“Insularin, a disintegrin from Bothrops insularis venom: Inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins”

Della-Casa, Maisa S.; Junqueira-de-Azevedo, Inácio L.M.; Butera, Diego; Clissa, Patrı́cia Bianca; Lopes, Daiana Silva; Serrano, Solange M.T.; Pimenta, Daniel C.; Magalhães, Geraldo Santana; Ho, Paulo Lee; Moura-da-Silva, Ana Maria

doi:10.1016/j.toxicon.2010.10.013

Cited by 21 publications

(18 citation statements)

References 45 publications

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“…Insularin (INS) was chosen as this toxin contains a high number of cysteines and its expression was only possible in fusion with glutathione S-transferase [38]. pSUMO Ulp1 -INS was transformed into BL21 Star ™ (DE3) cells and the expression was performed at 30 °C for four hours.…”

Section: Resultsmentioning

confidence: 99%

“…Previously, the expression of Insularin was only possible after its fusion with glutathione S-transferase (GST) [38]. However, the GST could not be removed, probably because the small thrombin cleavage site was sterically hindered.…”

Section: Discussionmentioning

confidence: 99%

“…The vector was also tested through the expression of Insularin, a disintegrin isolated from the viper Bothrops insularis that previously could not be expressed without a tag [38]. This class of toxins from viper venoms shows great potential to develop new anti-thrombotic [39] and antitumoral agents [40], but these molecules have been difficult to express in E. coli because of their high cysteine content [41,42].…”

Section: Introductionmentioning

confidence: 99%

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Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria

Shimokawa-Falcão

Caporrino

Barbaro

et al. 2017

Toxins

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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.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria

Shimokawa-Falcão

Caporrino

Barbaro

et al. 2017

Toxins

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“…Both N-terminal sequences were submitted to BLAST but neither shared homology with other snake venom protein. Interestingly, the primary sequence of BmooAi has the sequence APEN in the same position (residues 10–13) occupied by the identical sequence in Insularin, a disintegrin from B. insularis venom that inhibits platelet aggregation induced by ADP [28]. This finding deserves attention, but more studies are needed to elucidate the importance of this sequence for inhibition of platelet aggregation.…”

Section: Resultsmentioning

confidence: 99%

“…They typically have an RGD sequence that binds to integrin α IIb β 3 and other integrins inhibiting their functions [15, 28, 36]. The RGD sequence presents inhibitory activity on platelet aggregation induced by several agonists [6].…”

Section: Resultsmentioning

confidence: 99%

Purification and Characterization of BmooAi: A New Toxin fromBothrops moojeniSnake Venom That Inhibits Platelet Aggregation

Queiroz

Mamede

Morais

et al. 2014

BioMed Research International

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In this paper, we describe the purification/characterization of BmooAi, a new toxin from Bothrops moojeni that inhibits platelet aggregation. The purification of BmooAi was carried out through three chromatographic steps (ion-exchange on a DEAE-Sephacel column, molecular exclusion on a Sephadex G-75 column, and reverse-phase HPLC chromatography on a C2/C18 column). BmooAi was homogeneous by SDS-PAGE and shown to be a single-chain protein of 15,000 Da. BmooAi was analysed by MALDI-TOF Spectrometry and revealed two major components with molecular masses 7824.4 and 7409.2 as well as a trace of protein with a molecular mass of 15,237.4 Da. Sequencing of BmooAi by Edman degradation showed two amino acid sequences: IRDFDPLTNAPENTA and ETEEGAEEGTQ, which revealed no homology to any known toxin from snake venom. BmooAi showed a rather specific inhibitory effect on platelet aggregation induced by collagen, adenosine diphosphate, or epinephrine in human platelet-rich plasma in a dose-dependent manner, whereas it had little or no effect on platelet aggregation induced by ristocetin. The effect on platelet aggregation induced by BmooAi remained active even when heated to 100°C. BmooAi could be of medical interest as a new tool for the development of novel therapeutic agents for the prevention and treatment of thrombotic disorders.

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Novel disintegrin‐like peptides derived from an amphibian skin cDNA sequence of Hypsiboas punctatus

Pires

Tacca

Aslan

et al. 2021

Journal of Peptide Science

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Disintegrins comprise a family of small proteins that bind to and alter the physiological function of integrins, especially integrins that mediate platelet aggregation in blood. Here, we report a lysine-glycine-aspartic acid (KGD) disintegrin-like motif present in a 15-amino acid residue peptide identified in a cDNA library of the amphibian Hypsiboas punctatus skin. The original peptide sequence was used as a template from which five new analogs were designed, chemically synthesized by solid phase, and tested for disintegrin activity and tridimensional structural studies using NMR spectroscopy. The original amphibian peptide had no effect on integrin-mediated responses. Nevertheless, derived peptide analogs inhibited integrin-mediated platelet function, including platelet spreading on fibrinogen.

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“Insularin, a disintegrin from Bothrops insularis venom: Inhibition of platelet aggregation and endothelial cell adhesion by the native and recombinant GST-insularin proteins”

Cited by 21 publications

References 45 publications

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria

Toxin Fused with SUMO Tag: A New Expression Vector Strategy to Obtain Recombinant Venom Toxins with Easy Tag Removal inside the Bacteria

Purification and Characterization of BmooAi: A New Toxin fromBothrops moojeniSnake Venom That Inhibits Platelet Aggregation

Novel disintegrin‐like peptides derived from an amphibian skin cDNA sequence of Hypsiboas punctatus

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