Dataset Information

Simultaneous detection of multiple proteases using a non-array nanopore platform.

ABSTRACT: Multiplexing methods which are capable of measurement of multiple analytes in a single assay are of great importance in many fields. The conventional strategy for simultaneous detection of multiple species is to construct a sensor array. Herein, we report an innovative multiplex multi-analyte detection platform in a non-array format for protease measurement. By monitoring protease degradation of a single peptide substrate containing two cleavage sites for a disintegrin and metalloproteinase 10 (ADAM10) and a disintegrin and metalloproteinase 10 (ADAM17) in a single nanopore, simultaneous detection and quantification of these two model proteases in mixture samples could satisfactorily be accomplished. Our developed multiplexing sensing platform has the potential to be coupled with the traditional sensor array to further improve the multiplexing capability of the sensor, which may find useful applications in clinical diagnosis and prognosis.

SUBMITTER: Chen X

PROVIDER: S-EPMC8485758 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Simultaneous detection of multiple proteases using a non-array nanopore platform.

Chen Xiaohan X Zhang Youwen Y Guan Xiyun X

Nanoscale 20210803 32

Multiplexing methods which are capable of measurement of multiple analytes in a single assay are of great importance in many fields. The conventional strategy for simultaneous detection of multiple species is to construct a sensor array. Herein, we report an innovative multiplex multi-analyte detection platform in a non-array format for protease measurement. By monitoring protease degradation of a single peptide substrate containing two cleavage sites for a disintegrin and metalloproteinase 10 ( ...[more]

PMID: 34477641

Similar Datasets

Project description:BackgroundRecombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. Here we propose a new fusion platform for multiple proteases, by using diaminopropionate ammonia-lyase (DAL) as the fusion protein. It was based on the finding that a fused His6-tag could significantly decreases the activities of DAL from E. coli (eDAL) and Salmonella typhimurium (sDAL). Previously, we have shown that His6GST-tagged eDAL could be used to determine the activity of tobacco etch virus protease (TEVp) under different temperatures or in the denaturant at different concentrations. In this report, we will assay different tags and cleavage sequences on DAL for expressing yield in E. coli, stability of the fused proteins and performance of substrate of other common proteases.ResultsWe tested seven different protease cleavage sequences (rhinovirus 3C, TEV protease, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), three different tags (His6, GST, CBD and MBP) and two different DALs (eDAL and sDAL), for their performance as substrate to the seven corresponding proteases. Among them, we found four active DAL-fusion substrates suitable for TEVp, factor Xa, thrombin and DnaB intein. Enterokinase cleaved eDAL at undesired positions and did not process sDAL. Substitution of GST with MBP increase the expression level of the fused eDAL and this fusion protein was suitable as a substrate for analyzing activity of rhinovirus 3C. We demonstrated that SUMO protease Ulp1 with a N-terminal His6-tag or MBP tag displayed different activity using the designed His6SUMO-eDAL as substrate. Finally, owing to the high level of the DAL-fusion protein in E. coli, these protein substrates can also be detected directly from the crude extract.ConclusionThe results show that our designed DAL-fusion proteins can be used to quantify the activities of both sequence- and conformational-specific proteases, with sufficient substrate specificity.

Dataset Information

Simultaneous detection of multiple proteases using a non-array nanopore platform.

Publications

Simultaneous detection of multiple proteases using a non-array nanopore platform.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets