RESUMEN
Native Clostridium botulinum gene coding for type A neurotoxin has been used to construct recombinant derivatives coding separately for L and H polypeptide chains of the toxin. The gene derivatives have been cloned into an expression vector pET28b in E. coli BL21 (DE3) cells. The recombinant L and H proteins seem to be the major individual proteins after IPTG induction of the recombinant cells. Each of the proteins has been accumulated only in inclusion bodies. The recombinant L chain (but not H chain) has been successfully resolubilized. Each of the proteins contains six His residues on the N terminus which allows purification on Ni-agarose columns with high yield. No toxic effect has been observed for both L and H chains after injection of 10 micrograms of recombinant preparations purified from inclusion bodies. Moreover, the injection resulted in an increase in the titer of specific antibodies which protected mice from 1 DLM of type A native botulinum neurotoxin. Hence, the recombinant neurotoxin protein derivatives which are present in E. coli inclusion bodies can be a source of material for producing diagnostic and therapeutic sera against type A botulinum neurotoxin.
Asunto(s)
Toxinas Botulínicas/genética , Escherichia coli/genética , Secuencia de Bases , Toxinas Botulínicas/administración & dosificación , Toxinas Botulínicas/química , Toxinas Botulínicas/inmunología , Clonación Molecular , ADN Bacteriano , Proteínas Recombinantes/administración & dosificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunologíaRESUMEN
The causes of differences in the stability of CD4 receptor and diphtherial toxin based recombinant receptorotoxins synthesized in E.coli and differing by their primary structure were under study. Insertion of a CD4 receptor fragment, responsible for HIV binding, on the N terminus of hybrid protein was found to lead to a drastic reduction of the stability of the hybrid polypeptide in E.coli and to impossibility of obtaining full-size protein product. In vitro experiments on a model of human T lymphocyte culture demonstrated that recombinant receptorotoxin stably expressed in E.coli inhibited the cytopathic effect of type 1 HIV and the effect of syncytium formation induced by the virus.
Asunto(s)
Antivirales/farmacología , Antígenos CD4/genética , Toxina Diftérica/genética , Proteínas Recombinantes de Fusión/farmacología , Linfocitos T/inmunología , Antivirales/metabolismo , Línea Celular , Clonación Molecular , Efecto Citopatogénico Viral/efectos de los fármacos , Escherichia coli/genética , Células Gigantes , VIH-1/efectos de los fármacos , VIH-1/patogenicidad , VIH-1/fisiología , Humanos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
A number of vectors expressing in E. coli hybrid proteins (receptorotoxins) composed of diphtheria toxin lacking the C-terminal region and CD4-receptor fragment including N-terminal region of the natural protein have been constructed. The receptorotoxins consisting of the CD4 fragment in their N-terminal region were more stable. These recombinant receptorotoxins were cultured with HIV-infected Hut-78 cells and were shown to block HIV infection in vitro.
Asunto(s)
Antivirales/farmacología , Antígenos CD4/farmacología , Toxina Diftérica/farmacología , Proteínas Bacterianas/genética , Antígenos CD4/genética , Línea Celular , Toxina Diftérica/genética , Escherichia coli/genética , Regulación Bacteriana de la Expresión Génica/genética , Vectores Genéticos/genética , VIH-1/efectos de los fármacos , VIH-2/efectos de los fármacos , Humanos , Plásmidos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacología , Cultivo de VirusRESUMEN
By gene-engineering technique a chimeric protein made up of fragments of Staphylococcus aureus protein A and . Pseudomonas aeruginosa exotoxin A has been constructed. The chimeric protein was shown to preserve features characteristic of its both constituents--it ADP-ribosylates elongation factor 2 and binds to Ig. Cytotoxic properties of the chimeric protein were studied in two model systems. Treatment of target cells in both systems was performed successively with antibodies against corresponding antigens and after washing--with recombinant chimeric toxin which bound to antibodies on the surface of target cells. In the first model system human B-lymphoma cells (Daudi line) carrying Ig molecules on their surface were treated with polyclonal antibodies against human Ig L-chains. In the other system, human T-lymphoma cells (Jurkat line) were treated successively with monoclonal antibodies against cell surface CD5 antigen and further on--with polyclonal antibodies against mouse Ig. In both systems, only a slight inhibition of the target cells' growth was registered. The probable reasons of low cytotoxic activity of the chimeric protein and prospects of increasing it are discussed.
Asunto(s)
ADP Ribosa Transferasas , Toxinas Bacterianas , Supervivencia Celular/efectos de los fármacos , Exotoxinas/genética , Proteína Estafilocócica A/toxicidad , Factores de Virulencia , Adenosina Difosfato Ribosa/metabolismo , Catálisis , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Exotoxinas/farmacología , Genes Bacterianos , Cadenas Ligeras de Inmunoglobulina/metabolismo , Factor Tu de Elongación Peptídica/metabolismo , Plásmidos , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/toxicidad , Proteína Estafilocócica A/genética , Células Tumorales Cultivadas/efectos de los fármacos , Exotoxina A de Pseudomonas aeruginosaRESUMEN
The genes coding for Pseudomonas aeruginosa exotoxin-A and Staphylococcus aureus A-protein have been cloned. Different fragments of the genes were subcloned on the plasmid vestors. The plasmids pAPA4 and pAPA42 have been constructed. The plasmids are coding for the hybrid protein which consists of the immunoglobulin binding A-protein domain at its NH2-terminus and the catalytically active fragment of exotoxin-A at its COON-terminus. The hybrid gene is expressed in Escherichia coli cells under the control of lambda cro-gene expression elements (pAPA42) or lac-operon expression elements (pAPA4). The latter proved to be most productive.
Asunto(s)
ADP Ribosa Transferasas , Toxinas Bacterianas , Exotoxinas/biosíntesis , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , Proteína Estafilocócica A/biosíntesis , Factores de Virulencia , Exotoxinas/genética , Immunoblotting , Plásmidos , Proteínas Recombinantes/genética , Proteína Estafilocócica A/genética , Exotoxina A de Pseudomonas aeruginosaRESUMEN
The survey of the literature on the problem of structural and functional relationship of different parts of diphtheria toxin (i.e. in the binding of toxin to eucaryotic cells receptors, intracellular transport of a-fragment of diphtheria toxin and toxin-mediated ADP-ribosylation of EF2) is presented. Some data concerning structural similarities of A-fragment of diphtheria toxin and C-terminal part of Pseudomonas aeruginosa exotoxin A are presented.
Asunto(s)
Toxina Diftérica , Animales , Toxina Diftérica/farmacología , HumanosRESUMEN
Plasmids coding different nontoxic derivatives (toxoids) of the diphtheria toxin were constructed. A secretion of toxoids that carry a signal sequence was found in the periplasmic space of E. coli and Erwinia carotovora. Toxoids without a signal sequence appear in the cytoplasm. We believe that the toxoids secreted in E. coli and E. carotovora cells undergo a limited proteolysis. According to the molecular weights of the fragments there are three targets for proteolysis. One of them being just between A- and B-fragments of the diphtheria toxin. The others are localised in the B-fragment. The role of E. coli signal peptidase in the specific cutting is discussed.
Asunto(s)
Clonación Molecular , Toxina Diftérica/genética , Plásmidos , Secuencia de Aminoácidos , Toxina Diftérica/biosíntesis , Erwinia/genética , Escherichia coli/genética , Hidrólisis , Péptido Hidrolasas , Señales de Clasificación de Proteína/biosíntesis , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genéticaRESUMEN
A fragment of diphtheria toxin (tox) gene from beta 45 phage DNA was cloned on pUC19 plasmid in E. coli cells. The fragment is coding for toxA fragment of the toxin and contains the control region of the tox gene. The tox gene promoter is active in E. coli. The toxA protein is found mainly in periplasm of E. coli cells. The protein is enzymatically active in ADP-ribosilation of elongation factor 2 from eucaryotic cells. An in frame toxA-lacZ' fusion was constructed on pUC8 plasmid. The hybrid protein expresses both toxA and lacZ' activities. Two or seven base pairs were deleted from the central part of toxA gene by means of S1 nuclease digestion. Translation of hybrid toxA-lacZ' mRNA should be terminated downward the delections due to the frameshifts caused by them. Nevertheless, a functionally active alpha-peptide of beta-galactosidase is expressed by both the deletion fusions. The existence of another translational start site functioning in E. coli and located inside 3'-end region of toxA mRNA is suggested.