active ligase can be used to carry out protein conjugation. Ministry of Education (MOE) National Research Foundation (NRF) Published version This research was supported by the Academic Research Fund (AcRF) Tier 3 (MOE2016-T3-1-003) to the James P. Tam, JL, and CFL laboratories and National Research Foundation (NRF-CRP24-2020-0005). 2023-07-11T01:40:09Z 2023-07-11T01:40:09Z 2023 Journal Article Chua, N., Wong, Y. H., El Sahili, A., Liu, C. F. & Lescar, J. (2023). On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation. FEBS Open Bio, 13(6), 1095-1106. https://dx.doi.org/10.1002/2211-5463.13575 2211-5463 https://hdl.handle.net/10356/169274 10.1002/2211-5463.13575 36788723 2-s2.0-85152051757 6 13 1095 1106 en MOE2016-T3-1-003 NRF-CRP24-2020-0005 FEBS Open Bio © 2023 The Authors. FEBS Open Bio published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. application/pdf
institution Nanyang Technological University
building NTU Library
continent Asia
country Singapore
Singapore
content_provider NTU Library
collection DR-NTU
language English
topic Science::Biological sciences
Asparaginyl Endopeptidase
Peptideasparaginyl Ligase
spellingShingle Science::Biological sciences
Asparaginyl Endopeptidase
Peptideasparaginyl Ligase
Chua, Niying
Wong, Yee Hwa
El Sahili, Abbas
Liu, Chuan Fa
Lescar, Julien
On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation
description Peptide asparaginyl ligases (PALs) are precision tools for peptide cyclization, cell-surface labelling, protein semisynthesis and protein conjugation. PALs are expressed as inactive proenzymes requiring low pH activation. During activation, a large portion of the cap domain of the proenzyme that covers the substrate binding site is proteolytically removed, exposing the active site to solvent and releasing a population of heterogenous active enzymes. The availability of a readily active ligase not requiring acid activation and subsequent purification of active forms would facilitate manufacturing and streamline applications. Here, we engineered the OaAEP1b-C247A hyperactive ligase via serial truncations along the linker connecting the cap and core domain of the proenzyme. The recombinant expression of the truncated constructs was carried out in Escherichia coli. Following a solubilization/refolding protocol, one truncated construct termed 'OaAEP1b-C247A-∆351' could be overexpressed in the insoluble fraction, purified, and displayed a level of ligase activity comparable to the acid-activated OaAEP1b-C247A enzyme. This constitutively active protein can be stored for up to 2 years at -80 °C and readily used for peptide cyclization and protein conjugation. We were able to express and purify a stable constitutively active asparaginyl ligase that can be stored for months without significant activity loss. The removal of the low pH proenzyme activation step eliminates the heterogeneity introduced by this procedure. The yield of purified recombinant active ligase that can be routinely obtained per 100 mL of E. coli cell culture is about 0.9 mg. This recombinant active ligase can be used to carry out protein conjugation.
author2 School of Biological Sciences
author_facet School of Biological Sciences
Chua, Niying
Wong, Yee Hwa
El Sahili, Abbas
Liu, Chuan Fa
Lescar, Julien
format Article
author Chua, Niying
Wong, Yee Hwa
El Sahili, Abbas
Liu, Chuan Fa
Lescar, Julien
author_sort Chua, Niying
title On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation
title_short On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation
title_full On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation
title_fullStr On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation
title_full_unstemmed On the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation
title_sort on the design of a constitutively active peptide asparaginyl ligase for facile protein conjugation
publishDate 2023
url https://hdl.handle.net/10356/169274
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