What will the Next-Gen Protease activity detection be?

Fluorescent technologies enable the measurement of protease activities and the screening of compounds influencing protease activities. Fluorescence Resonance Energy Transfer (FRET) but also TR-FRET, Q-FRET together with Time Resolved Fluorescence (TRF) and Fluorescence Lifetime (FLT) are popular assays, in which protease substrates occupy a central place. These assays are based on the specific recognition and cleavage of a peptide sequence (substrate) by the protease of interest. If the substrate is not optimally designed (or too short), experimental output can be unrelevant: low selectivity and specificity, high background, false negatives or positives…). In addition, the data can also be affected by the reaction buffer (pH, compound solvent…).This lack of relevant biological information is at the origin the emergence of “Next-Gen” fluorescent protease substrates.

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Direct localization of MMP activity in 3D tumor invasion model

Recently, the non-FRET EnSens technology was launched for  in vitro assessment of specific protease activities (Enzium, Inc.). Up to date, the EnSens substates were validated for the detection of protease activity in microplate assays. Now, Enzium has extended the applicability of their Ensens method to 3 D live-cell imaging. On the top of this, they announce that the experimental procedure is easy and non-toxic for cell cultures and co-cultures. So how does live-cell imaging help you locate protease activities in in vitro tumor invasion assays ?

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Optimal substrate design for protease screening!

EnSens assays, a new protease activity assay technology, which are produced by  Enzium, were recently introduced to the European market. A number of  assays are available from catalog, such as activity assays for MMP-2, MMP-9, MMP-14, MMP-25, ADAM10, ADAM17, Factor Xa, Furin, and Thrombin.

But what about if what you’re looking for is not available? In this post, we’ll take a look at how you can get an optimal substrate for your protease of interest, even if it is not yet obtainable from catalog.

EnSens technology

First, let’s take a look at the EnSens assay technology itself. It’s based on a non-FRET technology and makes use of a protein substrate expressed in E. coli which is a modular dumbbell comprising two domains separated by a linker that contains a highly specific protease recognition sequence. One domain is the dye-binding domain, and the other is a blocking domain. When the linker is cleaved by a protease, separating the two domains, the binding domain is free to bind dye, constraining it and causing it to fluoresce (see Figure below).

EnSens principle

EnSens assays overcome limitations of FRET based protease assays

  • the substrate can incorporate full recognition site sequences – in FRET assays the recognition sites usually have to be shorter which can result in a number of false positives and negatives because the recognition site might not be highly selective.
  • The substrate is an scFv-based protein (single-chain variable fragment, an artificially generated antibody fragment) which confers high stability in vitro and in vivo.
  • The EnSens se-Red fluorophore is resistant to photobleaching up to 48 hours
  • The long emission wavelength (665nm) reduces interference from other assay components
  • The assay works stably in wide range of pH, salt, & solvent conditions
  • Substrate and fluorophore are separate in the systems, so the fluorophore is constantly renewed in solution
  • The system is characterized by high signal-to-noise ratios: 6-20x
  • The read-out can be done in standard plate or cuvette fluorimeters
  • The EnSens assay gives repeatable and reproducible kinetic activity curves from reagent to reagent

and finally (and this is where it gets interesting for non-catalog items!):

  • The substrate is modular, allowing easy swapping of protease recognition sites – thus customized substrates can be obtained if you know the recognition site of your protease of interest

So, you can get your customized protease substrate!

In other words: If you want to screen for modulators/inhibitors of a protease with a known recognition site, you simply need to provide the sequence of the site – and the substrate and the complete EnSens assay can be designed for you in cooperation with Enzium.

Interested in a highly specific protease assay for your screening? Get in touch through the form below, I’m sure a tailored solution can be found to match your needs exactly!

 

Measuring Furin – a protease to process & activate proteins

Furin belongs to the family of subtilisin-like proprotein convertases. These convertases are in charge activating latent precursor proteins, which are processed into their biologically active fragments. Furin functions as a serin endoprotease.

Furin activates a variety of proteins

Furin cleaves and thus activates a number of proteins such as…

  • Proparathyroid hormone
  • Transforming Growth Factor beta 1 precursur (TGF-ß1 precursor)
  • Proalbulmin
  • Pro-beta-Secretase
  • beta subunit of Pro-Nerve Growth Factor (NGF)
  • Von Willebrand Factor
  • Membranetype-1 Matrix Metalloprotease (MT1-MMP)

…. and a lot more. [Read more…]

MMP-25 activity assay – the first one on the market!

With the recent release of the first commercially available and ready-to-use assay to detect MMP-25 activity , Enzium has opened a new era for screening protease activities through a robust non-FRET experimental approach. A good opportunity to take a look at the EnSens technology.

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