RESUMO
The transcription factor nuclear factor kappaB (NFkappaB) is a key factor in the immune response triggered by a wide variety of molecules such as inflammatory cytokines, or some bacterial and viral products. This transcription factor represents a new target for the development of anti-inflammatory molecules, but this type of research is currently hampered by the lack of a convenient and rapid screening assay for NFkappaB activation. Indeed, NFkappaB DNA-binding capacity is traditionally estimated by radioactive gel shift assay. Here we propose a new DNA-binding assay based on the use of multi-well plates coated with a cold oligonucleotide containing the consensus binding site for NFkappaB. The presence of the DNA-bound transcription factor is then detected by anti-NFkappaB antibodies and revealed by colorimetry. This assay is easy to use, non-radioactive, highly reproducible, specific for NFkappaB, more sensitive than regular radioactive gel shift and very convenient for high throughput screening.
Assuntos
NF-kappa B/metabolismo , Elementos de Resposta/genética , Animais , Anticorpos/imunologia , Especificidade de Anticorpos , Sequência de Bases , Ligação Competitiva , Extratos Celulares , Linhagem Celular , Colorimetria , Sequência Consenso/genética , DNA/genética , DNA/metabolismo , Dimerização , Humanos , NF-kappa B/imunologia , Subunidade p50 de NF-kappa B , Sondas de Oligonucleotídeos/genética , Sondas de Oligonucleotídeos/metabolismo , Ligação Proteica , Coelhos , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
To obtain an expression vector that will optimize secretion of proteins with disulfide bridges in Escherichia coli, we fused the phoA gene, encoding the bacterial alkaline phosphatase (PhoA), to the sequence encoding the pectate lyase B signal sequence (PelBSS). We used an extensively degenerate pelBSS with silent mutations to study their effects on the production level and activity of PhoA. 11 representative clones differed by a factor of five between the lowest and the highest level of activity, and by a factor greater than seven for the production levels. The efficiency of translocation seems to be the result of an equilibrium between production and secretion levels that favours the secretion of active PhoA according to the competence of the fusion protein being translocated. Free energy calculations and the predicted mRNA secondary structures of the translation initiation regions showed that the high stability of the secondary structure decreased production and secretion levels of PhoA and vice versa. A stem-loop encompassing the degenerate positions downstream from the AUG start codon appears to be responsible for the differences in the production levels.
Assuntos
Fosfatase Alcalina/biossíntese , Proteínas de Bactérias/biossíntese , Cistina/metabolismo , Escherichia coli/metabolismo , Vetores Genéticos/genética , Polissacarídeo-Liases/genética , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes de Fusão/biossíntese , Fosfatase Alcalina/genética , Proteínas de Bactérias/genética , Sequência de Bases , Transporte Biológico , Indução Enzimática , Genes Bacterianos , Genes Sintéticos , Dados de Sequência Molecular , Mutação , Conformação de Ácido Nucleico , Processamento de Proteína Pós-Traducional , RNA Bacteriano/química , RNA Bacteriano/genética , RNA Mensageiro/química , RNA Mensageiro/genéticaRESUMO
Two monoclonal antibodies (mAbs), 5A4 and 6D6, directed against cortisol, have been obtained; 6D6 is used in an assay kit for cortisol. The antibodies also recognize other, structurally related steroids present in the sample assayed. To improve the specificity of the assay, we aimed to minimize the recognition of non-cortisol steroids by the two anti-cortisol mAbs. Our strategy consisted in constructing an efficient expression vector in E. coli which produced the single-chain variable fragment (scFv) of the mAbs in the periplasmic space. We demonstrated that temperature and inducer concentration of the bacterial culture influenced dramatically the yield of active scFv. From the nucleotide sequence we constructed a three-dimensional model of the two variable fragments in order to understand why related steroids are, or are not recognized by the antibody. For both antibodies, we have identified chemical groups which are probably involved in the binding of the steroid haptens and the antibodies. The hydrophobic pocket formed by the antibody comprises two or three tryptophan residues which can interact with the steroid nucleus by stacking. The serine at position 35 of the heavy chain is buried in the back of the pocket and can form a hydrogen bond with the 20-keto group of the cortisol. The stacking interactions and the hydrogen bond orient the steroid in the pocket. This reactivity of the binding site is sustained by the analysis of the cross-reactions of related steroids with the mAbs.
