RESUMO
Many animal toxins may target the same molecules that need to be controlled in certain pathologies; therefore, some toxins have led to the formulation of drugs that are presently used, and many other drugs are still under development. Nevertheless, collecting sufficient toxins from the original source might be a limiting factor in studying their biological activities. Thus, molecular biology techniques have been applied in order to obtain large amounts of recombinant toxins into Escherichia coli. However, most animal toxins are difficult to express in this system, which results in insoluble, misfolded, or unstable proteins. To solve these issues, toxins have been fused with tags that may improve protein expression, solubility, and stability. Among these tags, the SUMO (small ubiquitin-related modifier) has been shown to be very efficient and can be removed by the Ulp1 protease. However, removing SUMO is a labor- and time-consuming process. To enhance this system, here we show the construction of a bicistronic vector that allows the expression of any protein fused to both the SUMO and Ulp1 protease. In this way, after expression, Ulp1 is able to cleave SUMO and leave the protein interest-free and ready for purification. This strategy was validated through the expression of a new phospholipase D from the spider Loxosceles gaucho and a disintegrin from the Bothrops insularis snake. Both recombinant toxins showed good yield and preserved biological activities, indicating that the bicistronic vector may be a viable method to produce proteins that are difficult to express.
Assuntos
Cisteína Endopeptidases/genética , Proteína SUMO-1/genética , Animais , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/toxicidade , Plaquetas/efeitos dos fármacos , Bothrops , Venenos de Crotalídeos/genética , Venenos de Crotalídeos/toxicidade , Cisteína Endopeptidases/metabolismo , Desintegrinas/genética , Desintegrinas/toxicidade , Escherichia coli/genética , Humanos , Fosfolipase D/genética , Fosfolipase D/toxicidade , Agregação Plaquetária/efeitos dos fármacos , Inibidores da Agregação Plaquetária/toxicidade , Proteínas Recombinantes de Fusão/toxicidade , Proteína SUMO-1/metabolismo , Venenos de Aranha , AranhasRESUMO
Endostatin (ES) inhibits angiogenesis, reducing tumor growth in animal models. However, it has low therapeutic effect in human clinical trials. BAX is a member of the BCL-2 family of proteins; its proapoptotic (BH3) domain interacts with other members of the family in the cytoplasm, to induce apoptosis. Here, we fused the BAX BH3 domain with murine ES, to enhance ES potency. Endothelial cells specifically internalize the fusion protein ES-BAX. The presence of the BAX domain enhances endothelial cell death by apoptosis by 1.8-fold and diminishes microvessel outgrowth in the rat aortic ring assay by 6.5-fold. Daily injections of 15 µg of ES-BAX/g in tumor-bearing mice reduce tumor weight by 86.9% as compared with ES-treated animals. Co-immunoprecipitation assays confirmed that ES-BAX interacts with members of the BCL-2 family. Also, ES interacts with BCL-2, BCL-XL, and BAK in endothelial cell lysates, suggesting a potential new mechanism for the apoptosis induction by ES. The superiority of the ES-BAX antiangiogenic effect indicates that this fusion protein could be a promising therapeutic alternative to treat cancer.
Assuntos
Inibidores da Angiogênese/toxicidade , Apoptose/efeitos dos fármacos , Endostatinas/toxicidade , Proteína X Associada a bcl-2/metabolismo , Sequência de Aminoácidos , Inibidores da Angiogênese/uso terapêutico , Animais , Linhagem Celular Tumoral , Endostatinas/genética , Endostatinas/uso terapêutico , Escherichia coli/metabolismo , Neoplasias Renais/tratamento farmacológico , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Dados de Sequência Molecular , Células NIH 3T3 , Ligação Proteica , Estrutura Terciária de Proteína , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/uso terapêutico , Proteínas Recombinantes de Fusão/toxicidade , Transplante Homólogo , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/genéticaRESUMO
Evidence is accumulating that commonly used pesticides are linked to decline of pollinator populations; adverse effects of three neonicotinoids on bees have led to bans on their use across the European Union. Developing insecticides that pose negligible risks to beneficial organisms such as honeybees is desirable and timely. One strategy is to use recombinant fusion proteins containing neuroactive peptides/proteins linked to a 'carrier' protein that confers oral toxicity. Hv1a/GNA (Galanthus nivalis agglutinin), containing an insect-specific spider venom calcium channel blocker (ω-hexatoxin-Hv1a) linked to snowdrop lectin (GNA) as a 'carrier', is an effective oral biopesticide towards various insect pests. Effects of Hv1a/GNA towards a non-target species, Apis mellifera, were assessed through a thorough early-tier risk assessment. Following feeding, honeybees internalized Hv1a/GNA, which reached the brain within 1 h after exposure. However, survival was only slightly affected by ingestion (LD50>100 µg bee(-1)) or injection of fusion protein. Bees fed acute (100 µg bee(-1)) or chronic (0.35 mg ml(-1)) doses of Hv1a/GNA and trained in an olfactory learning task had similar rates of learning and memory to no-pesticide controls. Larvae were unaffected, being able to degrade Hv1a/GNA. These tests suggest that Hv1a/GNA is unlikely to cause detrimental effects on honeybees, indicating that atracotoxins targeting calcium channels are potential alternatives to conventional pesticides.
Assuntos
Abelhas/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/toxicidade , Inseticidas/toxicidade , Lectinas de Ligação a Manose/toxicidade , Lectinas de Plantas/toxicidade , Venenos de Aranha/toxicidade , Animais , Abelhas/crescimento & desenvolvimento , Bloqueadores dos Canais de Cálcio/metabolismo , Galanthus/química , Inseticidas/metabolismo , Larva/efeitos dos fármacos , Aprendizagem/efeitos dos fármacos , Lectinas de Ligação a Manose/genética , Lectinas de Ligação a Manose/metabolismo , Lectinas de Plantas/genética , Lectinas de Plantas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/toxicidade , Venenos de Aranha/genética , Venenos de Aranha/metabolismoRESUMO
Pulchellin is a type 2 ribosome-inactivating protein isolated from seeds of the Abrus pulchellus tenuiflorus plant. This study aims to obtain active and homogeneous protein for structural and biological studies that will clarify the functional aspects of this toxin. The DNA fragment encoding pulchellin A-chain was cloned and inserted into pGEX-5X to express the recombinant pulchellin A-chain (rPAC) as a fusion protein in Escherichia coli. The deduced amino acid sequence analyses of the rPAC presented a high sequential identity (> 86%) with the A-chain of abrin-c. The ability of the rPAC to depurinate rRNA in yeast ribosome was also demonstrated in vitro. In order to validate the toxic activity we promoted the in vitro association of the rPAC with the recombinant pulchellin binding chain (rPBC). Both chains were incubated in the presence of a reduced/oxidized system, yielding an active heterodimer (rPAB). The rPAB showed an apparent molecular mass of approximately 60 kDa, similar to the native pulchellin. The toxic activities of the rPAB and native pulchellin were compared by intraperitoneal injection of different dilutions into mice. The rPAB was able to kill 50% of the tested mice with doses of 45 microg x kg(-1). Our results indicated that the heterodimer showed toxic activity and a conformational pattern similar to pulchellin. In addition, rPAC produced in this heterologous system might be useful for the preparation of immunoconjugates with potential as a therapeutic agent.