Abstract
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.
Highlights
Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases
We developed four active diaminopropionate ammonia-lyase (DAL)-fusion substrates suitable for tobacco etch virus protease (TEVp), factor Xa, thrombin and DnaB intein, and summarized potential factors that may affect the development of other fusion protein substrates in the future
Design and overall results of the DAL-fusion substrate screen To determine if DAL-fusion can be used as substrate for other proteases and to select the optimal fusion construct, we designed a library of DAL-fusion constructs, by combining seven different protease cleavage sequences (R3P, TEVp, factor Xa, Ssp DnaB intein, Sce VMA1 intein, thrombin and enterokinase), four different tags (His6, glutathione S-transferase (GST), chitin binding domain (CBD), maltose-binding protein (MBP)) and two different DALs, total of seventeen constructs
Summary
Recombinant proteins fused with specific cleavage sequences are widely used as substrate for quantitatively analyzing the activity of proteases. 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). 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. 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
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