Bacterial Protease Inhibitors

Contents

1. Overview
2. Notes
3. Recipe

1. Overview

There are two major classes of proteases in E. coli: serine proteases and metalloproteases. The serine proteases are inhibited by AEBSF (a PMSF replacement) and benzamidine. The metalloproteases are inhibited by EDTA, which is not included in the cocktail. There are three uncharacterized cysteine proteases in E. coli, which should be inhibited by E-64. There are three aspartic proteases in E. coli as well, but they are membrane-bound enzymes that target signal sequences. We do not have inhibitors in our cocktail for these, nor do we plan to.

Danger

All of the inhibitors in the mix are toxic. Wear gloves.

2. Notes

Order replacements as follows:

• AEBSF: 500 mg bottle from Fisher Sci.
• E-64: 25 mg bottle from Sigma
• Benzamidine: big bottle from Fisher

The final, working concentrations of the protease inhibitors are:

• AEBSF: 0.5 mM
• E-64: 10 uM
• Benzamidine: 2 mM (this may not be necessary, but it is cheap)

Our protease inhibitor mix is prepared as a 100X stock in 10% DMSO and is stored in the -20˚C freezer, along with antibiotics, etc. The tops of the tubes should be labeled “PI”. Thaw the protease mix at room temperature at the same time that you thaw your frozen cell pellets. Do not thaw them in any type of heating device or warm water bath. To use the protease inhibitor cocktail, add 1/100th volume to your cells BEFORE you lyse them.

Also add EDTA at 0.5-1 mM (if running metal affinity column first) or 5 mM (if you are not).

DO NOT ADD MORE PROTEASE INHIBITOR THAN REQUIRED TO MAKE A 1X CONCENTRATION. Some of these reagents are very expensive and more is not better in this case.

3. Recipe

1. Take the AEBSF, E-64, and benzamidine out of the freezer and equilibrate to room temperature.
2. Put on gloves and add 3 mL DMSO to the E-64 bottle. Mix with a pipetman and transfer all you can to a 10 mL conical tube. Add another 3 mL DMSO to the bottle, mix, and transfer again. There should be no E-64 residue remaining. Add a final 1 mL DMSO to the tube for a total of 7 mL, making a 10 mM stock solution.
3. Weigh out 1.25 g benzamidine and transfer to a 50 ml conical tube.
4. Transfer the entire bottle (500 mg) AEBSF to the same 50 mL tube. Do this in a hood; the powder is toxic.
5. Add 35 mL MilliQ water and dissolve the two powders by gently inverting the tube.
6. Add 4 mL of 10 mM E-64 in DMSO and mix by inverting the tube.
7. Add water to 40 mL total and mix.
8. Pipet 5 ml aliquots into 15 ml tubes labeled “100X PI” and also labeled with the date. Store the tubes at -20˚C along with the antibiotic stocks.