Use of Recombinent Casein Peptides as Inhibitors of spPEP

Abstract

Eurygaster integreceps (Sunn pest) causes major damage in wheat grains by injecting their saliva into the grain degrading the gluten proteins. This saliva protein has been identified and characterized as a prolyl endoprotease (spPEP) in our lab (Darkoh et al, 2010). A natural, biological friendly inhibitor to the spPEP is being sought. Previous studies have shown that casein peptides generated by Lactobacillis species have inhibitory effects on mammalian and bacterial prolyl endoproteases (PEP). The Objective of this study was to clone the four bovine casein genes, α1, α2, β and κ for use in expressing each isomer to identify which peptides inhibit the spPEP. The nucleotide sequences corresponding to bovine casein isomers were identified from the NCBI database to design gene specific primers that would amplify the full length casein proteins. Total RNA was extracted from bovine mammary gland biopsy tissue kindly donated by Dr. Erin Connor, Research Leader, USDA‐ARS, Beltsville, MD and subsequent cDNA has been synthesized. Each casein subunit was cloned into the pGEM T‐Easy vector for sequencing. Verified sequences were cloned into the pET15b E.coli expression vector for protein expression and purification using IMAC chromatography. Purified proteins will be used for subsequent incubation with L. helveticus at varying times. The resulting peptide mixture will be incubated with the spPEP in the presence of the GPpNA substrate to determine which isomer/s contains the spPEP inhibitory sequences.Funding Sources: Office of Research and Sponsored Projects, Stephen F. Austin State University, Nacogdoches, TX and Ed and Gwen Cole, Nacogdoches, TX.