Investigating the proteases responsible for specific SNAP-25 fragmentation during bacterial production

SNAP-25 (synaptosomal-associated protein of 25 kDa) is a SNARE protein involved in vesicle fusion. When grown in bacteria, our lab and others consistently see recombinant SNAP-25A and SNAP-25B result in fragmentation. We have previously characterized the fragments with mass spectrometry and identified a consistent fragmentation pattern, indicative of non-random cleavage. The causes remain unknown, as they persist in spite of several different growth conditions and post-lysis treatments. We present results from growing and purifying SNAP-25B in various conditions to measure the effects on fragmentation, in order to understand the mechanism at play. Initial experiments showed our purified fusion protein being clipped in predictable patterns when allowed to incubate at room temperature. We suspected that there were external proteases cleaving SNAP-25B following bacterial cell lysis. We experimented with different incubation temperatures after purification, confirming proteolytic activity and increased fragmentation. Additionally, increased IPTG concentrations during bacterial cell growth ultimately resulted in greater SNAP-25B digestion. To denature and inhibit any potential pH sensitive proteases, we adjusted pHs, without observing significant fragmentation effects. Next, because SNAP-25B has multiple cysteine residues that respond to oxidation, we tested the purified protein under oxidizing and reducing conditions, with no observed effect. Treatment with a broad-spectrum protease inhibitor cocktail reduced cleavage. We compared the effects of this cocktail to individual protease inhibitors EDTA and AEBSF, and our data showed EDTA was effective in reducing fragmentation, while AEBSF was not, suggesting that a metalloprotease was involved in fragmentation.