• Analytical biochemistry · Feb 2009

    Comparative Study

    Direct comparison of bioluminescence-based resonance energy transfer methods for monitoring of proteolytic cleavage.

    • Helen Dacres, Mira M Dumancic, Irene Horne, and Stephen C Trowell.
    • Food Futures Flagship, CSIRO Entomology, Canberra, ACT, Australia. helen.dacres@csiro.au
    • Anal. Biochem. 2009 Feb 15; 385 (2): 194-202.

    AbstractBioluminescence resonance energy transfer (BRET) is a powerful tool for the study of protein-protein interactions and conformational changes within proteins. Two common implementations of BRET are BRET(1) with Renilla luciferase (RLuc) and coelenterazine h (CLZ, lambda(em) approximately 475 nm) and BRET(2) with the substrate coelenterazine 400a (CLZ400A substrate, lambda(em)=395 nm) as the respective donors. For BRET(1) the acceptor is yellow fluorescent protein (YFP) (lambda(em) approximately 535 nm), a mutant of green fluorescent protein (GFP), and for BRET(2) it is GFP(2) (lambda(em) approximately 515 nm). It is not clear from previous studies which of these systems has superior signal-to-background characteristics. Here we directly compared BRET(1) and BRET(2) by placing two different protease-specific cleavage sequences between the donor and acceptor domains. The intact proteins simulate protein-protein association. Proteolytic cleavage of the peptide linker simulates protein dissociation and can be detected as a change in the BRET ratios. Complete cleavage of its target sequence by thrombin changed the BRET(2) ratio by a factor of 28.9+/-0.2 (relative standard deviation [RSD], n=3) and changed the BRET(1) ratio by a factor of 3.05+/-0.07. Complete cleavage of a caspase-3 target sequence resulted in the BRET ratio changes by factors of 15.45+/-0.08 for BRET(2) and 2.00+/-0.04 for BRET(1). The BRET(2) assay for thrombin was 2.9 times more sensitive compared with the BRET(1) version. Calculated detection limits (blank signal+3sigma(b), where sigma(b)=standard deviation [SD] of blank signal) were 53 pM (0.002 U) thrombin with BRET(1) and 15 pM (0.0005 U) thrombin with BRET(2). The results presented here suggest that BRET(2) is a more suitable system than BRET(1) for studying protein-protein interactions and as a potential sensor for monitoring protease activity.

      Pubmed     Full text   Copy Citation     Plaintext  

      Add institutional full text...

    Notes

     
    Knowledge, pearl, summary or comment to share?
    300 characters remaining
    help        
    You can also include formatting, links, images and footnotes in your notes
    • Simple formatting can be added to notes, such as *italics*, _underline_ or **bold**.
    • Superscript can be denoted by <sup>text</sup> and subscript <sub>text</sub>.
    • Numbered or bulleted lists can be created using either numbered lines 1. 2. 3., hyphens - or asterisks *.
    • Links can be included with: [my link to pubmed](http://pubmed.com)
    • Images can be included with: ![alt text](https://bestmedicaljournal.com/study_graph.jpg "Image Title Text")
    • For footnotes use [^1](This is a footnote.) inline.
    • Or use an inline reference [^1] to refer to a longer footnote elseweher in the document [^1]: This is a long footnote..

    hide…