Directed evolution of the S1 subsite specificity of trypsin
Final Report Abstract
The final goal of this study relates to the identification of trypsin variants bearing an inverse stereospecificity compared to wild-type trypsin. For developing a powerful screening assay it was essential to design an adapted trypsin construct, suitable screening substrates as well as an efficient screening approach itself. In the first steps, a trypsin plasmid based on the pET22b(+), allowing for the periplasmatic expression of the trypsin variants, three randomized loops as well as a loop shuffling system were generated. Furthermore, the loop 172 of trypsin was modified in the starting trypsin construct to mediate an enhanced plasticity and reduced stringency of the protein structure. Due to the fact that the classical substrate Ac-D-Arg-pNA shows a high signal to noise ratio and unfavorable diffusion effects hindering the identification of clones expressing trypsin variants with a respective specificity, two alternative types of substrates have been established. The first one is based on activated indolyl esters allowing for a blue-white screening on solid LB-media according to the so called X-Gal assay. The second one is based on an internally quenched peptide substrate enabling the "one pot"-detection of two distinct enzyme specificities in liquid media. Due to the fact that the measurement of fluorescence as well as absorbance is affected by the nature of the LB media, the latter had to optimize regarding a reduced fluorescence and absorption background enabling equally high expression rates compared to the original culture medium. So far, trypsin variants with randomized loop 189 were completely screened regarding a D-Arg as well as D-Ala specificity. The size of the enzyme library covers about 95,000 variants screened in a 96-well plate format. Until now, three trypsin variants were identified to show an increased turnover of the internally quenched all-D-peptide substrate. Sequencing of the corresponding mutations within the randomized loop 189 has been done. Detailed analysis of the hits identified shall be performed via heterologous expression and enzyme kinetics. In parallel, the optimized expression and screening protocol will be used for the screening of enzyme libraries with randomized loop 215 and 226, respectively.
