02308nas a2200241 4500008004100000022001400041245015900055210006900214260007900283300001000362490000700372520137500379653002001754653001901774653001401793653001401807653001901821100002501840700003401865700002301899700001401922856013001936 2010 eng d a0196-978100aMolecular cloning, over expression, and activity studies of a peptidic HIV-1 protease inhibitor: designed synthetic gene to functional recombinant peptide0 aMolecular cloning over expression and activity studies of a pept a360 PARK AVE SOUTH, NEW YORK, NY 10010-1710 USAbELSEVIER SCIENCE INCcJAN a16-210 v313 a

The aspartic protease inhibitor (ATBI) purified from a Bacillus sp. is a potent inhibitor of several proteases including recombinant HIV-1 protease, pepsin, and fungal aspartic protease. In this study, we report the cloning, and over expression of a synthetic gene coding for ATBI in Escherichia coli and establish a purification Protocol. The ATBI molecule consists of eleven amino acids and is peptidic in nature. We used the peptide sequence data of ATBI to synthesize complementary oligonucleotides, which were annealed and subsequently cloned in-frame with the gene for glutathione-S-transferase (GST). The expression of the resulting fusion protein was induced in E. coli BL21-A1 cells using arabinose. The recombinant peptide was Purified using a reduced glutathione column, and cleaved with Factor Xa to remove the GST tag. The resultant product was further purified to homogeneity using RP-HPLC. Mass spectroscopy analysis revealed that the purified peptide had a molecular weight of 1186 Da which matches the theoretical molecular weight of the amino acids present in the synthetic gene. The recombinant peptide was found to be active in vitro against HIV-1 protease, pepsin, and fungal aspartic protease. The protocol described in this study may be used to clone pharmaceutically important peptide molecules. (C) 2009 Elsevier Inc. All rights reserved.

10aGateway cloning10aHIV-1 protease10ainhibitor10aProteases10aSynthetic gene1 aVathipadiekal, Vinod1 aUmasankar, Perunthottathu, K.1 aPatole, Milind, S.1 aRao, Mala uhttps://library.ncl.res.in/content/molecular-cloning-over-expression-and-activity-studies-peptidic-hiv-1-protease-inhibitor-002341nas a2200265 4500008004100000022001400041245012500055210006900180260005800249300000700307490000700314520143700321653001301758653002301771653001201794653001401806100001901820700002001839700001901859700001901878700001401897700002101911700002001932856012301952 2012 eng d a1536-244200aDevelopmental and digestive flexibilities in the midgut of a polyphagous pest, the cotton bollworm, helicoverpa armigera0 aDevelopmental and digestive flexibilities in the midgut of a pol aLIBRARY C327, TUCSON, AZ 85721 USAbUNIV ARIZONAcMAR a420 v123 a

Developmental patterns and survival of the cotton bollworm, Helicoverpa armigera Hubner (Lepidoptera: Noctuidae), a polyphagous insect pest, have been studied with reference to the effect of diet on major gut digestive enzymes (amylases, proteases, and lipases). Significant correlations between nutritional quality of the diet and larval and pupal mass were observed when H. armigera larvae were fed on various host plants viz. legumes (chickpea and pigeonpea), vegetables (tomato and okra), flowers (rose and marigold), and cereals (sorghum and maize). Larvae fed on diets rich in proteins and/or carbohydrates (pigeonpea, chickpea, maize, and sorghum) showed higher larval mass and developed more rapidly than larvae fed on diets with low protein and carbohydrate content (rose, marigold, okra, and tomato). Low calorific value diets like rose and marigold resulted in higher mortality (25-35%) of H. armigera. Even with highly varying development efficiency and larval/pupal survival rates, H. armigera populations feeding on different diets completed their life cycles. Digestive enzymes of H. armigera displayed variable expression levels and were found to be regulated on the basis of macromolecular composition of the diet. Post-ingestive adaptations operating at the gut level, in the form of controlled release of digestive enzymes, might be a key factor contributing to the physiological plasticity in H. armigera.

10aamylases10alarval performance10aLipases10aProteases1 aSarate, P., J.1 aTamhane, V., A.1 aKotkar, H., M.1 aRatnakaran, N.1 aSusan, N.1 aGupta, Vidya, S.1 aGiri, Ashok, P. uhttps://library.ncl.res.in/content/developmental-and-digestive-flexibilities-midgut-polyphagous-pest-cotton-bollworm-102205nas a2200229 4500008004100000022001400041245014400055210006900199260010600268300001000374490000800384520127700392653002001669653002501689653002901714653001401743653002601757100002201783700002101805700002001826856012901846 2014 eng d a0031-942200aDifferential antibiosis against helicoverpa armigera exerted by distinct inhibitory repeat domains of capsicum annuum proteinase inhibitors0 aDifferential antibiosis against helicoverpa armigera exerted by  aTHE BOULEVARD, LANGFORD LANE, KIDLINGTON, OXFORD OX5 1GB, ENGLANDbPERGAMON-ELSEVIER SCIENCE LTDcMAY a16-220 v1013 a

Plant defensive serine proteinase inhibitors (Pis) are known to have negative impact on digestive physiology of herbivore insects and thus have a crucial role in plant protection. Here, we have assessed the efficacy and specificity of three previously characterized inhibitory repeat domain (IRD) variants from Capsicum annuum PIs viz., IRD-7, -9 and -12 against gut proteinases from Helicoverpa armigera. Comparative study of in silico binding energy revealed that IRD-9 possesses higher affinity towards H. armigera serine proteinases as compared to IRD-7 and -12. H. armigera fed on artificial diet containing 5 TIU/g of recombinant IRD proteins exhibited differential effects on larval growth, survival rate and other nutritional parameters. Major digestive gut trypsin and chymotrypsin genes were down regulated in the IRD fed larvae, while few of them were up-regulated, this indicate alterations in insect digestive physiology. The results corroborated with proteinase activity assays and zymography. These findings suggest that the sequence variations among Pis reflect in their efficacy against proteinases in vitro and in vivo, which also could be used for developing tailor-made multi-domain inhibitor gene(s). (C) 2014 Elsevier Ltd. All rights reserved.

10aCapsicum annuum10aHelicoverpa armigera10aInhibitory repeat domain10aProteases10aProteinase inhibitors1 aJoshi, Rakesh, S.1 aGupta, Vidya, S.1 aGiri, Ashok, P. uhttps://library.ncl.res.in/content/differential-antibiosis-against-helicoverpa-armigera-exerted-distinct-inhibitory-repeat-002993nas a2200289 4500008004100000022001400041245017700055210006900232260008600301300001000387490000700397520189300404653002002297653002502317653001402342653002502356100002202381700001902403700002802422700001902450700002402469700001902493700002002512700002102532700002002553856013002573 2014 eng d a0739-110200aRemarkable efficiency of a pin-II proteinase inhibitor sans two conserved disulfide bonds is due to enhanced flexibility and hydrogen bond density in the reactive site loop0 aRemarkable efficiency of a pinII proteinase inhibitor sans two c a325 CHESTNUT ST, SUITE 800, PHILADELPHIA, PA 19106 USAbTAYLOR & FRANCIS INCcJAN a13-260 v323 a

Capsicum annuum (L.) expresses diverse potato type II family proteinase inhibitors comprising of inhibitory repeat domain (IRD) as basic functional unit. Most IRDs contain eight conserved cysteines forming four disulfide bonds, which are indispensible for their stability and activity. We investigated the functional significance of evolutionary variations in IRDs and their role in mediating interaction between the inhibitor and cognate proteinase. Among the 18 IRDs encoded by C. annuum, IRD-7, -9, and -12 were selected for further characterization on the basis of variation in their reactive site loop, number of conserved cysteine residues, and higher theoretical G(bind) for interaction with Helicoverpa armigera trypsin. Moreover, inhibition kinetics showed that IRD-9, despite loss of some of the disulfide bonds, was a more potent proteinase inhibitor among the three selected IRDs. Molecular dynamic simulations revealed that serine residues in the place of cysteines at seventh and eighth positions of IRD-9 resulted in an increase in the density of intramolecular hydrogen bonds and reactive site loop flexibility. Results of the serine residues chemical modification also supported this observation and provided a possible explanation for the remarkable inhibitory potential of IRD-9. Furthermore, this natural variant among IRDs showed special attributes like stability to proteolysis and synergistic inhibitory effect on other IRDs. It is likely that IRDs have coevolved selective specialization of their structure and function as a response towards specific insect proteases they encountered. Understanding the molecular mechanism of pest protease-plant proteinaceous inhibitor interaction will help in developing effective pest control strategies.An animated interactive 3D complement (I3DC) is available in Proteopedia at http://proteopedia.org/w/Journal:JBSD:39

10aCapsicum annuum10aHelicoverpa armigera10aProteases10aProteinase inhibitor1 aJoshi, Rakesh, S.1 aMishra, Manasi1 aTamhane, Vaijayanti, A.1 aGhosh, Anirban1 aSonavane, Uddhavesh1 aSuresh, C., G.1 aJoshi, Rajendra1 aGupta, Vidya, S.1 aGiri, Ashok, P. uhttps://library.ncl.res.in/content/remarkable-efficiency-pin-ii-proteinase-inhibitor-sans-two-conserved-disulfide-bonds-due-002460nas a2200241 4500008004100000022001400041245014600055210006900201260000900270300001100279490000800290520157900298653001901877653002901896653002301925653003201948653001401980653002701994100002402021700002202045700002002067856013102087 2024 eng d a0006-291X00aEngineering inhibitory repeat domains of Pin-II type proteinase inhibitors indicate their high structural-functional tolerance to mutagenesis0 aEngineering inhibitory repeat domains of PinII type proteinase i cNOV  a1508080 v7353 a

Plant proteinase inhibitors (PIs) are critical in defending against biotic stress. Most PIs contain an inhibitory repeat domain (IRD), which serves as the functional component, displaying a high degree of sequence and structural conservation. In this study, we examined the structural and functional resilience of IRDs using a combination of computational modeling and experimental validation. We have taken an evolution-based approach to enhance the PIs effectiveness of two previously identified Capsicum annuum IRDs, IRD4 and IRD10. Through in silico site-saturation mutagenesis of IRD4 and IRD10, we identified key sites associated with enhanced PI activity for targeted mutagenesis. Binding energy predictions for a mutant IRD library, tested against target proteases, suggested that positions R11 and N32 in IRD4 and N32 and H33 in IRD10 were promising candidates for further modification to improve inhibitory potential. Subsequent experimental validation revealed that the mutant proteins IRD4_R11K and IRD4_N32S exhibited stronger chymotrypsin inhibition than the wild-type (WT) IRD4. Similarly, the mutants IRD10_N32S and IRD10_H33 N demonstrated improved trypsin inhibition relative to the WT IRD10. These findings indicate that engineered IRD variants can tolerate structural changes while maintaining or enhancing their inhibitory activity against target proteases. Overall, this study demonstrates the potential of engineering PIs to increase their structural and functional resilience, offering new opportunities for biotechnological applications.

10aCapsicum annum10aInhibitory repeat domain10amolecular modeling10aplant proteinase inhibitors10aProteases10aSaturation mutagenesis1 aBarbole, Ranjit, S.1 aJoshi, Rakesh, S.1 aGiri, Ashok, P. uhttps://library.ncl.res.in/content/engineering-inhibitory-repeat-domains-pin-ii-type-proteinase-inhibitors-indicate-their-high