RESUMEN
Four different isoforms of the Voltage-Dependent Anion Channel (VDAC) have been identified in Arabidopsis plant cells. The electrophysiological characteristics of several VDAC channels from animal as well as plant cells are well documented, but those of this model plant are unknown. One isoform, AtVDAC-3 was obtained either directly by cell-free synthesis or produced in Escherichia coli, as inclusion bodies, and re-natured. An electrophysiological study of the purified proteins in planar lipid bilayers showed that both methods yielded proteins with similar channel activity. The characteristics of AtVDAC-3 are that of a bona fide VDAC-like channel.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Ingeniería de Proteínas/métodos , Canales Aniónicos Dependientes del Voltaje/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/aislamiento & purificación , Sistema Libre de Células , Fenómenos Electrofisiológicos , Escherichia coli/genética , Membrana Dobles de Lípidos , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Canales Aniónicos Dependientes del Voltaje/genética , Canales Aniónicos Dependientes del Voltaje/aislamiento & purificaciónRESUMEN
We study the electrophoretic blockades due to entries of partially unfolded proteins into a nanopore as a function of the concentration of the denaturing agent. Short and long pore blockades are observed by electrical detection. Short blockades are due to the passage of completely unfolded proteins, their frequency increases as the concentration of the denaturing agent increases, following a sigmoidal denaturation curve. Long blockades reveal partially folded conformations. Their duration increases as the proteins are more folded. The observation of a Vogel-Fulcher law suggests a glassy behavior.
Asunto(s)
Proteínas Portadoras/química , Proteínas Hemolisinas/química , Nanoestructuras/química , Pliegue de Proteína , Materiales Biomiméticos/química , Electroforesis/métodos , Proteínas de Escherichia coli/química , Guanidina/química , Proteínas de Unión a Maltosa , Conformación Proteica , Desnaturalización Proteica , Staphylococcus aureusRESUMEN
The Structural Proteomics In Europe (SPINE) programme is aimed at the development and implementation of high-throughput technologies for the efficient structure determination of proteins of biomedical importance, such as those of bacterial and viral pathogens linked to human health. Despite the challenging nature of some of these targets, 175 novel pathogen protein structures (approximately 220 including complexes) have been determined to date. Here the impact of several technologies on the structural determination of proteins from human pathogens is illustrated with selected examples, including the parallel expression of multiple constructs, the use of standardized refolding protocols and optimized crystallization screens.
Asunto(s)
Infecciones Bacterianas/metabolismo , Proteínas Bacterianas/química , Proteómica/métodos , Proteínas Virales/química , Virosis/metabolismo , Animales , Infecciones Bacterianas/microbiología , Humanos , Pliegue de Proteína , Virosis/virologíaRESUMEN
The protein FkpA from the periplasm of Escherichia coli exhibits both cis/trans peptidyl-prolyl isomerase (PPIase) and chaperone activities. The crystal structure of the protein has been determined in three different forms: as the full-length native molecule, as a truncated form lacking the last 21 residues, and as the same truncated form in complex with the immunosuppressant ligand, FK506. FkpA is a dimeric molecule in which the 245-residue subunit is divided into two domains. The N-terminal domain includes three helices that are interlaced with those of the other subunit to provide all inter-subunit contacts maintaining the dimeric species. The C-terminal domain, which belongs to the FK506-binding protein (FKBP) family, binds the FK506 ligand. The overall form of the dimer is V-shaped, and the different crystal structures reveal a flexibility in the relative orientation of the two C-terminal domains located at the extremities of the V. The deletion mutant FkpNL, comprising the N-terminal domain only, exists in solution as a mixture of monomeric and dimeric species, and exhibits chaperone activity. By contrast, a deletion mutant comprising the C-terminal domain only is monomeric, and although it shows PPIase activity, it is devoid of chaperone function. These results suggest that the chaperone and catalytic activities reside in the N and C-terminal domains, respectively. Accordingly, the observed mobility of the C-terminal domains of the dimeric molecule could effectively adapt these two independent folding functions of FkpA to polypeptide substrates.
Asunto(s)
Inmunofilinas/química , Proteínas de la Membrana/química , Isomerasa de Peptidilprolil , Tacrolimus/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Sitios de Unión , Catálisis , Cristalización , Cristalografía por Rayos X , Dimerización , Escherichia coli/metabolismo , Proteínas de Escherichia coli , Inmunofilinas/metabolismo , Ligandos , Proteínas de la Membrana/metabolismo , Chaperonas Moleculares , Datos de Secuencia Molecular , Periplasma , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Estructura Terciaria de Proteína , Eliminación de Secuencia , Homología de Secuencia de Aminoácido , Proteínas de Unión a Tacrolimus/química , Proteínas de Unión a Tacrolimus/metabolismoRESUMEN
Rapid Translation System (RTS) is a cell-free protein production system employing an enhanced Escherichia coli lysate to perform coupled in vitro transcription-translation reactions. A continuous supply of energy substrates, nucleotides and amino acids combined with the removal of by-products guarantees a high yield of protein production. The gene to express is either cloned into a plasmid vector or introduced as a PCR product amenable to automation. The main property of this alternative system to cellular expression systems is its open design allowing direct manipulation of the reaction conditions and applications that are impossible or difficult in cell-based systems. RTS offers new promising possibilities in the postgenomic era.
Asunto(s)
Ingeniería Genética/métodos , Biosíntesis de Proteínas , Proteínas Recombinantes/biosíntesis , Extractos Celulares , Escherichia coli , Humanos , Pliegue de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Factores de Tiempo , Transcripción GenéticaRESUMEN
The nature of molecular chaperones in the periplasm of Escherichia coli that assist newly translocated proteins to reach their native state has remained poorly defined. Here, we show that FkpA, a heat shock periplasmic peptidyl-prolyl cis/trans isomerase (PPIase), suppresses the formation of inclusion bodies from a defective-folding variant of the maltose-binding protein, MalE31. This chaperone-like activity of FkpA, which is independent of its PPIase activity, requires a full-length structure of the protein. In vitro, FkpA does not catalyse a slow rate-limiting step in the refolding of MalE31, but prevents its aggregation at stoichiometric amounts and promotes the reactivation of denaturated citrate synthase. We propose that FkpA functions as a chaperone for envelope proteins in the bacterial periplasm.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Inmunofilinas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos , Isomerasa de Peptidilprolil/metabolismo , Periplasma/metabolismo , Proteínas de Unión Periplasmáticas , Sitios de Unión/genética , Proteínas Portadoras/metabolismo , Dicroismo Circular , Citrato (si)-Sintasa/metabolismo , Escherichia coli/genética , Respuesta al Choque Térmico , Inmunofilinas/genética , Cuerpos de Inclusión , Proteínas de Unión a Maltosa , Proteínas de la Membrana/genética , Pliegue de Proteína , Estructura Secundaria de Proteína , Transporte de Proteínas , Eliminación de SecuenciaRESUMEN
We recently isolated a mutant of a human anti-beta-galactosidase single chain antibody fragment (scFv) able to fold at high levels in Escherichia coli cytoplasm. When targeted to the periplasm, this mutant and the wild-type scFv are both expressed at comparable levels in a soluble, active and oxidized form. If a reducing agent is added to the growth medium, only the mutant scFv is still able to fold, showing that in vivo aggregation is a direct consequence of the lack of disulphide bond formation and not of the cellular localization. In vitro denaturation/renaturation experiments show that the mutant protein is more stable than the wild-type scFv. Furthermore, refolding kinetics under reducing conditions show that the mutant folds faster than the wild-type protein. Aggregation does not proceed from the native or unfolded conformation of the protein, but from a species only present during the unfolding/refolding transition. In conclusion, the in vivo properties of the mutant scFv can be explained by, first, an increase in the stability of the protein in order to tolerate the removal of the two disulphide bonds and, second, a modification of its folding properties that reduces the kinetic competition between folding and aggregation of a reduced folding intermediate.
Asunto(s)
Citoplasma/metabolismo , Escherichia coli/metabolismo , Fragmentos de Inmunoglobulinas/biosíntesis , Fragmentos de Inmunoglobulinas/química , Pliegue de Proteína , Proteínas Recombinantes/biosíntesis , Disulfuros/química , Disulfuros/metabolismo , Escherichia coli/genética , Escherichia coli/ultraestructura , Humanos , Fragmentos de Inmunoglobulinas/genética , Fragmentos de Inmunoglobulinas/metabolismo , Cinética , Mutación , Oxidación-Reducción , Periplasma/metabolismo , Unión Proteica , Desnaturalización Proteica , Renaturación de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Solubilidad , Termodinámica , UreaRESUMEN
DegP (HtrA) is a periplasmic heat shock serine protease of Escherichia coli that degrades misfolded proteins at high temperatures. Biochemical and biophysical experiments have indicated that the purified DegP exists as a hexamer. To examine whether the PDZ domains of DegP were required for oligomerization, we constructed a DegP variant lacking both PDZ domains. This truncated variant, DegPDelta, exhibited no proteolytic activity but exerted a dominant-negative effect on growth at high temperatures by interfering with the functional assembly of oligomeric DegP. Thus, the PDZ domains contain information necessary for proper assembly of the functional hexameric structure of DegP.
Asunto(s)
Proteínas Bacterianas/química , Escherichia coli/enzimología , Proteínas de Choque Térmico , Proteínas Periplasmáticas , Serina Endopeptidasas/química , División Celular , Dimerización , Escherichia coli/genética , Espectrometría de Masas , Mutación , Conformación Proteica , Pliegue de Proteína , Serina Endopeptidasas/genética , Esferoplastos/enzimología , TemperaturaRESUMEN
We previously identified and characterized amino acid substitutions in a loop connecting helix I to strand B, the alphaI/betaB loop, of the N-domain that are critical for in vivo folding of the maltose-binding protein (MalE31). The tertiary context-dependence of this mutation in MalE folding was assessed by probing the tolerance of an equivalent alphabeta loop of the C-domain to the same amino acid substitutions (MalE219). Moving the loop mutation from the N- to the C-domain eliminated the in vivo misfolding step that led to the formation of inclusion bodies. In vitro, both loop variants exhibited an important decrease of stability, but their intrinsic tendency to aggregate was well correlated with their periplasmic fates in Escherichia coli. Furthermore, the noncoincidence of the unfolding and refolding transition curves and increase of light scattering during the refolding of MalE31 indicate that a competing off-pathway reaction could occurs on the folding pathway of this variant. These results strongly support the notion that the formation of super-secondary structures of the N-domain is a rate-limiting step in the folding pathway of MalE.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Portadoras/química , Proteínas de Escherichia coli , Proteínas de Transporte de Monosacáridos , Proteínas de Unión Periplasmáticas , Pliegue de Proteína , Estructura Terciaria de Proteína , Proteínas Bacterianas/química , Escherichia coli/química , Fluorescencia , Guanidina/farmacología , Cinética , Proteínas de Unión a Maltosa , Mutagénesis Sitio-Dirigida/genética , Mutación/genética , Estructura Secundaria de Proteína , Dispersión de RadiaciónRESUMEN
The periplasmic fates of misfolded MalE31, a defective folding mutant of the maltose-binding protein, were determined by manipulating two cellular activities affecting the protein folding pathway in host cells: (i) the malEp promoter activity, which is controlled by the transcriptional activator MalT, and (ii) the DegP and Protease III periplasmic proteolytic activity. At a low level of expression, the degradation of misfolded MalE31 was partially impaired in cells lacking DegP or Protease III. At a high level of expression, misfolded MalE31 rapidly formed periplasmic inclusion bodies and thus escaped degradation. However, the manipulated host cell activities did not enhance the production of periplasmic, soluble MalE31. A kinetic competition between folding, aggregation, and degradation is proposed as a general model for the biogenesis of periplasmic proteins.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Portadoras/química , Proteínas Portadoras/metabolismo , Proteínas de Unión al ADN , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Choque Térmico , Proteínas de Transporte de Monosacáridos , Periplasma/metabolismo , Proteínas de Unión Periplasmáticas , Proteínas Periplasmáticas , Pliegue de Proteína , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/genética , Escherichia coli/genética , Genotipo , Cinética , Maltosa/metabolismo , Proteínas de Unión a Maltosa , Metaloendopeptidasas/metabolismo , Modelos Químicos , Mutagénesis , Regiones Promotoras Genéticas , Desnaturalización Proteica , Serina Endopeptidasas/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Hybrid proteins were generated by inserting the penicillin-hydrolyzing enzyme, TEM beta-lactamase (Bla), into the maltodextrin-binding protein (MalE). The inserted Bla was functionally accommodated by MalE when it was placed within permissive sites. The maltose binding and penicillinase activities of purified hybrids were indistinguishable from those of the wild-type MalE and Bla proteins. Moreover, these hybrids displayed an additional unexpected property: maltose stabilized the active site of inserted Bla.
Asunto(s)
Proteínas Bacterianas/química , Proteínas Portadoras/química , Proteínas de Escherichia coli , beta-Lactamasas/química , Sitios de Unión , Escherichia coli/química , Escherichia coli/genética , Proteínas de Unión Periplasmáticas , Plásmidos , Pliegue de Proteína , beta-Lactamasas/metabolismoRESUMEN
We analysed the effects of the overproduction of parts or all of a multisubunit ATP-binding cassette (ABC) transporter, the MalFGK2 complex, involved in the uptake of maltose and maltodextrins in Escherichia coli. We found that production of the MalF protein alone was inducing the phtrA promoter, which is under the control of a recently discovered sigma factor, sigma24, involved in the response to extracytoplasmic stresses. The production level, stability and localization of MalF were not altered when produced without its partners, suggesting that the protein was correctly inserted in the membrane. Our results indicate that a large periplasmic loop located between the third and fourth transmembrane segment of MalF, the L3 loop, is responsible for phtrA induction: (i) deleted MalF proteins with no L3 loop or with a L3 loop lacking 120 amino acids do not induce the phtrA promoter; (ii) the export to the periplasm of the L3 loop alone or fused to MalE induces the phtrA promoter. Moreover, the proteolytic sensitivity of MalF is different when it is produced alone and when MalF and MalG are produced together, suggesting a change in the conformation and/or accessibility of MalF. These results suggest that some inner membrane proteins can be sensed outside the cytoplasm by a quality control apparatus or by the export machinery. Moreover, the observation of the phtrA induction by MalF could be a useful new tool for studying the insertion and assembly of the MalFGK2 complex.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Bacterianas/metabolismo , Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Respuesta al Choque Térmico , Maltosa/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Monosacáridos , Proteínas de Unión Periplasmáticas , Proteínas Bacterianas/genética , Proteínas Portadoras/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica , Proteínas de Unión a Maltosa , Proteínas de la Membrana/genética , Regiones Promotoras Genéticas , Conformación Proteica , Eliminación de Secuencia , Factor sigma/metabolismoRESUMEN
Heat shock response in Escherichia coli is autoregulated. Consistent with this, mutations in certain heat shock genes, such as dnaK, dnaJ, grpE or htrC lead to a higher constitutive heat shock gene expression at low temperatures. A similar situation occurs upon accumulation of newly synthesized peptides released prematurely from the ribosomes by puromycin. We looked for gene(s) which, when present in multicopy, prevent the constitutive heat shock response associated with htrC mutant bacteria or caused by the presence of puromycin. One such locus was identified and shown to carry the recently sequenced hslV hslU (clpQ clpY) operon. HslV/ClpQ shares a very high degree of homology with members of the beta-type subunit, constituting the catalytic core of the 20S proteasome. HslU/ClpY is 50% identical to the ClpX protein of E. coli, which is known to present large polypeptides to its partner, the ATP-independent proteolytic enzyme ClpP. We show that, in vivo, HslV and HslU interact and participate in the degradation of abnormal puromycylpolypeptides. Biochemical evidence suggests that HslV/ClpQ is an efficient peptidase whose activity is enhanced by HslU/CIpY in the presence of ATP.
Asunto(s)
Adenosina Trifosfatasas/metabolismo , Endopeptidasa Clp , Endopeptidasas/metabolismo , Proteínas de Escherichia coli , Escherichia coli/metabolismo , Proteínas de Choque Térmico/metabolismo , Pliegue de Proteína , Serina Endopeptidasas , Proteasas ATP-Dependientes , Adenosina Trifosfatasas/genética , Adenosina Trifosfatasas/aislamiento & purificación , Clonación Molecular , Concanavalina A/farmacología , Farmacorresistencia Microbiana/genética , Endopeptidasas/genética , Endopeptidasas/aislamiento & purificación , Escherichia coli/genética , Proteínas de Choque Térmico/genética , Hidrólisis , Operón , Puromicina/farmacología , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
The maltose-binding protein (MBP) of Escherichia coli is the periplasmic receptor of the maltose transport system. Previous studies have identified amino acid substitutions in an alpha/beta loop of the structure of MBP that are critical for the in vivo folding. To probe genetically the structural role of this surface loop, we generated a library in which the corresponding codons 32 and 33 of malE were mutagenized. The maltose phenotype, which correlates with a biologically active structure of MBP in the periplasm, indicated a considerable variability in the loop residues compatible with a correct in vivo folding pathway of the protein. By the same genetic screens, we characterized loop-variant MBPs associated with a defective periplasmic folding pathway and aggregated into inclusion bodies. Heat-shock induction with production of misfolded loop variants was examined using both lon-lacZ and htrA-lacZ fusions. We found that the extent of formation of inclusion bodies in the periplasm of E. coli, from misfolded loop variant MBPs, correlated with the level of heat-shock response regulated by the alternate heat-shock sigma factor, sigma 24.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Bacterianas , Proteínas de Unión al Calcio , Proteínas Portadoras/química , Proteínas de Escherichia coli , Cuerpos de Inclusión/química , Proteínas de Transporte de Monosacáridos , Proteínas de Unión Periplasmáticas , Proteínas Periplasmáticas , Proteasa La , Proteasas ATP-Dependientes , Secuencia de Aminoácidos , Proteínas Portadoras/genética , Bases de Datos Factuales , Escherichia coli , Proteínas de Choque Térmico/metabolismo , Calor , Cuerpos de Inclusión/ultraestructura , Operón Lac , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Serina Endopeptidasas/metabolismo , Relación Estructura-ActividadRESUMEN
A global search for extracytoplasmic folding catalysts in Escherichia coli was undertaken using different genetic systems that produce unstable or misfolded proteins in the periplasm. The extent of misfolding was monitored by the increased activity of the sigma E regulon that is specifically induced by misfolded proteins in the periplasm. Using multicopy libraries, we cloned two genes, surA and fkpA, that decreased the sigma E-dependent response constitutively induced by misfolded proteins. According to their sequences and their biochemical activities, SurA and FkpA belong to two different peptidyl prolyl isomerase (PPI) families. Interestingly, surA was also selected as a multicopy suppressor of a defined htrM (rfaD) null mutation. Such mutants produce a defective lipopolysaccharide that is unable to protect outer membrane proteins from degradation during folding. The SurA multicopy suppression effect in htrM (rfaD) mutant bacteria was directly associated with its ability to catalyse the folding of outer membrane proteins immediately after export. Finally, Tn10 insertions were isolated, which led to an increased activity of the sigma E regulon. Such insertions were mapped to the dsb genes encoding catalysts of the protein disulphide isomerase (PDI) family, as well as to the surA, fkpA and ompH/skp genes. We propose that these three proteins (SurA, FkpA and OmpH/Skp) play an active role either as folding catalysts or as chaperones in extracytoplasmic compartments.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas Portadoras , Proteínas de Unión al ADN/genética , Proteínas de Escherichia coli , Escherichia coli/química , Proteínas de Choque Térmico , Inmunofilinas , Proteínas de la Membrana/genética , Chaperonas Moleculares , Isomerasa de Peptidilprolil , Proteínas Periplasmáticas , Pliegue de Proteína , Proteínas de la Membrana Bacteriana Externa/análisis , Proteínas de la Membrana Bacteriana Externa/química , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas Bacterianas/fisiología , Carbohidrato Epimerasas/genética , Detergentes/farmacología , Escherichia coli/efectos de los fármacos , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/fisiología , Prueba de Complementación Genética , Isomerasas/genética , Lipopolisacáridos , Mutación , Proteína Disulfuro Isomerasas , Proteínas Recombinantes de Fusión , Regulón/genética , Serina Endopeptidasas/genética , Factor sigma/fisiología , Supresión Genética , Factores de Transcripción/fisiologíaRESUMEN
The KAL gene is responsible for the X-chromosome linked form of Kallmann's syndrome in humans. Upon transfection of CHO cells with a human KAL cDNA, the corresponding encoded protein, KALc, was produced. This protein is N-glycosylated, secreted in the cell culture medium, and is localized at the cell surface. Several lines of evidence indicate that heparan-sulfate chains of proteoglycan(s) are involved in the binding of KALc to the cell membrane. Polyclonal and monoclonal antibodies to the purified KALc were generated. They allowed us to detect and characterize the protein encoded by the KAL gene in the chicken central nervous system at late stages of embryonic development. This protein is synthesized by definite neuronal cell populations including Purkinje cells in the cerebellum, mitral cells in the olfactory bulbs and several subpopulations in the optic tectum and the striatum. The protein, with an approximate molecular mass of 100 kDa, was named anosmin-1 in reference to the deficiency of the sense of smell which characterizes the human disease. Anosmin-1 is likely to be an extracellular matrix component. Since heparin treatment of cell membrane fractions from cerebellum and tectum resulted in the release of the protein, we suggest that one or several heparan-sulfate proteoglycans are involved in the binding of anosmin-1 to the membranes in vivo.
Asunto(s)
Encéfalo/metabolismo , Proteínas de la Matriz Extracelular/genética , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Pollos , Cricetinae , Proteínas de la Matriz Extracelular/análisis , Proteínas de la Matriz Extracelular/aislamiento & purificación , Técnicas de Transferencia de Gen , Humanos , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/análisis , Proteínas del Tejido Nervioso/aislamiento & purificación , Cromosoma XRESUMEN
The maltose-binding protein (MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. We have examined the characteristics of a Mal- mutant of malE corresponding to the double substitution Gly32 --> Asp/Ile33 --> Pro, MalE31, previously obtained by random mutagenesis. In vivo, the MalE31 precursor is efficiently processed, but the mature protein forms inclusion bodies in the periplasm. Furthermore, the accumulation of insoluble MalE31 is independent of its cellular localization; MalE31 lacking its signal sequence forms inclusion bodies in the cytoplasm. The native MalE31 protein can be purified by affinity chromatography from inclusion bodies after denaturation by 8 M urea. The renatured protein exhibits full maltose binding affinity (Kd= 9 x 10(-7) M), suggesting that its folded structure is similar to that of the wild-type protein. Unfolding/refolding experiments show that MalE31 is less stable (-5. 5 kcal/mol) than the wild-type protein (-9.5 kcal/mol) and that folding intermediates have a high tendency to form aggregates. In conclusion, the observed phenotype of cells expressing malE31 can be explained by a defective folding pathway of the protein.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Proteínas Bacterianas/química , Proteínas Portadoras/química , Proteínas de Escherichia coli , Proteínas de Transporte de Monosacáridos , Proteínas de Unión Periplasmáticas , Secuencia de Bases , Compartimento Celular , Cartilla de ADN/química , Escherichia coli , Proteínas de Unión a Maltosa , Datos de Secuencia Molecular , Unión Proteica , Pliegue de Proteína , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , SolubilidadRESUMEN
We genetically introduced two different viral CD4+ T cell epitopes within two internal sites of the Escherichia coli maltose-binding (MalE) protein. Affinity-purified hybrid MalE proteins were used to analyze the influence of the molecular environment on the presentation of inserted epitope to T cells. In the first model, the 120 to 132 PreS T cell epitope was inserted alone or with its C-terminal B cell epitope (132-145) at site 133 or 303 of MalE. The maltose-binding protein with PreS peptide inserts expressing the 120 to 132 sequence were able to induce in vivo and in vitro peptide-specific T cell response, whatever the length and the position of the insert. In the second model, the 103 to 115 T cell epitope from the C3 region of poliovirus type 1 (PV1) was inserted, with various flanking sequences, either at site 133 or 303 of MalE protein. The longer C3:86 to 115 insert induced poliovirus-specific T cell responses at both sites of MalE, whereas the C3:93 to 115 insert did it only at site 303 but not at site 133. Moreover, C3:103 to 115 specific T cell hybridomas discriminated between the processed peptides generated from the different chimeric proteins, as a result of differences in the length and the position of the inserted sequence. Therefore, in this experimental model the loss of in vivo immunogenicity of an antigenic determinant within a chimeric protein is related to the activation of a reduced T cell repertoire. These observations involve important consequences for the engineering of recombinant vaccines.
Asunto(s)
Transportadoras de Casetes de Unión a ATP , Linfocitos T CD4-Positivos/inmunología , Proteínas de Escherichia coli , Proteínas de Transporte de Monosacáridos , Proteínas de Unión Periplasmáticas , Proteínas Virales/inmunología , Secuencia de Aminoácidos , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/inmunología , Cápside/genética , Cápside/inmunología , Proteínas de la Cápside , Proteínas Portadoras/genética , Proteínas Portadoras/inmunología , Epítopos/genética , Escherichia coli/genética , Escherichia coli/inmunología , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Hibridomas/inmunología , Activación de Linfocitos , Proteínas de Unión a Maltosa , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos DBA , Datos de Secuencia Molecular , Poliovirus/genética , Poliovirus/inmunología , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/inmunología , Proteínas Virales/genéticaRESUMEN
The maltose binding protein (MBP or MalE) of Escherichia coli is the periplasmic component of the transport system for malto-oligosaccharides. It is synthesized in the cytoplasm with an N-terminal signal peptide that is cleaved upon export. We examined whether active MBP could assemble into an active protein in bacteria, from N- and COOH-terminal complementary protein fragments encoded by distinct, engineered segments of its structural gene. We found export and functional periplasmic assembly of MBP fragments, despite the complex polypeptide chain topology of this protein, if two conditions were satisfied. First, each of the two fragments must carry a signal peptide. Second, the boundaries between the two fragments must correspond to a permissive site within the protein. Functional assembly of active MBP occurred in five cases where these conditions were met: sites after residues 133, 161, 206, 285 and 303; but not in three other cases where the break junction corresponded to a non-permissive site: after residues 31, 120 and 339. Thus, permissive sites which were initially characterized because they could accept extensive genetic insertion/deletion modifications without loss of most biological properties provide a means of defining complementing protein fragments. This observation opens a way to study genetically the relationships between protein export and folding into the periplasm.