Methods have been elaborated for rapid location of biologically active bacterial endotoxin components on preparative thin layer chromatography (tlc) by Limulus lysate clotting assay. The separation of the components f...
详细信息
Methods have been elaborated for rapid location of biologically active bacterial endotoxin components on preparative thin layer chromatography (tlc) by Limulus lysate clotting assay. The separation of the components from silicic acid is not necessary. Further assays were established for the semiquantitative estimation of endotoxic activities (Shwartzman reactivity, chick embryo lethality, and nonspecific tumor resistance enhancement) without complete removal of silicic acid. Quantitative chemical analytical procedures were also elaborated to determine the molar ratios of the biologically active components separated and detected in the tlc system. These included phosphorus, hexosamine, heptose, 2 keto 3 deoxyoctonate, and long chain carboxylic acid content measurements. Here again, the chemical determinations were carried out in the presence of silicic acid.
Recombinant proteins overexpressed in and purified from Escherichia coli contain impurities that are toxic in biological assays. The application of affinity purification procedures is often not sufficient to remove th...
详细信息
Recombinant proteins overexpressed in and purified from Escherichia coli contain impurities that are toxic in biological assays. The application of affinity purification procedures is often not sufficient to remove these toxic components, We here describe a simple and fast, one-step protocol to remove these impurities highly efficiently. Four recombinant proteins were overexpressed in E, coli as His-tagged fusion proteins and purified by immobilized metal chelate affinity chromatography on Ni-NTA beads. Depending on the protein, the composition of the lysis buffer, and the washing protocol, various impurities appeared to be present in the purified protein preparations. Here we show how the use of 60% isopropanol during washing steps removed most of these contaminants from the end products, In addition to the removal of proteins that aspecifically adhere to the beads or to the tagged protein, this procedure was particularly useful in removing endotoxins, Moreover, we show that detergents such as NP-40, that are necessarily employed during lysis, are also efficiently removed, Finally, we show that proteins are able to refold correctly after isopropanol treatment. Thus, the resulting end products contain significantly less contaminating E. coli proteins, endotoxins, and detergents. (C) 2000 Academic Press.
Cry1Ac from Bacillus thuringiensis ssp. kurstaki HD-73 is a pore-forming protein specifically toxic to lepidopteran insect larvae. It binds to the cell-surface receptor aminopeptidase N in Manduca sexta midgut via the...
详细信息
Cry1Ac from Bacillus thuringiensis ssp. kurstaki HD-73 is a pore-forming protein specifically toxic to lepidopteran insect larvae. It binds to the cell-surface receptor aminopeptidase N in Manduca sexta midgut via the sugar N-acetyl-D-galactosamine (GalNAc). By using 1,3-diaminopropane (DAP) as the buffer throughout protoxin activation and chromatography on Q-Sepharose at pH 10.3, trypsin-activated Cry1Ac has been purified in a monomeric state, which was crucial to obtaining single crystals of Cry1Ac and of the Cry1Ac-GalNAc complex. Crystals of Cry1Ac alone are triclinic, with unit-cell parameters a=51.78, b=113.23, c=123.41 Angstrom, alpha =113.11, beta =91.49, gamma =100.46 degrees;those of the Cry1Ac-GalNAc complex show P2(1) symmetry, with unit-cell parameters a=121.36, b=51.19, c=210.56 Angstrom, beta =105.75 degrees. Data sets collected to 2.36 and 2.95 Angstrom resolution, respectively, show that both crystal forms contain four molecules of the 66 kDa toxin in the asymmetric unit and have related packing arrangements. The deaggregating effect of DAP may be explained by its capacity for bivalent hydrogen bonding and hydrophobic interactions at protein interfaces.
A new cvy1I-type gene, cry1Id1, was cloned from a B. thuringiensis isolate, and its nucleotide sequence was determined. The deduced amino acid sequence of Cry1Id1 is 89.7%, 87.2%, and 83.4% identical to the Cry1Ia, Cr...
详细信息
A new cvy1I-type gene, cry1Id1, was cloned from a B. thuringiensis isolate, and its nucleotide sequence was determined. The deduced amino acid sequence of Cry1Id1 is 89.7%, 87.2%, and 83.4% identical to the Cry1Ia, Cry1Ib, and Cry1Ic proteins, respectively. The upstream sequence of the cry1Id1 structural gene was not functional as promoter in B. subtilis. The Cry1Id1 protein, purified from recombinant E. coli cells, had a toxicity comparable to that of Cry1Ia against Plutella xylostella, but it was significantly less active than Cry1Ia against Bombyx mori. Cry1Id1 was not active against the coleopteran insect, Agelastica coerulea.
暂无评论