Eurygaster integriceps Puton, commonly known as sunn pest, is a major pest of wheat in Northern Africa, the Middle East and Eastern Europe. This insect injects a prolyl endoprotease into the wheat, destroying the gluten. The purpose of this study was to clone the full length cDNA of the sunn pest prolyl endoprotease (spPEP) for expression in E. coli and to compare the amino acid sequence of the enzyme to other known PEPs in both phylogeny and potential tertiary structure. Sequence analysis shows that the 5ꞌ UTR contains several putative transcription factor binding sites for transcription factors known to be expressed in Drosophila that might be useful targets for inhibition of the enzyme. The spPEP was first identified as a prolyl endoprotease by Darkoh et al., 2010. The enzyme is a unique serine protease of the S9A family by way of its substrate recognition of the gluten proteins, which are greater than 30 kD in size. At 51% maximum identity to known PEPs, homology modeling using SWISS-MODEL, the porcine brain PEP (PDB: 2XWD) was selected in the database of known PEP structures, resulting in a predicted tertiary structure 99% identical to the porcine brain PEP structure. A Km for the recombinant spPEP was determined to be 210 ± 53 µM for the zGly-Pro-pNA substrate in 0.025 M ethanolamine, pH 8.5, containing 0.1 M NaCl at 37 °C with a turnover rate of 172 ± 47 µM Gly-Pro-pNA/s/µM of enzyme.
Chaperone assist in protein folding. The purpose of this study were to 1) determine if co‐expression of Pseudomonas aeruginosa GroESL or Trigger factor with prolyl endo protease (PEP) cDNA in Escherichia coli system resulted in increase amount of soluble active enzyme 2) determine if two different temperatures, 37°C for 24 hours and biphasic temperature incubation of 37°C for 6 hours followed by overnight in 28°C would increase in amount of soluble active PEP and 3) to identify which chaperone mechanism results in highest expression of active protein. GroESL and TF chaperones were cloned into pLys‐S and pLys‐E plasmid. The resulting pGROELS‐S/E plasmid or pTF‐S/E plasmid was co‐transformed into E. coli. Western Blot techniques were used to determine the level of protein expression. Enzyme activity was assayed using a synthetic chromagenic peptide. Biphasic temperature growth resulted in higher level of active than constant 37°C. pGroESL‐ E and pTF‐S resulted in highest increase in amount of soluble rPEP as compared to amount found. Expression in presence of pGroESL‐E and pTF‐S resulted in the highest levels of active soluble enzyme. P. aeruginosa GroESL and TF chaperones co‐express and assist in folding successfully expression of a large insect PEP in E. coli.Funding: Office of Research and Sponsored Projects, Stephen F Austin State University; Ed and Gwen Cole, Nacogdoches, TX.
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