RESUMO
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.
Assuntos
Toxinas Botulínicas/genética , Escherichia coli/genética , Sequência de Bases , Toxinas Botulínicas/administração & dosagem , Toxinas Botulínicas/química , Toxinas Botulínicas/imunologia , Clonagem Molecular , DNA Bacteriano , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologiaRESUMO
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.
Assuntos
Antivirais/farmacologia , Antígenos CD4/genética , Toxina Diftérica/genética , Proteínas Recombinantes de Fusão/farmacologia , Linfócitos T/imunologia , Antivirais/metabolismo , Linhagem Celular , Clonagem Molecular , Efeito Citopatogênico Viral/efeitos dos fármacos , Escherichia coli/genética , Células Gigantes , HIV-1/efeitos dos fármacos , HIV-1/patogenicidade , HIV-1/fisiologia , Humanos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
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.
Assuntos
Antivirais/farmacologia , Antígenos CD4/farmacologia , Toxina Diftérica/farmacologia , Proteínas de Bactérias/genética , Antígenos CD4/genética , Linhagem Celular , Toxina Diftérica/genética , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Vetores Genéticos/genética , HIV-1/efeitos dos fármacos , HIV-2/efeitos dos fármacos , Humanos , Plasmídeos/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Cultura de VírusRESUMO
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.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas , Sobrevivência Celular/efeitos dos fármacos , Exotoxinas/genética , Proteína Estafilocócica A/toxicidade , Fatores de Virulência , Adenosina Difosfato Ribose/metabolismo , Catálise , Eletroforese em Gel de Poliacrilamida , Escherichia coli/genética , Exotoxinas/farmacologia , Genes Bacterianos , Cadeias Leves de Imunoglobulina/metabolismo , Fator Tu de Elongação de Peptídeos/metabolismo , Plasmídeos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/toxicidade , Proteína Estafilocócica A/genética , Células Tumorais Cultivadas/efeitos dos fármacos , Exotoxina A de Pseudomonas aeruginosaRESUMO
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.
Assuntos
ADP Ribose Transferases , Toxinas Bacterianas , Exotoxinas/biossíntese , Engenharia de Proteínas , Proteínas Recombinantes/biossíntese , Proteína Estafilocócica A/biossíntese , Fatores de Virulência , Exotoxinas/genética , Immunoblotting , Plasmídeos , Proteínas Recombinantes/genética , Proteína Estafilocócica A/genética , Exotoxina A de Pseudomonas aeruginosaRESUMO
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.
Assuntos
Toxina Diftérica , Animais , Toxina Diftérica/farmacologia , HumanosRESUMO
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.
Assuntos
Clonagem Molecular , Toxina Diftérica/genética , Plasmídeos , Sequência de Aminoácidos , Toxina Diftérica/biossíntese , Erwinia/genética , Escherichia coli/genética , Hidrólise , Peptídeo Hidrolases , Sinais Direcionadores de Proteínas/biossíntese , Sinais Direcionadores de Proteínas/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaRESUMO
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.
