02554nas a2200253   4500008004100000022001400041245014600055210006900201260007300270300001000343490000800353520157900361653002201940653001501962653002601977653002002003653003802023653001502061100002802076700002002104700002402124700002102148856013102169       2007                        eng d  a0378-111900aDiverse forms of pin-II family proteinase inhibitors from capsicum annuum adversely affect the growth and development of helicoverpa armigera0 aDiverse forms of pinII family proteinase inhibitors from capsicu  aPO BOX 211, 1000 AE AMSTERDAM, NETHERLANDSbELSEVIER SCIENCE BVcNOV  a29-380 v4033 a<p>Novel forms of Pin-II type proteinase inhibitor (PIs) cDNAs (CanPIs) having three or four inhibitory repeat domains (IRD) were isolated from the developing green fruits of Capsicum annuum. Deduced amino acid (aa) sequences of the CanPIs showed up to 15% sequence divergence among each other or reported inhibitors (CanPI-1 AF039398, CanPI-2 AF221097). Amino acid sequence analysis of these CanPIs revealed that three IRD Pls have trypsin inhibitory sites, while four IRD CanPIs have both trypsin and chymotrypsin inhibitory sites. Four CanPIs, two having three IRD (CanPI-3 AY986465 and CanPI-5 DQ005912) and two having four IRD (CanPI-7 DQ005913 and CanPI-9 DQ005915), were cloned in Pichia pastoris to express recombinant CanPIs. Recombinant CanPIs inhibited 90% of bovine trypsin (TI), while chymotrypsin inhibition (0) varied with the number of chymotrypsin inhibitory sites in the CanPIs. Recombinant inhibitors inhibited over 70% of the gut proteinase activity of Helicoverpa armigera. H. armigera larvae fed on recombinant CanPIs individually incorporated into artificial diet, showed 35% mortality; in addition, weight gain in H. armigera larvae and pupae was severely reduced compared to controls. Of the four CanPIs, CanPI-7, which has two sites for TI and CI, was the only one to have a consistently antagonistic effect on H. armigera growth and development. We conclude that among the four recombinant Pls tested, CanPIs containing diverse IRDs are best suited for developing insect-resistant transgenic plants. (c) 2007 Elsevier B.V. All rights reserved.</p>10ainsect resistance10aIP repeats10aPI-insect interaction10aPichia pastoris10apotato type 2 inhibitors (Pot-II)10aSolanaceae1 aTamhane, Vaijayantl, A.1 aGiri, Ashok, P.1 aSainani, Mohini, N.1 aGupta, Vidya, S.  uhttps://library.ncl.res.in/content/diverse-forms-pin-ii-family-proteinase-inhibitors-capsicum-annuum-adversely-affect-growth-002158nas a2200205   4500008004100000022001400041245009900055210006900154260000800223300001200231490000800243520140900251653003101660653002001691653003001711100002201741700003301763700002601796856013001822       2019                        eng d  a0141-813000aZebrafish acid ceramidase: expression in Pichia pastoris GS115and biochemical characterization0 aZebrafish acid ceramidase expression in Pichia pastoris GS115and  cFEB  a587-5930 v1223 a<p>Acid ceramidase (N-acylsphingosine deacylase EC 3.5.1.23; AC) catalyzes the hydrolysis of ceramide into sphingosine (SPH) and free fatty acid. Zebrafish acid ceramidase (AC) has 60% homology with the human AC). Mutations in the human AC gene asah1 are known to cause Farber disease and spinal muscular atrophy with progressive myoclonic epilepsy. Zebrafish AC was overexpressed in Pichia pastoris by inserting asah1b gene into the genome. The majority of the overexpressed enzyme was secreted into the culture medium and purified to apparent homogeneity by stepwise chromatography. The recombinant protein was glycosylated precursor, that further undergoes limited autoproteolytic processing into two subunits (alpha and beta) which are visible in SDS-PAGE. The zebrafish AC is heterodimer associated with an inter-subunit disulfide bond. SDS-PAGE estimated the mass of native enzyme to be approximately 50 kDa &amp; size exclusion chromatography estimated the mass of the active enzyme as approximately 100 kDa, suggesting the formation of a dimer of heterodimers. The protein was secreted as a mixture of processed and unprocessed forms in the culture media. A preliminary characterization of purified zebrafish AC was done by an enzyme assay. The zebrafish AC expressed in Pichia pastoris would be used for further structural and functional analysis. (C) 2018 Elsevier B.V. All rights reserved.</p>10aAutoproteolytic processing10aPichia pastoris10aZebrafish acid ceramidase1 aRajput, Vijay, B.1 aKarthikeyan, Muthukumarasamy1 aRamasamy, Sureshkumar  uhttps://library.ncl.res.in/content/zebrafish-acid-ceramidase-expression-pichia-pastoris-gs115and-biochemical-characterization