Serine protease-specific protease inhibitors

[BiochemistInTraining]

Hi,

 

I'm an undergraduate student currently doing a one year research placement as part of my degree.

 

According to recent literature, a cytokine I'm working with is cleaved into a more active form by at least two serine proteases, but cleaved and inactivated by a cysteine protease. In order to obtain a native source of the cytokine, I culture a human cell line and then lyse the cells using multiple freeze-thawing cycles (the cells do not secrete the cytokine in question). I've found that adding a universal protease inhibitor cocktail to the culture during the freeze-thawing process greatly reduces cleavage of the cytokine (shown by IP) and its activity (measured using a reliable and well established reporter gene assay).

 

Now I would like to lyse the cells in the presence of inhibitors which only inhibit serine proteases, but I am unsure which ones to use, and how likely they are to inhibit the serine proteases I'm interested in (I can't find any specific inhibitors for these) while at the same time having minimal effects on the activity of other classes of proteases, particularly cytseine proteases.

[CharonY]

I am not absolutely certain about the specificity, so you should research it a bit more but IIRC inhibitors that are mostly selective for serine proteases include AEBSF, and PMSF (though both also inhibit papain, and potentially some other cysteine proteases). Then there is aprotinin, TLCK and TPCK which tend to be a bit more narrow in use.

 

Obviously, if your protease is somewhat more exotic some testing is likely to be needed. And as I said, this is just stuff that I vaguely remember so it may be somewhat inaccurate.

[BabcockHall]

Stay away from E-64 and leupeptin. Look into regenerating cysteine protease activity after treatment with PMSF. I think dithiothreitol will work, but I don't have a citation.

Edited April 11, 2013 by BabcockHall