ABSTRACT
A common strategy for the production and purification of recombinant proteins is to fuse a tag to the protein terminal residues and employ a "tag-specific" ligand for fusion protein capture and purification. In this work, we explored the effect of two tryptophan-based tags, NWNWNW and WFWFWF, on the expression and purification of Green Fluorescence Protein (GFP) used as a model fusion protein. The titers obtained with the expression of these fusion proteins in soluble form were 0.11mgml-1 and 0.48mgml-1 for WFWFWF and NWNWNW, respectively. A combinatorial library comprising 64 ligands based on the Ugi reaction was prepared and screened for binding GFP-tagged and non-tagged proteins. Complementary ligands A2C2 and A3C1 were selected for the effective capture of NWNWNW and WFWFWF tagged proteins, respectively, in soluble forms. These affinity pairs displayed 106M-1 affinity constants and Qmax values of 19.11±2.60ugg-1 and 79.39ugg-1 for the systems WFWFWF AND NWNWNW, respectively. GFP fused to the WFWFWF affinity tag was also produced as inclusion bodies, and a refolding-on column strategy was explored using the ligand A4C8, selected from the combinatorial library of ligands but in presence of denaturant agents.
Subject(s)
Chromatography, Affinity/methods , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/isolation & purification , Ligands , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/isolation & purification , Tryptophan/chemistry , Tryptophan/isolation & purification , Amino Acid Sequence , Green Fluorescent Proteins/biosynthesis , Inclusion Bodies/metabolism , Protein Denaturation , Protein Refolding , Recombinant Fusion Proteins/biosynthesis , SolubilityABSTRACT
Organisms are often exposed to different types of ionizing radiation that, directly or not, will promote damage to DNA molecules and/or other cellular structures. Because of that, organisms developed a wide range of response mechanisms to deal with these threats. Endonuclease III is one of the enzymes responsible to detect and repair oxidized pyrimidine base lesions. However, the effect of radiation on the structure/function of these enzymes is not clear yet. Here, we demonstrate the effect of UV-C radiation on E. coli endonuclease III through several techniques, namely UV-visible, fluorescence and Mössbauer spectroscopies, as well as SDS-PAGE and electrophoretic mobility shift assay. We demonstrate that irradiation with a UV-C source has dramatic consequences on the absorption, fluorescence, structure and functionality of the protein, affecting its [4Fe-4S] cluster and its DNA-binding ability, which results in its inactivation. An UV-C radiation-induced conversion of the [4Fe-4S](2+) into a [2Fe-2S](2+) was observed for the first time and proven by Mössbauer and UV-visible analysis. This work also shows that the DNA-binding capability of endonuclease III is highly dependent of the nuclearity of the endogenous iron-sulfur cluster. Thus, from our point of view, in a cellular context, these results strengthen the argument that cellular sensitivity to radiation can also be due to loss of radiation-induced damage repair ability.
Subject(s)
Deoxyribonuclease (Pyrimidine Dimer)/radiation effects , Escherichia coli Proteins/radiation effects , Iron-Sulfur Proteins/radiation effects , Ultraviolet Rays , DNA/metabolism , DNA Repair , Deoxyribonuclease (Pyrimidine Dimer)/chemistry , Deoxyribonuclease (Pyrimidine Dimer)/genetics , Deoxyribonuclease (Pyrimidine Dimer)/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Iron-Sulfur Proteins/chemistry , Iron-Sulfur Proteins/metabolism , Plasmids , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Recombinant Proteins/radiation effects , Spectrum AnalysisABSTRACT
A novel affinity "tag-receptor" pair was developed as a generic platform for the purification of fusion proteins. The hexapeptide RKRKRK was selected as the affinity tag and fused to green fluorescent protein (GFP). The DNA fragments were designed, cloned in Pet-21c expression vector and expressed in E. coli host as soluble protein. A solid-phase combinatorial library based on the Ugi reaction was synthesized: 64 affinity ligands displaying complementary functionalities towards the designed tag. The library was screened by affinity chromatography in a 96-well format for binding to the RKRKRK-tagged GFP protein. Lead ligand A7C1 was selected for the purification of RKRKRK fusion proteins. The affinity pair RKRKRK-tagged GFP with A7C1 emerged as a promising solution (Ka of 2.45×10(5) M(-1) ). The specificity of the ligand towards the tag was observed experimentally and theoretically through automated docking and molecular dynamics simulations.
Subject(s)
Affinity Labels/isolation & purification , Chromatography, Affinity/methods , Green Fluorescent Proteins/isolation & purification , Oligopeptides/isolation & purification , Recombinant Fusion Proteins/isolation & purification , Affinity Labels/chemistry , Affinity Labels/metabolism , Amino Acid Sequence , Binding Sites , Cloning, Molecular , Escherichia coli/genetics , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/genetics , Ligands , Molecular Dynamics Simulation , Oligopeptides/chemistry , Oligopeptides/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/geneticsABSTRACT
Ferritins are ubiquitous and can be found in practically all organisms that utilize Fe. They are composed of 24 subunits forming a hollow sphere with an inner cavity of ~80 Å in diameter. The main function of ferritin is to oxidize the cytotoxic Fe(2+) ions and store the oxidized Fe in the inner cavity. It has been established that the initial step of rapid oxidation of Fe(2+) (ferroxidation) by H-type ferritins, found in vertebrates, occurs at a diiron binding center, termed the ferroxidase center. In bacterial ferritins, however, X-ray crystallographic evidence and amino acid sequence analysis revealed a trinuclear Fe binding center comprising a binuclear Fe binding center (sites A and B), homologous to the ferroxidase center of H-type ferritin, and an adjacent mononuclear Fe binding site (site C). In an effort to obtain further evidence supporting the presence of a trinuclear Fe binding center in bacterial ferritins and to gain information on the states of the iron bound to the trinuclear center, bacterial ferritin from Desulfovibrio vulgaris (DvFtn) and its E130A variant was loaded with substoichiometric amounts of Fe(2+), and the products were characterized by Mössbauer and EPR spectroscopy. Four distinct Fe species were identified: a paramagnetic diferrous species, a diamagnetic diferrous species, a mixed valence Fe(2+)Fe(3+) species, and a mononuclear Fe(2+) species. The latter three species were detected in the wild-type DvFtn, while the paramagnetic diferrous species was detected in the E130A variant. These observations can be rationally explained by the presence of a trinuclear Fe binding center, and the four Fe species can be properly assigned to the three Fe binding sites. Further, our spectroscopic data suggest that (1) the fully occupied trinuclear center supports an all ferrous state, (2) sites B and C are bridged by a µ-OH group forming a diiron subcenter within the trinuclear center, and (3) this subcenter can afford both a mixed valence Fe(2+)Fe(3+) state and a diferrous state. Mechanistic insights provided by these new findings are discussed and a minimal mechanistic scheme involving O-O bond cleavage is proposed.
Subject(s)
Ceruloplasmin/metabolism , Ferritins/metabolism , Ferrous Compounds/metabolism , Bacterial Proteins/chemistry , Ceruloplasmin/chemistry , Desulfovibrio vulgaris/metabolism , Electron Spin Resonance Spectroscopy , Ferritins/chemistry , Ferrous Compounds/chemistryABSTRACT
A gene encoding Bfr (bacterioferritin) was identified and isolated from the genome of Desulfovibrio vulgaris cells, and overexpressed in Escherichia coli. In vitro, H(2)O(2) oxidizes Fe(2+) ions at much higher reaction rates than O(2). The H(2)O(2) oxidation of two Fe(2+) ions was proven by Mössbauer spectroscopy of rapid freeze-quenched samples. On the basis of the Mössbauer parameters of the intermediate species we propose that D. vulgaris Bfr follows a mineralization mechanism similar to the one reported for vertebrate H-type ferritins subunits, in which a diferrous centre at the ferroxidase site is oxidized to diferric intermediate species, that are subsequently translocated into the inner nanocavity. D. vulgaris recombinant Bfr oxidizes and stores up to 600 iron atoms per protein. This Bfr is able to bind DNA and protect it against hydroxyl radical and DNase deleterious effects. The use of H(2)O(2) as an oxidant, combined with the DNA binding and protection activities, seems to indicate a DPS (DNA-binding protein from starved cells)-like role for D. vulgaris Bfr.
Subject(s)
Bacterial Proteins/metabolism , Cytochrome b Group/metabolism , DNA, Bacterial/metabolism , Desulfovibrio vulgaris/metabolism , Ferritins/metabolism , Iron/metabolism , Bacterial Proteins/drug effects , Bacterial Proteins/genetics , Cloning, Molecular , Cytochrome b Group/drug effects , Cytochrome b Group/genetics , DNA-Binding Proteins/metabolism , Escherichia coli/genetics , Ferritins/drug effects , Ferritins/genetics , Hydrogen Peroxide/pharmacology , Oxidation-Reduction , Oxidative Stress , Spectrophotometry, Ultraviolet , Spectroscopy, MossbauerABSTRACT
OBJECTIVE: The purpose of this study was to compare the maximum exposure and extent of bioavailability of two lithium carbonate (CAS 554-13-2) containing 300 mg tablet formulations (test and reference) for oral administration. METHOD: This bioequivalence study was conducted in a 2-period crossover design with a washout phase of 7 days. Plasma samples were obtained by blood sampling over 72 h in each period. Twenty-four healthy volunteers of both genders participated in the trial. Samples were analyzed by a flame atomic absorption spectrometer. Resulting Li+ concentrations were used for determination of the pharmacokinetic parameters AUC(last), AUC(inf) and C(max). RESULTS: 90 % confidence intervals for AUC(last), AUC(inf) and C(max) were 96.81-107.44%, 98.44-109.54% and 98.60-111.33%, respectively. CONCLUSION: All 90% and 95% confidence intervals were inside the limits defined by the FDA Guidance for Industry (80%-125%) and thus stated that test and reference formulation may be accepted as bioequivalent, with regard to both, maximum exposure and extent of bioavailability.
Subject(s)
Antimanic Agents/administration & dosage , Antimanic Agents/pharmacokinetics , Lithium Carbonate/administration & dosage , Lithium Carbonate/pharmacokinetics , Adult , Antimanic Agents/blood , Area Under Curve , Calibration , Chemistry, Pharmaceutical , Cross-Over Studies , Double-Blind Method , Female , Humans , Lithium Carbonate/blood , Male , Middle Aged , Quality Control , Therapeutic EquivalencyABSTRACT
O lítio (Li+) é a terapia mais adequada na profilaxia de pacientes bipolares na gravidez e puerpério. Contudo, seu impacto sobre o comportamento nos descendentes tem sido pouco estudado. Avaliar as possíveis alterações no desenvolvimento cognitivo dos filhotes fêmeas, após tratamento perinatal com lítio em doses profiláticas. Ratas Winstar foram tratadas com solução de LiCI ou NaCI 10mM/L, ad libitum, como única fonte de bebida durante a gravidez e lactação. Após o nascimento, foi aplicada a adoção cruzada dos filhotes, formando os grupos experimentais (10 ninhadas cada): 1) Controle - salina durante os períodos pré e pós-natal; 2) LiPós - Li+ durante o período pós-natal; 3) LiPré - Li+ durante o período pré-natal; 4) Li - Li+ durante os períodos pré e pós-natal. Os filhotes (um por ninhada) foram submetidos aos seguintes testes de aprendizado e memória em idade adulta: teste de fuga (TF), esquiva ativa sinalizada (EAS) e esquiva passiva (EP). Utilizou-se o teste de campo-aberto (CA) para avaliação da atividade exploratória. Foram encontradas diferenças significantes apenas na resposta de EP no grupo Li (Li=24,4 por cento x Controle=70,1 por cento, Anova & Student - Newman-Keuls; **p<0,01). Os resultados sugerem que a exposição ao lítio durante o desenvolvimento pode ter efeitos duradouros sobre o aprendizado, tais como na aquisição e atenção. Foi necessária a exposição durante a gestação e lactação para a produção de efeitos detectáveis em ratas adultas. São necessários posteriores estudos para investigar os circuitos neurais afetados pela exposição perinatal ao lítio.
Subject(s)
Animals , Female , Rats , Behavior, Animal , Learning , Lithium , Memory , Pregnancy , Rats, WistarABSTRACT
Os sais de lítio (Li+) säo usados para o tratamento profilático do transtorno bipolar. Algumas vezes, é necessária a continuaçäo do tratamento mesmo durante a gravidez, uma vez que o risco de morbidade materna é alto. Objetivos: Avaliar o impacto da exposiçäo perinatal ao lítio em nível profilático. A susceptibilidade da prole feminina de ratos para o desenvolvimento de ansiedade e depressäo foi verificada em idade adulta. Métodos: Fêmeas Wistar beberam cloreto de lítio 10mMol ou cloreto de sódio 10mMol, ad libitum, como única fonte de bebida, nos períodos pré e/ou pós-natal (lactaçäo). Após o nascimento, os filhotes sofreram adoçäo cruzada para formar os quatro grupos experimentais (n=10 ninhadas cada): a) Controle - salina durante os períodos pré e pós-natal; b) LiPos - Li+ durante o período pós-natal; c) LiPre - Li+ durante o período pré-natal e d) Li - Li+ durante os períodos pré e pós-natal. Resultados: Näo se observou alteraçäo na atividade locomotora e em comportamentos associados à ansiedade, no teste do labirinto em cruz elevado (Anova e teste de Student-Newman-Keuls, p>0,05). O tratamento com lítio também näo provocou diferenças significantes na latência de fuga em ratos desamparados (Anova e Student-Newman-Keuls, p>0,05). Conclusäo: Os resultados sugerem que o tratamento perinatal com lítio näo altera intensidade da depressäo comportamental e ansiedade, induzidas em idade adulta.
