DNA and heparin chaperone the refolding of purified recombinant replication protein A subunit 1 from Leishmania amazonensis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Refolding and purification of bothropstoxin-1, a Lys49 - Phospholipase A(2) homologue, expressed as inclusion bodies in Escherichia coli

Hydrolysis of phospholipids by Group II phospholipase A(2) enzymes involves a nucleophilic attack on the sn-2 ester bond by the His48 residue and stabilization of the reaction intermediate by a Ca2+ ion cofactor bound to the Asp49 residue in the protein active site region, Bothropstoxin-I (BthTX-I) is a PLA, variant present in the venom of the snake Bothrops jararacussu which shows a Asp49 to Lys substitution and which lacks hydrolytic activity yet damages artificial membranes by a noncatalytic Ca2+-independent mechanism. In order to better characterize this unusual mechanism of membrane damage, we have established an expression system for BthTX-I in Escherichia coli. The DNA-coding sequence for BthTX-I was subcloned into the vector pET11-d, and the BthTX-I was expressed as inclusion bodies in E, coli BL21(DE3). The native BthTX-I contains seven disulfide bonds, and a straightforward protocol has been developed to refold the recombinant protein at high protein concentration in the presence of surfactants using a size-exclusion chromatography matrix. After refolding, recovery yields of 2.5% (corresponding to 4-5 mg of refolded recombinant BthTX-I per liter of bacterial culture) were routinely obtained. After refolding, identical fluorescent and circular dichroism spectra were obtained for the recombinant BthTX-I compared to those of the native protein. Furthermore, the native and refolded recombinant protein demonstrated identical membrane-damaging properties as evaluated by measuring the release of an entrapped fluorescent marker from liposomes, (C) 2001 Academic Press.

The Araguaia fold belt, Brazil: A reactivated Brasiliano-Pan-African cycle (550 Ma) geosuture

The Araguaia-Tocantins geosuture, which separates the Araguaia Fold Belt (AFB) from the Archean Amazonian Craton, was active in the late Middle Proterozoic. The Baixo Araguaia Supergroup was deposited, consisting of the Estrondo Group (lower quartzites with intercalated schists), Xambioá Formation (schists), and Canto da Vazante Formation (upper feldspathic schists); and the Tocantins Group consisting of the Couto Magalhaës Formation (phyllites, quartzites, slates, limestones, and metacherts) and Pequizeiro Formation (upper chlorite schists); and associated mafic-ultramafic bodies. The deformational history includes four regional phases of deformation within this supracrustal sequence: recumbent folds with vergence to the west; refolding with a N-S trend; an intense crenulation episode; and late thrusting from east to west. Metamorphism is of intermediate or intermediate-high pressure type with garnet, biotite, chlorite, and sericite isograds succeeded by a slightly or non-metamorphosed zone, from east to west. Rocks surrounding sparse gneissic-cored domes contain isograds of staurolite, kyanite, and fibrolite. These isograds are believed to be associated with the 1100 Ma Uruaçuano event. The Brasiliano Orogeny strongly affected the AFB with displacements due to transcurrent reactivation of great and old faults of the basement, slight folding in the supracrustal sequence, intrusion of small granite bodies, and development of domes with associated normal faults. The area underlain by the Estrondo Group was uplifted at this time, causing the deposition of the Rio das Barreiras polymictic conglomerate of the central area. K-Ar and Rb-Sr analyses date this thermo-tectonic event at 550 ± 100 Ma. The Archean basement is exposed in the cores of domes as a granite-gneiss association, the Colméia complex, which shows thermo-tectonic features that may be interpreted as polycyclic imprints (Jequié, Transamazonian?, Uruaçuano, and Brasiliano Events). © 1989.

Espectroscopia de correlação bidimensional generalizada e sample-sample aplicada ao estudo da tripsina pancreática bovina

Pós-graduação em Biofísica Molecular - IBILCE

Biologia estrutural: expressão e caracterização estrutural da proteína 25K do Cole latent virus

Pós-graduação em Microbiologia - IBILCE

Structural and functional characterization of the chaperone Hsp70 from sugarcane. Insights into conformational changes during cycling from cross-linking/mass spectrometry assays

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Expressão, purificação e caracterização estrutural de proteínas codificadas por dois fitovírus

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Expression, purification and characterization of cold shock protein A of Corynebacterium pseudotuberculosis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)