scholarly journals Purification of a protein from Bacillus thuringiensis toxic to larvae of lepidoptera

1968 ◽  
Vol 106 (2) ◽  
pp. 445-454 ◽  
Author(s):  
K. E. Cooksey
Keyword(s):  
Bacillus Thuringiensis ◽  
Gel Filtration ◽  
Pieris Brassicae ◽  
Digestion Product ◽  
Toxic Protein ◽  
Protein Toxin ◽  
Ammonium Sulphate Precipitation ◽  
Alkaline Conditions ◽  
Crystal Toxin

The protein toxin of the parasporal body or crystal of Bacillus thuringiensis (Mattés isolate) has been purified severalfold by a combination of Sephadex G-200 gel filtration and ammonium sulphate precipitation. It has been shown that the use of highly alkaline conditions for dissolution of the crystals does not lead to serious artifacts. The crystal toxin has been shown to be quantitatively related to the crystal antigen. It is possible that there is a second distinct toxin present in the crystal and this too can be detected by its antigenic reaction. Purified toxic protein has been hydrolysed in vitro by regurgitated Pieris brassicae gut enzymes, chymotrypsin, trypsin and subtilisin. In each case the digest contained a product that was still antigenic, had mol.wt. about 40000 and was toxic to P. brassicae larvae. Smaller toxic molecules (mol.wt. approx. 10000) that did not react as antigens were also produced by proteolysis. It is possible that these smaller molecules were hydrolytic products of the larger digestion product.

scholarly journals Role of Receptors in Bacillus thuringiensis Crystal Toxin Activity

2007 ◽  
Vol 71 (2) ◽  
pp. 255-281 ◽  
Author(s):  
Craig R. Pigott ◽  
David J. Ellar
Keyword(s):  
Bacillus Thuringiensis ◽  
Structural Features ◽  
Small Subset ◽  
Cry Toxins ◽  
Toxin Resistance ◽  
Sequence Of Events ◽  
Crystal Toxin

SUMMARY Bacillus thuringiensis produces crystalline protein inclusions with insecticidal or nematocidal properties. These crystal (Cry) proteins determine a particular strain's toxicity profile. Transgenic crops expressing one or more recombinant Cry toxins have become agriculturally important. Individual Cry toxins are usually toxic to only a few species within an order, and receptors on midgut epithelial cells have been shown to be critical determinants of Cry specificity. The best characterized of these receptors have been identified for lepidopterans, and two major receptor classes have emerged: the aminopeptidase N (APN) receptors and the cadherin-like receptors. Currently, 38 different APNs have been reported for 12 different lepidopterans. Each APN belongs to one of five groups that have unique structural features and Cry-binding properties. While 17 different APNs have been reported to bind to Cry toxins, only 2 have been shown to mediate toxin susceptibly in vivo. In contrast, several cadherin-like proteins bind to Cry toxins and confer toxin susceptibility in vitro, and disruption of the cadherin gene has been associated with toxin resistance. Nonetheless, only a small subset of the lepidopteran-specific Cry toxins has been shown to interact with cadherin-like proteins. This review analyzes the interactions between Cry toxins and their receptors, focusing on the identification and validation of receptors, the molecular basis for receptor recognition, the role of the receptor in resistant insects, and proposed models to explain the sequence of events at the cell surface by which receptor binding leads to cell death.

scholarly journals Lipolytic Potential ofAspergillus japonicusLAB01: Production, Partial Purification, and Characterisation of an Extracellular Lipase

2014 ◽  
Vol 2014 ◽  
pp. 1-11 ◽  
Author(s):  
Lívia Tereza Andrade Souza ◽  
Jamil S. Oliveira ◽  
Vera L. dos Santos ◽  
Wiliam C. B. Regis ◽  
Marcelo M. Santoro ◽  
...  
Keyword(s):  
Room Temperature ◽  
Gel Filtration ◽  
Industrial Applications ◽  
Partial Purification ◽  
Extracellular Lipase ◽  
Inhibitory Effects ◽  
Ammonium Sulphate Precipitation ◽  
Lipase Enzyme ◽  
Alkaline Conditions ◽  
Triton X

Lipolytic potential ofAspergillus japonicusLAB01 was investigated by describing the catalytic properties and stability of a secreted extracellular lipase. Enzyme production was considered high under room temperature after 4 days using sunflower oil and a combination of casein with sodium nitrate. Lipase was partially purified by 3.9-fold, resulting in a 44.2% yield using ammonium sulphate precipitation (60%) quantified with Superose 12 HR gel filtration chromatography. The activity of the enzyme was maximised at pH 8.5, and the enzyme demonstrated stability under alkaline conditions. The optimum temperature was found to be 45°C, and the enzyme was stable for up to 100 minutes, with more than 80% of initial activity remaining after incubation at this temperature. Partially purified enzyme showed reasonable stability with triton X-100 and was activated in the presence of organic solvents (toluene, hexane, and methanol). Among the tested ions, only Cu2+, Ni2+, and Al3+showed inhibitory effects. Substrate specificity of the lipase was higher for C14 among various p-nitrophenyl esters assayed. TheKMandVmaxvalues of the purified enzyme for p-nitrophenyl palmitate were 0.13 mM and 12.58 umol/(L·min), respectively. These features render a novel biocatalyst for industrial applications.

Structurally related Bacillus thuringiensis delta-endotoxins display major differences in insecticidal activity in vivo and in vitro

1986 ◽  
Vol 84 (1) ◽  
pp. 221-236
Author(s):  
B.H. Knowles ◽  
P.H. Francis ◽  
D.J. Ellar
Keyword(s):  
Bacillus Thuringiensis ◽  
Cell Lines ◽  
Insecticidal Activity ◽  
Pieris Brassicae ◽  
Cross Reaction ◽  
Insect Cell Lines ◽  
Insecticidal Toxicity ◽  
Insect Gut

Many strains within the 22 serotypes of Bacillus thuringiensis produce crystal delta-endotoxins with slight differences in their insecticidal toxicity spectrum in vivo. Since the basis of this specificity is unknown, we chose to compare the activity of delta-endotoxins from three strains: B. thuringiensis var. kurstaki HD-1, var. aizawai HD-249 and var. thuringiensis HD-350, both in vivo and on insect cell lines in vitro. Immunoblotting with antisera to activated var. kurstaki P1 lepidopteran toxin revealed antigenic cross-reaction with the 130 X 10(3) Mr toxin of var. aizawai, and with polypeptides of 130 and 138 (X 10(3)) Mr from var. thuringiensis. In addition, crystals from var. kurstaki and var. aizawai contained an antigenically related 63 X 10(3) Mr protein that did not cross-react with antisera to the 130 X 10(3) Mr component. Bioassays on Pieris brassicae larvae (Lepidoptera) and Aedes aegypti larvae (Diptera) indicated that the 130 X 10(3) Mr protein of var. kurstaki, and the 138 plus 130 X 10(3) Mr components of var. thuringiensis killed only P. brassicae, while the 130 X 10(3) Mr protein of var. aizawai and the 63 X 10(3) Mr proteins of var. aizawai and var. kurstaki were toxic to both P. brassicae and A. aegypti. Activation of the 130 and 138 (X 10(3)) Mr proteins of the three varieties of B. thuringiensis with insect gut proteases yielded active products of 50–60 (X 10(3)) Mr. Assay of these products on a range of lepidopteran and dipteran cell lines revealed very different toxicity spectra: var. kurstaki killed only one lepidopteran line, var. thuringiensis killed two lepidopteran lines, while var. aizawai was cytolytic to all of the lepidopteran and most of the dipteran cell lines tested, reflecting its broader spectrum in vivo. Thus we have shown that antigenic cross-reaction of B. thuringiensis delta-endotoxins does not necessarily imply a similar toxicity spectrum in vivo or in vitro.

scholarly journals Phycocyanin Extracted from Oscillatoria minima Shows Antimicrobial, Algicidal, and Antiradical Activities: In silico and In vitro Analysis

2020 ◽  
Vol 19 (3) ◽  
pp. 240-253 ◽  
Author(s):  
Vaishali C. Venugopal ◽  
Abhimanyu Thakur ◽  
Latha K. Chennabasappa ◽  
Gaurav Mishra ◽  
Kunal Singh ◽  
...  
Keyword(s):  
In Silico ◽  
Radical Scavenging ◽  
Gel Filtration ◽  
Qualitative Evaluation ◽  
Exchange Chromatography ◽  
Alternative Source ◽  
Ammonium Sulphate Precipitation ◽  
In Silico Studies ◽  
Vitro Analysis

Background: Phycocyanin is an algae-derived protein, which binds to pigment for harvesting light. It has been reported in various different species, including that of red algae, dinoflagellates, and cryptophyta. Importantly, phycocyanin has enormous applications, including cosmetic colorant, food additive, biotechnology, diagnostics, fluorescence detection probe, an anticancer agent, anti-inflammatory, immune enhancer, etc. In addition, several different algae were utilized for the isolation of cyano-phycocyanin (C-PC), but most of the purification methods consist of several steps of crude extraction. Aim: To isolate C-PC from a new source of microalgae with better purity level and to evaluate its antimicrobial, algicidal, and antiradical activities. Methods: Biological activity, permeability, pharmacokinetics, and toxicity profile of C-PC were predicted by in silico studies. C-PC was purified and isolated by using ammonium sulphate precipitation, ion-exchange chromatography and gel-filtration chromatography. C-PC was characterized by SDS-PAGE and elution profile (purity ratio) analysis. Antimicrobial and algicial activities of C-PC were evaluated by the microtitre plate based assays. Antiradical activity of C-PC was evaluated by DPPH- and ABTS*+ radical scavenging assays. Conclusion: C-PC was extracted from Oscillatoria minima for the first time, followed by its quantitative as well qualitative evaluation, indicating a new alternative source of this important protein. Furthermore, the antimicrobial, algicidal, and antiradical activities of the isolated C-PC extract have been demonstrated by both in silico as well as in vitro methods.

Purification of the Antihemophilic Factor by Gel Filtration on Agarose

1969 ◽  
Vol 22 (03) ◽  
pp. 577-583 ◽  
Author(s):  
M.M.P Paulssen ◽  
A.C.M.G.B Wouterlood ◽  
H.L.M.A Scheffers
Keyword(s):  
Factor Viii ◽  
Plasma Proteins ◽  
Large Scale ◽  
Gel Filtration ◽  
Large Scale Preparation ◽  
Scale Preparation ◽  
Antihemophilic Factor

SummaryFactor VIII can be isolated from plasma proteins, including fibrinogen by chromatography on agarose. The best results were obtained with Sepharose 6B. Large scale preparation is also possible when cryoprecipitate is separated by chromatography. In most fractions containing factor VIII a turbidity is observed which may be due to the presence of chylomicrons.The purified factor VIII was active in vivo as well as in vitro.

Half-Life of Platelet Factor 4 (PF-4) in Plasma and Platelets from Macaca Mulatta

1977 ◽  
Vol 37 (01) ◽  
pp. 073-080 ◽  
Author(s):  
Knut Gjesdal ◽  
Duncan S. Pepper
Keyword(s):  
Macaca Mulatta ◽  
Half Life ◽  
Human Platelet ◽  
Gel Filtration ◽  
Platelet Factor 4 ◽  
Active Protein ◽  
Platelet Factor ◽  
Membrane Bound

SummaryHuman platelet factor 4 (PF-4) showed a reaction of complete identity with PF-4 from Macaca mulatta when tested against rabbit anti-human-PF-4. Such immunoglobulin was used for quantitative precipitation of in vivo labelled PF-4 in monkey serum. The results suggest that the active protein had an intra-platelet half-life of about 21 hours. In vitro 125I-labelled human PF-4 was injected intravenously into two monkeys and isolated by immuno-precipita-tion from platelet-poor plasma and from platelets disrupted after gel-filtration. Plasma PF-4 was found to have a half-life of 7 to 11 hours. Some of the labelled PF-4 was associated with platelets and this fraction had a rapid initial disappearance rate and a subsequent half-life close to that of plasma PF-4. The results are compatible with the hypothesis that granular PF-4 belongs to a separate compartment, whereas membrane-bound PF-4 and plasma PF-4 may interchange.

Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

1987 ◽  
Vol 114 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Chohei Shigeno ◽  
Itsuo Yamamoto ◽  
Shegiharu Dokoh ◽  
Megumu Hino ◽  
Jun Aoki ◽  
...  
Keyword(s):  
Bone Resorption ◽  
High Performance ◽  
Basic Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Factor ◽  
Human Tumours ◽  
Acid Stable ◽  
Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

Biochemical and pharmacokinetic characterisation of two PEGylated variants of dipetarudin

2009 ◽  
Vol 102 (09) ◽  
pp. 454-459 ◽  
Author(s):  
Anne Koehler ◽  
Goetz Nowak ◽  
Mercedes López
Keyword(s):  
Gel Filtration ◽  
Pharmacokinetic Profile ◽  
Peripheral Compartment ◽  
Therapeutic Application ◽  
Similar Activity ◽  
Elimination Half ◽  
K 12 ◽  
Extravascular Compartment

SummaryDipetarudin was coupled to polyethylene glycol (PEG)-5000 residues in order to improve its pharmacokinetic profile and to enhance its anticoagulant efficacy. The resulting compounds, mono-and di-PEGylated dipetarudin were purified by gel filtration. Mono-PEGylated dipetarudin exhibited similar activity like its non-conjugated equivalent both in vitro and in vivo. However, di-PEGylated dipetarudin showed longer distribution and elimination half-lives and higher area under the time-concentration curve in comparison with the unmodified inhibitor which may be attributed to decreased renal clearance. Futhermore, ratio k 12/k 21 decreased when the number of PEG chains coupled to dipetarudin increased. It means that the intercompartment transfer of dipetarudin, characterised by a fast distribution and a high retention in the peripheral compartment, is reverted by coupling to PEG. Thus, the transfer of mono-PEGylated dipetarudin between these compartments is similar in both senses and the transfer of di-PEGylated dipetarudin is slower from vascular to extravascular compartment than vice versa. Our results show that di-PEGylated dipetarudin produces a better and longer anticoagulant effect than unmodified dipetarudin which is a desirable attribute for future therapeutic application.

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