As I read that, designing assay for cysteine protease is a bit challenging compare to other proteases, I am wondering why it is so ? Many research articles follows fluorescence based assay for this by modification in its substrate. People attaches reporters and quenchers in the peptidomimetic substrates to read out the signal after protease cleavage. Considering this, my question is how i can design an assay for this using fluorophores/or any other methods available. I went through some papers but not clearly described. What measures I have to take into account. Should I also find the Scissile site of the enzyme prior to design the substrate, if yes then by which technique i can assess this. Waiting for your valuable suggestions. Thank you very much in advance.
Kind regards.
Are you investigating a novel protease, or one that has already been described in the literature? If it is a known enzyme, then there is probably already information in the literature about its physiological substrate.
If it is a novel protease, it might be best first to find out what its physiological substrate is, and where it cleaves that substrate. Once you know what the substrate is, you can purify the substrate protein and use mass spectrometry to identify the cleavage site of that substrate. After that, you can have unlabeled synthetic peptides made that include the sequence around the physiological substrate with variations to find out more about the selectivity of the enzyme, if you wish, still using mass spectrometry or just HPLC to follow the reaction. When you need to set up a high-throughput method to use for identifying and studying inhibitors, you can use that information to design a FRET substrate, which is a peptide that contains a fluorescent donor and acceptor on either side of the cleavage site. It may be possible to use a peptide substrate with only an aminomethylcoumarin C-terminus immediately after the cleavage site, if the enzyme will recognize it. This is simpler that a FRET substrate, but it is likely to be a poorer substrate.
Dear Prem
as ADAM wrote, it is important to understand if:
1) Are are characterizing a novel protease or one alredy reported in literature?=
2) Are you interested to desing functional assay to determin your protease specificity or just to assess if your recombinant protease is folded and active?
If you are characterizing a novel protease and you would like to have some data regarding its specifity anf you have one fluorescence multiplate reader available one interesting tool to have some informations is the JPT protease substrate SET (a Collection of 360 internally quenched (Dabcyl/EDANS) peptides) could be interesting. It is quite expensive but can provide to you a lot information in very short time.
On the contrary, if you would like just to test if your protease is active but you are nor interested to know the specificity, you can try to test a kit with less specificity (e.g https://www.thermofisher.com/order/catalog/product/E33758). Of course in this case is important that you have a very pure protease to be sure that the substrate digestion cannot be due to impurity in your sample.
Regarding the fact that the cisteines are more difficult than the other, i think that it is important to find the right condictions. For example. You can work with out DTT or do you need to had some reducing agent to be sure that the catalitic cisteine is free and able to react?
good luck
Manuele
Hi Prem, Cys proteinases function much like Ser proteinases. The major, and obvious difference, is the nucleophile in the active site that reacts with the substrate peptide bond.
If you know the substrate specificity of your Cys proteinase then you can use synthetic substrates for Ser proteinases.
The challenging part is to keep the active site -SH in a reduced state. It is more susceptible to oxidation than the active site Ser.
If you purify and keep the enzyme with low levels of reducing agents (DTT or BME) then you should be fine.
Dear Adam B Shapiro Thank you so much for your kind and prompt help as usual. I really appreciate your suggestions which have helped me, throughout my Ph.D. tenure. Yes I am going to start working on a novel cysteine protease. So, if I have to know the cleavage site through the mass spectrometry, then I have to first cleave the protein though the protease and then subjct the mixture in mass spect. So, which type of approach I should take during determination of cleavage site from a whole protein. Because during mass spect. the protein has to be trypsinised for fragmentation. Please suggest the way. Thank you.
It is not necessary to digest the purified substrate protein with trypsin. It is possible to do intact protein mass spectrometry using electrospray ionization, which allows you to precisely measure the mass of the entire substrate protein before cleavage, and the fragments afterwards. The data should make it possible to determine the site of cleavage precisely based on the known sequence of the substrate protein.
Further validation of the cleavage site can be attained by N-terminal sequencing of the C-terminal fragment. Actually, this alone should be sufficient to identify the cleavage site, no MS required.
You could also use proteomics to identify the cleavage site following trypsin digestion, as long as the cleavage site is not at Lys or Arg, by detecting the two new fragments that appear only after treatment with the new protease.
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