Elucidation and control of low and high active populations of alkaline phosphatase molecules for quantitative digital bioassay.
alkaline phosphatase
cell-free protein synthesis
digital bioassay
microdevice
protein maturation
single enzyme activity
Journal
Protein science : a publication of the Protein Society
ISSN: 1469-896X
Titre abrégé: Protein Sci
Pays: United States
ID NLM: 9211750
Informations de publication
Date de publication:
08 2021
08 2021
Historique:
revised:
27
04
2021
received:
04
02
2021
accepted:
02
05
2021
pubmed:
7
5
2021
medline:
1
2
2022
entrez:
6
5
2021
Statut:
ppublish
Résumé
Alkaline phosphatase (ALP), a homo-dimeric enzyme has been widely used in various bioassays as disease markers and enzyme probes. Recent advancements of digital bioassay revolutionized ALP-based diagnostic assays as seen in rapid growth of digital ELISA and the emerging multiplex profiling of single-molecule ALP isomers. However, the intrinsic heterogeneity found among ALP molecules hampers the ALP-based quantitative digital bioassays. This study aims quantitative analysis of single-molecule activities of ALP from Escherichia coli and reveals the static heterogeneity in catalytic activity of ALP with two distinct populations: half-active and fully-active portions. Digital assays with serial buffer exchange uncovered single-molecule Michaelis-Menten kinetics of ALP; half-active molecules have halved values of the catalytic turnover rate, k
Identifiants
pubmed: 33955095
doi: 10.1002/pro.4102
pmc: PMC8284569
doi:
Substances chimiques
Escherichia coli Proteins
0
Alkaline Phosphatase
EC 3.1.3.1
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
1628-1639Informations de copyright
© 2021 The Authors. Protein Science published by Wiley Periodicals LLC on behalf of The Protein Society.
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