## 4 Conclusions  
The primary focus of the present study includes obtaining the complete sequence of the PEP cDNA, cloning and expression of a recombinant PEP. The initial experiments performed for the isolation of total RNA, enrichment of mRNA and cDNA synthesis suggested that mRNA coding for the sum pest PEP is considerably low or nil in the nymphs and overwintering adults. Lack of PEP in the overwintering adults was expected, as the overwintered adults do not feed. The nymphs feed on grains, so it was expected to find some levels of PEP cDNA present; however, none of the PCR reactions using the nymph cDNA resulted in any amplicons using any of the primers. The spPEP enzyme appears to be expressed in high amounts only in the actively feeding adults.  
The sequence results showed the spPEP to be at most 56% identical to other known PEP amino acid sequences. The spPEP amino acid sequence was most similar to the _Daphnia pulex_, as evidenced on the phylogenetic tree, and least similar to the bacterial PEPs, which were the most divergent. The mammalian PEPs were separated by several nodes on the tree from the spPEP, but were included in the top 25 hits obtained from BLASTX. The presence of a 5' UTR might be important in regulation of protein expression and is being further investigated. Upon translation of the ORF sequence, the highly conserved catalytic triad SDH is present, indicating that the sum pest PEP might act similarly to other PEPs. Homology modeling showed that the spPEP could accommodate a tertiary structure almost identical to the porcine brain PEP known to hydrolyze oligopeptides shorter than 30 amino acids in length. The reason for the ability of the spPEP to cleave high molecular weight peptides, such as the glutenins and gliadins, still remains unexplained. The recombinant enzyme shows recognition of both the GPpNA peptide and its natural substrates, the glutenins and gliadins. Now that active recombinant spPEP can be expressed and purified, investigations to identify inhibitors specific for the spPEP can begin. Additionally, the enzyme can be purified in large enough amounts to determine the three-dimensional structure of the enzyme in  
Figure 8: SDS PAGE analysis of the digestion of total gluten by spPEP. spPEP (60 \(\upmu\)g) was incubated with 400 \(\upmu\)g of total gluten for a total of 90 min at 37 \({}^{\circ}\)C. Every 15 min, a volume of the reaction that would contain 50 \(\upmu\)g of gluten was removed, added to 6\(\times\) Laemmli sample buffer, heated and loaded onto the gel. The molecular weight marker was the Kaleidoscope marker from Bio-Rad Laboratories.  
the presence of inhibitors, peptides and its natural substrates, the glutens. Future studies are underway to investigate how this PEP can be so similar in catalytic activity of short peptides as other PEPs, yet be so unique in recognizing gluten proteins ranging in molecular weights up to 140 kD.  
We would like to acknowledge USAID linkage funds from the International Center for Agricultural Research in the Dry Areas, the Office of Sponsored Research, Stephen F. Austin State University, Nacogdoches, TX, and [PERSON] for funding this work.  
[PERSON] contributed all of the bugs for this work and provided access to his laboratory at the International Center for Agricultural Research in the Dry Areas for [PERSON] to carry out all of the RNA work. All other authors contributed to this work as part of their Master of Science in Biotechnology degree at Stephen F. Austin State University, Nacogdoches, TX, in the laboratory of [PERSON].  
The authors declare no conflict of interest.