title: A new fusion protein platform for quantitatively measuring activity of multiple proteases.
abstract: BACKGROUND
  Recombinant 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.
  
  
  RESULTS
  We 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.
  
  
  CONCLUSIONS
  The 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.
authors.forename: Chengdong
authors.initials: C
authors.lastname: Zhou
authors.forename: Yanping
authors.initials: Y
authors.lastname: Yan
authors.forename: Jie
authors.initials: J
authors.lastname: Fang
authors.forename: Beijiu
authors.initials: B
authors.lastname: Cheng
authors.affiliation: Key Laboratory of Crop Biology of Anhui Province, Anhui Agricultural University, 130#, Changjiang West Road, Hefei City, Anhui Province 230036, PR, China. fanjun@ahau.edu.cn.
authors.forename: Jun
authors.initials: J
authors.lastname: Fan
mesh_terms: D000086042:3C Viral Proteases; D020816:Amino Acid Motifs / Q000235:genetics; D000642:Ammonia-Lyases / Q000235:genetics / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000378:metabolism; D010450:Endopeptidases / Q000378:metabolism; D004765:Enteropeptidase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D015951:Factor Xa / Q000378:metabolism; D005982:Glutathione Transferase / Q000235:genetics / Q000378:metabolism; D006639:Histidine / Q000235:genetics / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000235:genetics / Q000302:isolation & purification; D012486:Salmonella typhimurium / Q000201:enzymology; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism; D014764:Viral Proteins / Q000378:metabolism
chemical_list: C471213:His-His-His-His-His-His; D058619:Maltose-Binding Proteins; D009842:Oligopeptides; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D006639:Histidine; D005982:Glutathione Transferase; D010450:Endopeptidases; C099488:TEV protease; D013917:Thrombin; D015951:Factor Xa; D004765:Enteropeptidase; D003546:Cysteine Endopeptidases; D000086042:3C Viral Proteases; D000642:Ammonia-Lyases; C023147:diaminopropionate ammonia-lyase