RESUMEN
Genetic defects in the Wnt-1 signaling pathway contribute to human tumor progression and are especially prevalent in colorectal cancer. We screened mouse C57MG cells to isolate mRNAs induced by Wnt-1 and identified Stra6, an mRNA known to be up-regulated by retinoic acid. Up-regulation of Stra6 mRNA was also observed in hyperplastic mammary tissue and mammary gland tumors from transgenic mice expressing Wnt-1 and in human tumors that frequently harbor defects in Wnt-1 signaling. Stimulation of C57MG cells with retinoic acid plus Wnt-1 resulted in expression of Stra6 transcript to levels greatly exceeding that observed with either stimulus alone. This synergy could be explained in part by the up-regulation of retinoic acid receptor-gamma that was observed in response to Wnt-1 signaling. Accordingly, treatment of human colorectal cancer cell lines with retinoic acid resulted in the up-regulation of Stra6 mRNA and accumulation of Stra6 protein at the cell membrane. The data support a model in which Wnt-1 signaling synergizes with retinoids to activate retinoic acid receptor-gamma-responsive genes in human cancers.
Asunto(s)
Antineoplásicos/farmacología , Neoplasias del Colon/genética , Proteínas de la Membrana/biosíntesis , Proteínas de la Membrana/genética , Proteínas Proto-Oncogénicas/fisiología , Tretinoina/farmacología , Proteínas de Pez Cebra , Adenocarcinoma/genética , Adenocarcinoma/metabolismo , Animales , Cromosomas Humanos Par 15 , Neoplasias del Colon/metabolismo , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Mamarias Experimentales/genética , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Proteínas Proto-Oncogénicas/biosíntesis , Proteínas Proto-Oncogénicas/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal/fisiología , Células Tumorales Cultivadas , Proteínas Wnt , Proteína Wnt1RESUMEN
We report identification of interleukin (IL)-17E, a novel member of the IL-17 family of cytokines. IL-17E is a ligand for the recently identified protein termed EVI27/IL-17BR, which we term IL-17 receptor homolog 1 (IL-17Rh1) in light of the multiple reported ligand-receptor relationships. Murine EVI27 was identified through its location at a common site of retroviral integration in BXH2 murine myeloid leukemias. IL-17Rh1 shows highest level expression in kidney with moderate expression in multiple other organs, whereas IL-17E mRNA was detected at very low levels in several peripheral tissues. IL-17E induces activation of NF-kappaB and stimulates production of the proinflammatory chemokine IL-8.
Asunto(s)
Interleucina-17/genética , Interleucina-17/metabolismo , Receptores de Interleucina/metabolismo , Proteínas Recombinantes/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Secuencia Conservada , Femenino , Biblioteca de Genes , Humanos , Interleucina-17/química , Interleucina-8/biosíntesis , Riñón/inmunología , Leucemia Experimental/inmunología , Leucemia Experimental/virología , Masculino , Ratones , Datos de Secuencia Molecular , FN-kappa B/metabolismo , Especificidad de Órganos , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , Receptores de Interleucina/genética , Receptores de Interleucina-17 , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Transcripción Genética , Transfección , Integración ViralRESUMEN
The Escherichia coli tryptophan (trp) promoter has been used extensively for the high level production of proteins on a small and large scale. This regulated promoter is readily available, relatively easy to turn on, and can be used in essentially any E. coli host background. This article gives a detailed use of the trp promoter including the design of expression vectors, subsequent culture conditions for promoter induction, and, finally, a protocol for the most common way of detecting the newly synthesized protein of interest. Its successful use for heterologous protein expression, however, sometimes requires consideration of parameters other than transcription such as translation initiation, translation elongation, and proteolysis. In this respect we offer guidance in getting through these post-transcriptional problems, which can occur with the use of any promoter.
Asunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Triptófano , Bacteriófago M13 , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos/genética , Indoles/farmacología , Operón , Biosíntesis de Proteínas , Transcripción GenéticaRESUMEN
Melanoma growth stimulating activity (MGSA) and IL-8 are related chemokines that are potent chemoattractants and activators of neutrophils both in vitro and in vivo. Increasing evidence suggests that these molecules play an important role in inflammation; thus, antagonists of their action could be useful therapeutically as antiinflammatory agents. We have generated an MGSA mutant, H19A, that shows a dissociation between receptor binding and biologic activity. The biologic activity of the H19A mutant is between 133-fold and 282-fold less potent than that of wild-type MGSA measured by three independent assays of neutrophil function, i.e., elastase release chemotaxis and the up-regulation of CD18. In addition, pretreatment of cells with the H19A mutant inhibited the ability of MGSA to induce elastase release and chemotaxis and to increase intracellular calcium. However, competition binding studies in cells transfected with the CXCR2 receptor and in neutrophils demonstrate that the receptor affinity of the H19A mutant is only 13-fold less than that of wild-type MGSA. These studies suggest that the mutant MGSA is defective in activating signaling through the receptor and indicate that binding to the receptor is not sufficient to activate a biologic response. The dissociation between receptor binding and activation for this mutant suggests that it should be possible to design antagonists of MGSA that may be of clinical utility.
Asunto(s)
Sustitución de Aminoácidos , Quimiocinas CXC , Factores Quimiotácticos/farmacología , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Receptores de Quimiocina/antagonistas & inhibidores , Receptores de Interleucina/antagonistas & inhibidores , Antígenos CD/genética , Antígenos CD/metabolismo , Señalización del Calcio/efectos de los fármacos , Línea Celular , Quimiocina CXCL1 , Factores Quimiotácticos/genética , Quimiotaxis de Leucocito/efectos de los fármacos , Citocalasina B/farmacología , Sustancias de Crecimiento/genética , Humanos , Interleucina-8/farmacología , Elastasa de Leucocito/metabolismo , Neutrófilos/efectos de los fármacos , Neutrófilos/fisiología , Receptores de Quimiocina/genética , Receptores de Quimiocina/metabolismo , Receptores de Interleucina/genética , Receptores de Interleucina/metabolismo , Receptores de Interleucina-8A , Receptores de Interleucina-8B , Proteínas Recombinantes de Fusión/antagonistas & inhibidores , Proteínas Recombinantes de Fusión/metabolismo , Transducción de Señal/fisiología , TransfecciónAsunto(s)
Clonación Molecular/métodos , Escherichia coli/genética , Operón/genética , Regiones Promotoras Genéticas , Proteínas Recombinantes/biosíntesis , Triptófano , Expresión Génica , Regulación Bacteriana de la Expresión Génica , Vectores Genéticos/genética , Indoles/farmacología , Biosíntesis de Proteínas , Transcripción GenéticaRESUMEN
A method for enhancing the secretion of heterologous proteins in Escherichia coli by optimizing, as opposed to simply maximizing, the translational level of a given protein is described. Random alteration of the translational initiation region (TIR) of the Heat-Stable Enterotoxin II (STII) signal sequence resulted in a library of vectors with varied translational strengths. Subsequent screening of this library using E. coli alkaline phosphatase as a reporter led to the selection of several TIR variants covering a 10-fold range of translational strength. These TIR variants, in combination with several previously generated variants, are shown to dramatically improve the secretion of a number of heterologous proteins. In fact, the heterologous proteins tested required a narrow translational range for optimal high-level secretion into the periplasm. Interestingly, the secretion of two native E. coli proteins was unaffected by TIR strength when tested over an identical range. The dependence of secretion on a narrow translational level demonstrates its critical role in the secretion of heterologous proteins in E. coli.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Periplasmáticas , Biosíntesis de Proteínas , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Fosfatasa Alcalina/biosíntesis , Proteínas Bacterianas/biosíntesis , Toxinas Bacterianas/biosíntesis , Toxinas Bacterianas/genética , Secuencia de Bases , Biotecnología , ADN Bacteriano/genética , Enterotoxinas/biosíntesis , Enterotoxinas/genética , Genes Reporteros , Variación Genética , Vectores Genéticos , Datos de Secuencia MolecularRESUMEN
Alanine scanning mutagenesis of the charged amino acids of melanoma growth stimulating activity (MGSA) was used to identify specific residues that are involved in binding to the human erythrocyte Duffy antigen/chemokine receptor (DARC) and to the type B interleukin-8 receptor (IL-8RB) on neutrophils. Receptor binding and biological studies with the alanine scan mutants of MGSA demonstrate that MGSA binds to DARC and the IL-8RB through distinct binding regions. One of the MGSA mutants, E6A, binds to human erythrocytes and is able to inhibit malaria invasion as efficiently as wild type MGSA but has a severely reduced ability to bind to or signal through the IL-8RB. Mutant chemokines like E6A could prove to be useful therapeutically for the design of receptor blocking drugs that inhibit erythrocyte invasion by Plasmodium vivax malaria.
Asunto(s)
Alanina , Quimiocinas CXC , Factores Quimiotácticos/metabolismo , Factores Quimiotácticos/farmacología , Eritrocitos/parasitología , Sustancias de Crecimiento/metabolismo , Sustancias de Crecimiento/farmacología , Péptidos y Proteínas de Señalización Intercelular , Neutrófilos/fisiología , Plasmodium knowlesi/patogenicidad , Receptores de Citocinas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Quimiocina CXCL1 , Factores Quimiotácticos/química , Clonación Molecular , Secuencia Conservada , Sistema del Grupo Sanguíneo Duffy/metabolismo , Eritrocitos/efectos de los fármacos , Escherichia coli , Sustancias de Crecimiento/química , Humanos , Riñón , Cinética , Malaria/sangre , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas de Neoplasias/metabolismo , Proteínas de Neoplasias/farmacología , Activación Neutrófila , Neutrófilos/efectos de los fármacos , Plasmodium knowlesi/efectos de los fármacos , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Homología de Secuencia de Aminoácido , TransfecciónRESUMEN
Human melanoma growth stimulating activity (MGSA) is a mitogenic factor first identified in the conditioned media of human melanoma cells. Structurally, MGSA belongs to a superfamily of proteins that includes interleukin-8 (IL-8) and platelet factor 4. These proteins are involved in inflammatory processes, and an understanding of their mechanism of action should provide insight into their pathophysiology. In this study, we report the high level expression of recombinant human MGSA in Escherichia coli. The structure was confirmed by mass spectrometry and NH2-terminal amino acid sequencing. Receptor binding studies were carried out in a human melanoma cell line, Hs294T, and in U937 cells. Direct binding experiments with 125I-MGSA in Hs294T cells have allowed us to identify a novel MGSA receptor in these cells, with a KD of 3.9-4.25 nM and approximately 52,960-67,758 binding sites/cell. These MGSA-binding sites were specific and could not be displaced by unlabeled IL-8. The MGSA receptor in these cells is biologically active, and the addition of ligand induces cellular proliferation in a dose-dependent manner. In U937 cells, unlabeled IL-8 and MGSA were able to completely displace radiolabeled IL-8. Scatchard analysis of the displacement binding data was consistent with binding to a single class of binding sites, and the calculated KD values were 2.4 +/- 0.6 nM for IL-8 and 3.2 +/- 0.80 nM for MGSA. Treatment of U937 cells with IL-8 or MGSA produced a rapid increase in Ca2+ flux; however, subsequent incubation with either ligand failed to produce any further Ca2+ flux. The IL-8 receptor in U937 cells was covalently labeled with 125I-IL-8 to reveal a protein with a molecular mass of 69 kDa.
Asunto(s)
Quimiocinas CXC , Factores Quimiotácticos/aislamiento & purificación , Factores Quimiotácticos/metabolismo , Sustancias de Crecimiento/aislamiento & purificación , Sustancias de Crecimiento/metabolismo , Péptidos y Proteínas de Señalización Intercelular , Proteínas de Neoplasias/aislamiento & purificación , Receptores de Superficie Celular/metabolismo , Receptores de Citocinas , Secuencia de Aminoácidos , Sitios de Unión , Western Blotting , Calcio/metabolismo , División Celular/efectos de los fármacos , Quimiocina CXCL1 , Factores Quimiotácticos/genética , Factores Quimiotácticos/farmacología , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Escherichia coli , Sustancias de Crecimiento/genética , Sustancias de Crecimiento/farmacología , Humanos , Interleucina-8/farmacología , Cinética , Espectrometría de Masas , Melanoma , Peso Molecular , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fragmentos de Péptidos/aislamiento & purificación , Plásmidos , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Células Tumorales CultivadasAsunto(s)
Proteínas Bacterianas/genética , Expresión Génica , Secuencia de Aminoácidos , Secuencia de Bases , Clonación Molecular/métodos , Escherichia coli/genética , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesis , Mapeo RestrictivoRESUMEN
Tissue factor (TF) is a 263 amino acid membrane-bound procoagulant protein that serves as a cofactor for the serine protease factor VII (fVII). Recombinant human TF (rTF) produced in both human kidney 293 cells and Escherichia coli has been immunoaffinity purified by using a TF-specific monoclonal antibody. Recombinant TF produced in 293 cells is glycosylated and migrates on reducing SDS-PAGE with an apparent molecular weight (Mr) of 45K. Some interchain disulfide-bonded rTF dimers are observed under nonreducing conditions. The E. coli produced rTF has a molecular weight of 33K and 35K, with the 33K band missing nine amino acids at the carboxy terminus. Although the E. coli produced rTF does not contain any carbohydrate, it is fully functional in both a chromogenic assay and a one-stage prothrombin time assay. A variant has been constructed wherein the cytoplasmic cysteine (residue 245) has been mutagenized to a serine residue. The amount of disulfide-linked aggregates is dramatically reduced following immunoaffinity purification of this four-cysteine variant (C2455), which is active in the chromogenic and prothrombin time assays.
Asunto(s)
Tromboplastina/aislamiento & purificación , Secuencia de Aminoácidos , Secuencia de Bases , Células Cultivadas , Bromuro de Cianógeno , Cisteína/metabolismo , Citoplasma/metabolismo , Escherichia coli/metabolismo , Humanos , Inmunoensayo , Riñón/metabolismo , Datos de Secuencia Molecular , Plásmidos , Tiempo de Protrombina , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/farmacología , Tromboplastina/farmacología , TripsinaRESUMEN
The coding region for the structural and nonstructural polypeptides of the type A12 foot-and-mouth disease virus genome has been identified by nucleotide sequencing of cloned DNA derived from the viral RNA. In addition, 704 nucleotides in the 5' untranslated region between the polycytidylic acid tract and the probable initiation codon of the first translated gene, P16-L, have been sequenced. This region has several potential initiation codons, one of which appears to be a low-frequency alternate initiation site. The coding region encompasses 6,912 nucleotides and ends in a single termination codon, UAA, located 96 nucleotides upstream from a 3'-terminal polyadenylic acid tract. Microsequencing of radiolabeled in vivo and in vitro translation products identified the genome position of the major foot-and-mouth disease virus proteins and the cleavage sites recognized by the putative viral protease and an additional protease(s), probably of cellular origin, to generate primary and functional foot-and-mouth disease virus polypeptides.
Asunto(s)
Aphthovirus/genética , Genes Virales , Péptidos/análisis , Proteínas Virales/análisis , Secuencia de Aminoácidos , Secuencia de Bases , Peso Molecular , ARN Viral/análisis , Proteínas Estructurales ViralesRESUMEN
The nucleotide sequences have been determined and compared from cloned cDNA genes coding for the foot-and-mouth disease virus (FMDV) immunogenic capsid protein, VP1, from eight different A subtypes: A5 Westerwald/58, A12 119ab (large plaque variant), A22 550 USSR/65, A24 Cruzeiro Brazil/55, A27 Cundinamarca Colombia/76, A32 Venezuela/70, A Venceslau Brazil/76, and A Argentina/79. We have also found sequence variations among different cDNA clones of the A5 and A24 subtypes. There are regions of nucleotide sequence within the VP1 gene that vary considerably among the subtypes as well as other regions that remain relatively constant. One highly variable region (codons 130-171) encodes amino acids previously identified as being exposed on the virus surface and constituting an important immunogenic site of the virus. There potentially exist secondary structures within the viral RNA sequences that code for this immunogenic site that could decrease the fidelity of replication at this sequence. The rapid generation of FMDV variants encouraged by such structures in the RNA could work together with various selective pressures to explain the observed accumulation of immunologically distinct viruses of the FMDV A type.
Asunto(s)
Aphthovirus/genética , Genes Virales , Proteínas Virales/genética , Aphthovirus/inmunología , Secuencia de Bases , ADN/genética , Variación Genética , Conformación de Ácido Nucleico , ARN Viral/genética , Proteínas Virales/inmunología , Proteínas Estructurales ViralesRESUMEN
Four groups of 9 cattle each were vaccinated with 10, 50, 250, or 1,250 micrograms of foot-and-mouth disease (FMD) virus A12 VP1 fusion protein that was produced in Escherichia coli and emulsified in an oil adjuvant. The groups given the 10 and 50 micrograms of antigen were revaccinated at 15 weeks and were challenge exposed at 30 weeks; 5 of 9 and 7 of 9 cattle, respectively, were protected from FMD virus infection. The remaining 2 groups, vaccinated with 250 or 1,250 micrograms of antigen, were revaccinated at 32 weeks and were challenge exposed at 45 weeks; 8 of 9 and 9 of 9 cattle, respectively, were protected. The results indicated that the biosynthetic polypeptide FMD vaccine was effective using vaccination intervals frequently followed with conventional whole-virus vaccines.
Asunto(s)
Aphthovirus/inmunología , Enfermedades de los Bovinos/prevención & control , Fiebre Aftosa/prevención & control , Proteínas Virales/inmunología , Vacunas Virales/inmunología , Animales , Anticuerpos Antivirales/biosíntesis , Bovinos , Enfermedades de los Bovinos/inmunología , ADN Recombinante , Relación Dosis-Respuesta Inmunológica , Fiebre Aftosa/inmunología , Inmunidad , Inmunización Secundaria/veterinaria , Masculino , Vacunación/veterinaria , Proteínas Virales/genética , Proteínas Estructurales ViralesRESUMEN
The Escherichia coli lac operator has been placed on the 3' side of the promoter for the penicillinase gene of Bacillus licheniformis, creating a hybrid promoter controllable by the E. coli lac repressor. The E. coli lac repressor gene has been placed under the control of a promoter and ribosome-binding site that allows expression in Bacillus subtilis. When the penicillinase gene that contains the lac operator is expressed in B. subtilis on a plasmid that also produces the lac repressor, the expression of the penicillinase gene can be modulated by isopropyl beta-D-thiogalactoside (IPTG), an inducer of the lac operon in E. coli. A similar system was constructed from a promoter of the B. subtilis phage SPO-1 and the leukocyte interferon A gene, which allowed the controlled expression of interferon in B. subtilis. These two examples show that a functional control system can be introduced into B. subtilis from E. coli.
Asunto(s)
Bacillus subtilis/genética , Escherichia coli/genética , Operón , Proteínas Represoras/genética , Factores de Transcripción/genética , Bacillus/genética , ADN Recombinante , Inducción Enzimática , Regulación de la Expresión Génica , Interferón Tipo I/genética , Penicilinasa/genéticaRESUMEN
A versatile vector for cloning DNA fragments containing promoter activity in Bacillus subtilis was derived from plasmids pBR322, pUB110 and pC194. Selection is based on chloramphenicol resistance which is dependent upon the introduction of DNA fragments allowing expression of a chloramphenicol acetyl transferase gene. The plasmid contains a second selectable marker, neomycin resistance, and contains functional origins of replication for both B. subtilis and Escherichia coli.
Asunto(s)
Bacillus subtilis/genética , Vectores Genéticos , Operón , Cloranfenicol/farmacología , Clonación Molecular , ADN Bacteriano/genética , Farmacorresistencia Microbiana , Genes Bacterianos , PlásmidosRESUMEN
A gene has been constructed which codes for an analog of human proinsulin in which the normal 35-amino acid connecting peptide is replaced by a "mini-C" peptide of six amino acids (Arg-Arg-Gly-Ser-Lys-Arg). The gene, composed of oligonucleotide fragments synthesized by the triester method, was cloned and expressed as a beta-galactosidase hybrid protein. The proinsulin analog was separated from beta-galactosidase by cyanogen bromide cleavage and purified. Controlled disulfide exchange in the S-sulfonate of the analog generated a molecule having high-pressure liquid chromatography (HPLC) and radioimmunoassay (RIA) behavior consistent with a proinsulin-like structure.
Asunto(s)
Genes Sintéticos , Oligopéptidos/genética , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Clonación Molecular , Bromuro de Cianógeno , Escherichia coli/genética , Insulina/análisis , Oligopéptidos/biosíntesis , Oligopéptidos/aislamiento & purificación , Plásmidos , Conformación Proteica , RadioinmunoensayoRESUMEN
DNA coding for human growth hormone was constructed by using chemically synthesised DNA in conjunction with enzymatically prepared cDNA. This 'hybrid' gene was expressed in Escherichia coli under the control of the lac promoter. A polypeptide was produced having the size and immunological properties characteristic of mature human growth hormone.