title: Cloning, Expression, Sequence Analysis and Homology Modeling of the Prolyl Endoprotease from Eurygaster integriceps Puton.
abstract: 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.
authors.affiliation: Department of Biology and Biotechnology, Stephen F. Austin State University, P.O. Box 13003, SFA Station, Nacogdoches, TX 75962, USA. yendamuri.ravi@gmail.com.
authors.forename: Ravi Chandra
authors.initials: RC
authors.lastname: Yandamuri
authors.affiliation: Department of Biology and Biotechnology, Stephen F. Austin State University, P.O. Box 13003, SFA Station, Nacogdoches, TX 75962, USA. ranjeeta_gautam2000@yahoo.com.
authors.forename: Ranjeeta
authors.initials: R
authors.lastname: Gautam
authors.affiliation: University of Texas Health Science Center, School of Public Health, Center for Infectious Diseases, 1200 Hermann Pressler Dr., RAS E715, Houston, TX 77030, USA. cdarkoh@hotmail.com.
authors.forename: Charles
authors.initials: C
authors.lastname: Darkoh
authors.affiliation: Department of Biology and Biotechnology, Stephen F. Austin State University, P.O. Box 13003, SFA Station, Nacogdoches, TX 75962, USA. vanithareddy006@yahoo.com.
authors.forename: Vanitha
authors.initials: V
authors.lastname: Dareddy
authors.affiliation: Biodiversity and Integrated Gene Management Program, International Center for Agricultural Research in the Dry Areas (ICARDA), Rabat Office P.O. Box 6299 Rabat-Instituts, Rabat, Morocco. M.BOHSSINI@CGIAR.ORG.
authors.forename: Mustapha
authors.initials: M
authors.lastname: El-Bouhssini
authors.affiliation: Department of Biology and Biotechnology, Stephen F. Austin State University, P.O. Box 13003, SFA Station, Nacogdoches, TX 75962, USA. bclack@sfasu.edu.
authors.forename: Beatrice A
authors.initials: BA
authors.lastname: Clack
keywords: Hemiptera; Scutelleridae; cDNA; gluten; homology modeling; insect; prolyl endoprotease; serine protease; wheat