Cloning, characterization, expression analysis and inhibition studies of a novel gene encoding Bowman-Birk type protease inhibitor from rice bean.

This paper presents the first study describing the isolation, cloning and characterization of a full length gene encoding Bowman-Birk protease inhibitor (RbTI) from rice bean (Vigna umbellata). A full-length protease inhibitor gene with complete open reading frame of 327 bp encoding 109 amino acids was cloned from rice bean seeds using degenerate primer set. BlastP search revealed that the RbTI encoded amino acid of approx 13.0 kDa and shared 99% homology each with BBI from Phaseolus parvulus, Vigna trilobata and Vigna vexilata. Phylogenetic tree also showed close relationship of RbTI with BBI from other members of Leguminaceae family. RbTI gene was further confirmed as intronless (GenBank accession no. KJ159908). The secondary and 3D-structural models for the RbTI were predicted with homology modeling. qRT-PCR studies revealed the highest RbTI expression in the seeds nearing maturity, whereas the low expression of the gene was noticed in young leaves. The isolated RbTI was successfully expressed in Escherichiacoli and the highest expression was recorded after 5.5h of induction. Study on the inhibitory activity of expressed protein against the gut proteases of Hessian fly larvae revealed 87% inhibition. The novel RbTI gene will further broaden the pool of plant defense genes and could be an ideal choice for developing transgenic crops resistant to insect pests with high economic value. In addition, it has the potential to be used as a probe for selection of insect- and pathogen-resistant genotypes.

Z α1-antitrypsin confers a proinflammatory phenotype that contributes to chronic obstructive pulmonary disease.

RATIONALE: Severe α1-antitrypsin deficiency caused by the Z variant (Glu342Lys; ZZ-AT) is a well-known genetic cause for emphysema. Although severe lack of antiproteinase protection is the critical etiologic factor for ZZ-AT-associated chronic obstructive pulmonary disease (COPD), some reports have suggested enhanced lung inflammation as a factor in ZZ-AT homozygotes. OBJECTIVES: To provide molecular characterization of inflammation in ZZ-AT. METHODS: Inflammatory cell and cytokine profile (nuclear factor-κB, IL-6, tumor necrosis factor-α), intracellular polymerization of Z-AT, and endoplasmic reticulum (ER) stress markers (protein kinase RNA-like ER kinase, activator transcription factor 4) were assessed in transgenic mice and transfected cells in response to cigarette smoke, and in explanted lungs from ZZ and MM individuals with severe COPD. MEASUREMENTS AND MAIN RESULTS: Compared with M-AT, transgenic Z-AT mice lungs exposed to cigarette smoke had higher levels of pulmonary cytokines, neutrophils, and macrophages and an exaggerated ER stress. Similarly, the ER overload response was greater in lungs from ZZ-AT homozygotes with COPD, and was particularly found in pulmonary epithelial cells. Cigarette smoke increased intracellular Z-AT polymers, ER overload response, and proinflammatory cytokine release in Z-AT-expressing pulmonary epithelial cells, which could be prevented with an inhibitor of polymerization, an antioxidant, and an inhibitor of protein kinase RNA-like ER kinase. CONCLUSIONS: We show here that aggregation of intracellular mutant Z-AT invokes a specific deleterious cellular inflammatory phenotype in COPD. Oxidant-induced intracellular polymerization of Z-AT in epithelial cells causes ER stress, and promotes excess cytokine and cellular inflammation. This pathway is likely to contribute to the development of COPD in ZZ-AT homozygotes, and therefore merits further investigation.

Cloning, expression and mutational studies of a trypsin inhibitor that retains activity even after cyanogen bromide digestion.

A winged bean trypsin inhibitor (WbTI-2) of molecular mass ∼20kDa, has been cloned and expressed in Escherichiacoli with full activity like the one from seed protein. It completely inhibits trypsin at an enzyme:inhibitor molar ratio of 1:2. PCR with cDNA and genomic DNA using same primers produced about 550 base pair product, which indicated it to be an intronless gene. Through site-directed mutagenesis, the Arg64 has been confirmed as the P1 residue. For the presence of five methionine residues in WbTI-2, cyanogen bromide (CNBr) digestion was carried out. Out of three fragments the one (about 65% of original size) containing the reactive site loop retained 50% activity.

Molecular characterization, expression and function analysis of a five-domain Kazal-type serine proteinase inhibitor from pearl oyster Pinctada fucata.

Serine proteinase inhibitors represent an expanding superfamily of endogenous inhibitors that are regulate proteolytic events and involved in a variety of physiological and immunological processes. A five-domain Kazal-type serine proteinase inhibitor (poKSPI) was identified and characterized from pearl oyster Pinctada fucata based on expressed sequence tag (EST) analysis. The full-length cDNA was 737 bp with an open reading frame (ORF) 660 bp encoding a 219 amino acid protein a theoretical molecular weight (Mw) of 23.3 kDa and an isoelectric point (pI) of 8.40. A putative signal peptide of 19 amino acid residues and five tandem Kazal domains were identified. Four of the Kazal domains had the highly conserved motif sequences with six cysteine residues responsible for the formation of disulfide bridges. The deduced amino acid sequence of the poKSPI shared high homology with KSPIs from Hirudo medicinalis. The poKSPI mRNA could be detected in all examined tissues, the expression level of the poKSPI mRNA was the highest in mantle and gonad, while the lowest in haemocyte and intestine. After LPS challenge, the expression level of the poKSPI mRNA in digestive gland was significantly up-regulated at 4 h post-challenge and reached the peak at 12 h post-challenge, which was 4.23-fold higher than control group; the expression level of the poKSPI mRNA in gill was also significantly up-regulated at 8 and 12 h post-challenge, which were 4.48 and 2.26-fold higher than control group. After Vibrio alginolyticus challenge, the expression levels of the poKSPI mRNA in digestive gland were significantly up-regulated at 8, 12, 48 and 72 h post-challenge, which were 1.70, 1.79, 3.89 and 5.69-fold higher than control group, respectively; the expression level of the poKSPI mRNA in gill was significantly up-regulated at 24 h post-challenge, which was 5.30-fold higher than control group. The recombinant poKSPI protein could inhibit chymotrypsin and trypsin activities in dose-dependent manner, when the ratios of rpoKSPI to chymotrypsin and trypsin were 36:1 and 72:1, respectively, the proteinase activities of chymotrypsin and trypsin could be almost completely inhibited, but the rpoKSPI could not inhibit subtilisin.

Reconstructed mung bean trypsin inhibitor targeting cell surface GRP78 induces apoptosis and inhibits tumor growth in colorectal cancer.

Glucose regulated protein 78 (GRP78) has been reported to be present on cell membranes of cancer cells but not the normal cells, serving as a potential anti-cancer target. In the present study, a fusion protein containing the GRP78 binding peptide WIFPWIQL and the active fragment of mung bean trypsin inhibitor was constructed, and its targeted anti-tumor effects were investigated both in vitro and in vivo. The results showed that the fusion protein specifically inhibited growth and induced apoptosis in colorectal cancer cells but not in the normal cells. Mechanistically, these anti-tumor effects were attributed to induction of G1 phase arrest and activation of multiple apoptotic pathways. Importantly, the fusion protein could also suppress the growth of xenografted human colorectal carcinoma in vivo. Our study reveals that this fusion protein may be developed as a therapeutic agent for treatment of colon cancer, and holds important implications for developing other anti-cancer peptide drugs.

Modified Puestow procedure for the management of chronic pancreatitis in children.

PURPOSE: To present our experience with the modified Puestow procedure in the management of children with chronic pancreatitis. METHODS: Retrospective chart review of patients treated between 2003 and 2012. RESULTS: Six patients underwent a modified Puestow procedure (lateral pancreaticojejunostomy) for the management of chronic pancreatitis, three females and three males. Four patients had hereditary pancreatitis (three with confirmed N34S mutation in the SPINK1 gene), one patient had chronic pancreatitis of unknown etiology, and one patient with annular pancreas developed obstructive chronic pancreatitis. The pancreatic duct was dilated in all cases, with a maximum diameter of 5 to 10mm. Median time between onset of pain and surgery was 4 years (range: 1-9). Median age at surgery was 7.5 years (range: 5-15). Median hospital stay was 12 days (range: 9-28). Median follow up was 4.5 years (range: 5 months to 9 years). All patients had temporary postoperative improvement of their abdominal pain. In two patients the pain recurred at 6 months and 2 years postoperatively and eventually required total pancreatectomy to treat intractable pain, 3 and 8 years after surgery. Two patients were pain free for two years and subsequently developed occasional episodes of pain. The two most recent patients are pain free at 1 year (obstructive chronic pancreatitis) and 5 months (hereditary pancreatitis) follow-up. Two patients developed type I diabetes mellitus 10 and 12 months postoperatively (one with hereditary and one with idiopathic chronic pancreatitis). CONCLUSION: We conclude that the modified Puestow procedure in children is feasible and safe. It seems to provide definitive pain control and prevent further damage to the pancreas in patients with obstructive chronic pancreatitis. However, in patients with hereditary pancreatitis, pain control outcomes are variable and the operation may not abrogate the progression of disease to pancreatic insufficiency.

The trypsin inhibitor panulirin regulates the prophenoloxidase-activating system in the spiny lobster Panulirus argus.

The melanization reaction promoted by the prophenoloxidase-activating system is an essential defense response in invertebrates subjected to regulatory mechanisms that are still not fully understood. We report here the finding and characterization of a novel trypsin inhibitor, named panulirin, isolated from the hemocytes of the spiny lobster Panulirus argus with regulatory functions on the melanization cascade. Panulirin is a cationic peptide (pI 9.5) composed of 48 amino acid residues (5.3 kDa), with six cysteine residues forming disulfide bridges. Its primary sequence was determined by combining Edman degradation/N-terminal sequencing and electrospray ionization-MS/MS spectrometry. The low amino acid sequence similarity with known proteins indicates that it represents a new family of peptidase inhibitors. Panulirin is a competitive and reversible tight-binding inhibitor of trypsin (Ki = 8.6 nm) with a notable specificity because it does not inhibit serine peptidases such as subtilisin, elastase, chymotrypsin, thrombin, and plasmin. The removal of panulirin from the lobster hemocyte lysate leads to an increase in phenoloxidase response to LPS. Likewise, the addition of increasing concentrations of panulirin to a lobster hemocyte lysate, previously depleted of trypsin-inhibitory activity, decreased the phenoloxidase response to LPS in a concentration-dependent fashion. These results indicate that panulirin is implicated in the regulation of the melanization cascade in P. argus by inhibiting peptidase(s) in the pathway toward the activation of the prophenoloxidase enzyme.

Buckwheat trypsin inhibitor enters Hep G2 cells by clathrin-dependent endocytosis.

Recombinant buckwheat trypsin inhibitor (rBTI) was studied to evaluate if it could enter cancer cells and to determine the mechanism. Fluorescein isothiocyanate-labelled buckwheat trypsin inhibitor (FITC-BTI) entered Hep G2 cells in a concentration-dependent manner. FITC-BTI colocalised with labelled transferrin (Tf) in the punctate structure, implying that rBTI enters Hep G2 cells by clathrin-dependent endocytosis. Incubation of Hep G2 cells with different chemical inhibitors abolished diffuse, but not punctate fluorescence, thus indicating that membrane potential plays a critical role in this process. Impairment of clathrin-mediated endocytosis by RNAi with clathrin heavy chain greatly reduced or completely abolished both diffuse and punctate fluorescence, further supporting a theory of a single route of endocytosis. Consistent with our working hypothesis, Hep G2 cells which were arrested in the M phase did not show any vesicular or diffuse FITC-BTI. We conclude from these results that both endocytosis and membrane potential are required for rBTI entry into Hep G2 cells.

Probing the physical determinants of thermal expansion of folded proteins.

The magnitude and sign of the volume change upon protein unfolding are strongly dependent on temperature. This temperature dependence reflects differences in the thermal expansivity of the folded and unfolded states. The factors that determine protein molar expansivities and the large differences in thermal expansivity for proteins of similar molar volume are not well understood. Model compound studies have suggested that a major contribution is made by differences in the molar volume of water molecules as they transfer from the protein surface to the bulk upon heating. The expansion of internal solvent-excluded voids upon heating is another possible contributing factor. Here, the contribution from hydration density to the molar thermal expansivity of a protein was examined by comparing bovine pancreatic trypsin inhibitor and variants with alanine substitutions at or near the protein-water interface. Variants of two of these proteins with an additional mutation that unfolded them under native conditions were also examined. A modest decrease in thermal expansivity was observed in both the folded and unfolded states for the alanine variants compared with the parent protein, revealing that large changes can be made to the external polarity of a protein without causing large ensuing changes in thermal expansivity. This modest effect is not surprising, given the small molar volume of the alanine residue. Contributions of the expansion of the internal void volume were probed by measuring the thermal expansion for cavity-containing variants of a highly stable form of staphylococcal nuclease. Significantly larger (2-3-fold) molar expansivities were found for these cavity-containing proteins relative to the reference protein. Taken together, these results suggest that a key determinant of the thermal expansivities of folded proteins lies in the expansion of internal solvent-excluded voids.

Neonicotinoid insecticides alter induced defenses and increase susceptibility to spider mites in distantly related crop plants.

BACKGROUND: Chemical suppression of arthropod herbivores is the most common approach to plant protection. Insecticides, however, can cause unintended, adverse consequences for non-target organisms. Previous studies focused on the effects of pesticides on target and non-target pests, predatory arthropods, and concomitant ecological disruptions. Little research, however, has focused on the direct effects of insecticides on plants. Here we demonstrate that applications of neonicotinoid insecticides, one of the most important insecticide classes worldwide, suppress expression of important plant defense genes, alter levels of phytohormones involved in plant defense, and decrease plant resistance to unsusceptible herbivores, spider mites Tetranychus urticae (Acari: Tetranychidae), in multiple, distantly related crop plants. METHODOLOGY/PRINCIPAL FINDINGS: Using cotton (Gossypium hirsutum), corn (Zea mays) and tomato (Solanum lycopersicum) plants, we show that transcription of phenylalanine ammonia lyase, coenzyme A ligase, trypsin protease inhibitor and chitinase are suppressed and concentrations of the phytohormone OPDA and salicylic acid were altered by neonicotinoid insecticides. Consequently, the population growth of spider mites increased from 30% to over 100% on neonicotinoid-treated plants in the greenhouse and by nearly 200% in the field experiment. CONCLUSIONS/SIGNIFICANCE: Our findings are important because applications of neonicotinoid insecticides have been associated with outbreaks of spider mites in several unrelated plant species. More importantly, this is the first study to document insecticide-mediated disruption of plant defenses and link it to increased population growth of a non-target herbivore. This study adds to growing evidence that bioactive agrochemicals can have unanticipated ecological effects and suggests that the direct effects of insecticides on plant defenses should be considered when the ecological costs of insecticides are evaluated.

Genomic and structural characterization of Kunitz-type peptide LmKTT-1a highlights diversity and evolution of scorpion potassium channel toxins.

BACKGROUND: Recently, a new subfamily of long-chain toxins with a Kunitz-type fold was found in scorpion venom glands. Functionally, these toxins inhibit protease activity and block potassium channels. However, the genomic organization and three-dimensional (3-D) structure of this kind of scorpion toxin has not been reported. PRINCIPAL FINDINGS: Here, we characterized the genomic organization and 3-D nuclear magnetic resonance structure of the scorpion Kunitz-type toxin, LmKTT-1a, which has a unique cysteine pattern. The LmKTT-1a gene contained three exons, which were interrupted by two introns located in the mature peptide region. Despite little similarity to other Kunitz-type toxins and a unique pattern of disulfide bridges, LmKTT-1a possessed a conserved Kunitz-type structural fold with one α-helix and two ß-sheets. Comparison of the genomic organization, 3-D structure, and functional data of known toxins from the α-KTx, ß-KTx, γ-KTx, and κ-KTx subfamily suggested that scorpion Kunitz-type potassium channel toxins might have evolved from a new ancestor that is completely different from the common ancestor of scorpion toxins with a CSα/ß fold. Thus, these analyses provide evidence of a new scorpion potassium channel toxin subfamily, which we have named δ-KTx. CONCLUSIONS/SIGNIFICANCE: Our results highlight the genomic, structural, and evolutionary diversity of scorpion potassium channel toxins. These findings may accelerate the design and development of diagnostic and therapeutic peptide agents for human potassium channelopathies.

Ovalbumin-related protein X is a heparin-binding ov-serpin exhibiting antimicrobial activities.

Ovalbumin family contains three proteins with high sequence similarity: ovalbumin, ovalbumin-related protein Y (OVAY), and ovalbumin-related protein X (OVAX). Ovalbumin is the major egg white protein with still undefined function, whereas the biological activity of OVAX and OVAY has not yet been explored. Similar to ovalbumin and OVAY, OVAX belongs to the ovalbumin serine protease inhibitor family (ov-serpin). We show that OVAX is specifically expressed by the magnum tissue, which is responsible for egg white formation. OVAX is also the main heparin-binding protein of egg white. This glycoprotein with a predicted reactive site at Lys(367)-His(368) is not able to inhibit trypsin, plasmin, or cathepsin G with or without heparin as a cofactor. Secondary structure of OVAX is similar to that of ovalbumin, but the three-dimensional model of OVAX reveals the presence of a cluster of exposed positive charges, which potentially explains the affinity of this ov-serpin for heparin, as opposed to ovalbumin. Interestingly, OVAX, unlike ovalbumin, displays antibacterial activities against both Listeria monocytogenes and Salmonella enterica sv. Enteritidis. These properties partly involve heparin-binding site(s) of the molecule as the presence of heparin reverses its anti-Salmonella but not its anti-Listeria potential. Altogether, these results suggest that OVAX and ovalbumin, although highly similar in sequence, have peculiar sequential and/or structural features that are likely to impact their respective biological functions.

A spider-derived Kunitz-type serine protease inhibitor that acts as a plasmin inhibitor and an elastase inhibitor.

Kunitz-type serine protease inhibitors are involved in various physiological processes, such as ion channel blocking, blood coagulation, fibrinolysis, and inflammation. While spider-derived Kunitz-type proteins show activity in trypsin or chymotrypsin inhibition and K(+) channel blocking, no additional role for these proteins has been elucidated. In this study, we identified the first spider (Araneus ventricosus) Kunitz-type serine protease inhibitor (AvKTI) that acts as a plasmin inhibitor and an elastase inhibitor. AvKTI possesses a Kunitz domain consisting of a 57-amino-acid mature peptide that displays features consistent with Kunitz-type inhibitors, including six conserved cysteine residues and a P1 lysine residue. Recombinant AvKTI, expressed in baculovirus-infected insect cells, showed a dual inhibitory activity against trypsin (K(i) 7.34 nM) and chymotrypsin (K(i) 37.75 nM), defining a role for AvKTI as a spider-derived Kunitz-type serine protease inhibitor. Additionally, AvKTI showed no detectable inhibitory effects on factor Xa, thrombin, or tissue plasminogen activator; however, AvKTI inhibited plasmin (K(i) 4.89 nM) and neutrophil elastase (K(i) 169.07 nM), indicating that it acts as an antifibrinolytic factor and an antielastolytic factor. These findings constitute molecular evidence that AvKTI acts as a plasmin inhibitor and an elastase inhibitor and also provide a novel view of the functions of a spider-derived Kunitz-type serine protease inhibitor.

Trypsin and chymotrypsin inhibitor peptides from the venom of Chinese Daboia russellii siamensis.

Two trypsin inhibitors and one chymotrypsin inhibitor from Chinese Daboia russellii siamensis venom, denoted as CBPTI-1, CBPTI-2 and CBPTI-3 were purified, characterized and cloned from lyophilized venom-derived cDNA libraries. The N-terminus of CBPTI-1 was modified and not amenable to Edman degradation sequencing, however an internal partial sequence was found to be SGRCRGHLRRIYYNPDSNKCE. The N-termini of CBPTI-2 and CBPTI-3 were unmodified and their partial sequences were established as HDRPTFCNLAPESGRCRAH and HDRPKFCYLPADPGECMAYIRSFYYDS respectively. From cloning studies CBPTI-1 was found to consist of 66 amino acid residues, while CBPTI-2 and CBPTI-3 precursors consist of 60 amino acid residues, including 6 cysteine residues. Another cDNA sequence (CBPTI-4) was also obtained. Alignment of cDNA sequences showed that CBPTI-3 exhibited similar sequence homology to CBPTI-4 cDNA except for an 8 nucleotide deletion in the open-reading frame. CBPTI-1 and CBPTI-2 were demonstrated to be potent trypsin inhibitors, but were also shown to be effectively potent in chymotrypsin inhibition. The K(i) values of CBPTI-1 and CBPTI-2 for trypsin inhibition were 4.07 × 10(-7) M and 6.66 × 10(-7) M, respectively, and they were non-competitive in their activity. CBPTI-3 showed chymotrypsin inhibition activity with a K(i) value of 2.55 × 10(-9) M, but did not show trypsin inhibitor activity.

OsNPR1 negatively regulates herbivore-induced JA and ethylene signaling and plant resistance to a chewing herbivore in rice.

NPR1 (a non-expressor of pathogenesis-related genes1) has been reported to play an important role in plant defense by regulating signaling pathways. However, little to nothing is known about its function in herbivore-induced defense in monocot plants. Here, using suppressive substrate hybridization, we identified a NPR1 gene from rice, OsNPR1, and found that its expression levels were upregulated in response to infestation by the rice striped stem borer (SSB) Chilo suppressalis and rice leaf folder (LF) Cnaphalocrocis medinalis, and to mechanical wounding and treatment with jasmonic acid (JA) and salicylic acid (SA). Moreover, mechanical wounding induced the expression of OsNPR1 quickly, whereas herbivore infestation induced the gene more slowly. The antisense expression of OsNPR1 (as-npr1), which reduced the expression of the gene by 50%, increased elicited levels of JA and ethylene (ET) as well as of expression of a lipoxygenase gene OsHI-LOX and an ACC synthase gene OsACS2. The enhanced JA and ET signaling in as-npr1 plants increased the levels of herbivore-induced trypsin proteinase inhibitors (TrypPIs) and volatiles, and reduced the performance of SSB. Our results suggest that OsNPR1 is an early responding gene in herbivore-induced defense and that plants can use it to activate a specific and appropriate defense response against invaders by modulating signaling pathways.

Structural-functional insights of single and multi-domain Capsicum annuum protease inhibitors.

Pin-II protease inhibitors (PIs) are the focus of research interest because of their large structural-functional diversity and relevance in plant defense. Two representative Capsicum annuum PI genes (CanPI-15 and -7) comprising one and four inhibitory repeat domains, respectively, were expressed and recombinant proteins were characterized. ß-Sheet and unordered structure was found predominant in CanPI-15 while -7 also displayed the signatures of polyproline fold, as revealed by circular dichroism studies. Inhibition kinetics against bovine trypsin indicated three times higher potency of CanPI-7 (K(i)~57 µM) than -15 (~184 µM). Activity and structural stability of these CanPIs were revealed under various conditions of pH, temperature and denaturing agent. Structure prediction, docking studies with proteases and mass spectroscopy revealed the organization of multiple reactive site loops of multi domain PIs in space as well as the steric hindrances imposed while binding to proteases due to their close proximity.

Wheat amylase trypsin inhibitors drive intestinal inflammation via activation of toll-like receptor 4.

Ingestion of wheat, barley, or rye triggers small intestinal inflammation in patients with celiac disease. Specifically, the storage proteins of these cereals (gluten) elicit an adaptive Th1-mediated immune response in individuals carrying HLA-DQ2 or HLA-DQ8 as major genetic predisposition. This well-defined role of adaptive immunity contrasts with an ill-defined component of innate immunity in celiac disease. We identify the α-amylase/trypsin inhibitors (ATIs) CM3 and 0.19, pest resistance molecules in wheat, as strong activators of innate immune responses in monocytes, macrophages, and dendritic cells. ATIs engage the TLR4-MD2-CD14 complex and lead to up-regulation of maturation markers and elicit release of proinflammatory cytokines in cells from celiac and nonceliac patients and in celiac patients' biopsies. Mice deficient in TLR4 or TLR4 signaling are protected from intestinal and systemic immune responses upon oral challenge with ATIs. These findings define cereal ATIs as novel contributors to celiac disease. Moreover, ATIs may fuel inflammation and immune reactions in other intestinal and nonintestinal immune disorders.

Molecular cloning and insecticidal effect of Inga laurina trypsin inhibitor on Diatraea saccharalis and Heliothis virescens.

Native Inga laurina (Fabaceae) trypsin inhibitor (ILTI) was tested for anti-insect activity against Diatraea saccharalis and Heliothis virescens larvae. The addition of 0.1% ILTI to the diet of D. saccharalis did not alter larval survival but decreased larval weight by 51%. The H. virescens larvae that were fed a diet containing 0.5% ILTI showed an 84% decrease in weight. ILTI was not digested by the midgut proteinases of either species of larvae. The trypsin levels were reduced by 55.3% in the feces of D. saccharalis and increased by 24.1% in the feces of H. virescens. The trypsin activity in both species fed with ILTI was sensitive to the inhibitor, suggesting that no novel proteinase resistant to ILTI was induced. Additionally, ILTI exhibited inhibitory activity against the proteinases present in the larval midgut of different species of Lepidoptera. The organization of the ilti gene was elucidated by analyzing its corresponding genomic sequence. The recombinant ILTI protein (reILTI) was expressed and purified, and its efficacy was evaluated. Both native ILTI and reILTI exhibited a similar strong inhibitory effect on bovine trypsin activity. These results suggest that ILTI presents insecticidal properties against both insects and may thus be a useful tool in the genetic engineering of plants.

[Large-scale purification and acute toxicity of cowpea trypsin inhibitor].

OBJECTIVE: To provide the acute toxicity data of cowpea trypsin inhibitor (CpTI) using recombinant protein purified from E. coli. METHODS: Recombinant CpTI protein was expressed and purified from E. coli. Bacterial recombinant plasmid was transformed into E. coli and the transformed cells were induced with IPTG. The expressed CpTI protein was purified by hydrophobic interaction chromatography and anion exchange chromatography. Sixty mice, randomly assigned to 6 groups, were administrated 10.0, 4.64, 2.15 and 1.00 g/kg BW of CpTI or 5.00 g/kg BW of BSA control protein or sterile water respectively by oral gavage. RESULTS: All animals survived with no significant change in body weight and food consumption throughout the study. Macroscopic necropsy examination on day 15 revealed no gross pathological lesions in any of the animals. The maximum tolerated dose (MTD) of CpTI was more than 10.0 g/kg body weight in mice. CONCLUSION: No toxicity of CpTI protein was found in ICR mice model.