Masters Thesis

Monitoring proteolytic efficiency of engineered trypsin via chymotrypsinogen activity assay

Protease therapeutics have been on the rise and have gained recognition for having diverse clinical applications. One major complication faced is the abundance of protease inhibitors that serve to regulate proteolytic activity resulting in a short therapeutic half-life. Previous research with the model serine protease trypsin showed that residues 39 and 60 play a key role in protease: inhibitor binding. Compared to wild type, four trypsin single variants (Y39A, Y39F, K60A, K60V) which were all catalytically similar using a synthetic substrate displayed altered.sensitivity towards bovine pancreatic trypsin inhibitor (BPTI) compared to wild type. In order to ascertain the viability of these engineered variants for inhibitor resistance in vivo, the interactions between naturally occurring macro molecular substrates were evaluated in this study. Variant Y39A displayed the highest kcat/KM (pM'1Min-1) at 3.34 ± 0.06, while K60V displayed at 0.58 ± 0.01. Variant Y39A activated the most chymotrypsinogen (Cg) with a total 30% activation 1.48 ± 0.05 jvM (out of 5 pM) while K60V had the lowest with 14% or 0.70 ± 0.04 pM. With 20 nM BPTI, the K60A variant had an activation drop of 49% while K60V dropped by 16%. At 30 nM, 50 nM and 100nM BPTI, the Y39F variant displayed greater activation than wild-type and the K60 variants in the presence of BPTI. At higher concentrations of BPTI, the K60 variants s have become more sensitive to inhibition, along with having lower kcat/KM and less total chymotrypsin activation. Overall K60 variants seemed to be less proteolytically efficient towards macromolecular substrates with less resistance towards inhibition, while Y39F displayed promising results in both studies.

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