Expression, Refolding, and Activity of a Recombinant Nonhemorrhagic Snake Venom Metalloprotease

Selistre-de-Araújo, Heloísa Sobreiro; Souza, Eduardo Lorensi de; Beltramini, Leila Maria; Ownby, Charlotte L.; Souza, Dulce Helena Ferreira de

doi:10.1006/prep.2000.1225

Cited by 25 publications

(6 citation statements)

References 34 publications

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“…This amount can be considered quite satisfactory for a refolded protein. The data reported in the literature show a smaller, but equally efficient, yield compared to the one obtained in the present study, such as 0.01 mgAEmL )1 for recombinant snake venom metalloprotease [22] and 0.008 mgAEmL )1 for recombinant human promatrilysin [23]. Figure 3 shows the CD spectra for Th and Ch of the Nfrutalin, Ufrutalin, Rfrutalin and Mfrutalin forms.…”

Section: R E S U L T S a N D Discussioncontrasting

confidence: 51%

Unfolding and refolding studies of frutalin, a tetrameric d‐galactose binding lectin

Campana

Moraes

Monteiro-Moreira

et al. 2002

European Journal of Biochemistry

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Protein refolding is currently a fundamental problem in biophysics and molecular biology. We have studied the refolding process of frutalin, a tetrameric lectin that presents structural homology with jacalin but shows a more marked biological activity. The initial state in our refolding puzzle was that proteins were unfolded after thermal denaturation or denaturation induced by guanidine hydrochloride, and under both conditions, frutalin was refolded. The denaturation curves, measured by fluorescence emission, gave values of conformational stability of 17.12 kJAEmol )1 and 12.34 kJAEmol, in the presence and absence of D-galactose, respectively. Native, unfolded, refolded frutalin and a distinct molecular form denoted misfolded, were separated by size-exclusion chromatography (SEC) on Superdex 75.The native and unfolded samples together with the fractions separated by SEC were also analyzed for heamagglutination activity by CD and fluorescence spectroscopy. The secondary structure content of refolded frutalin estimated from the CD spectra was found to be close to that of the native molecule. All the results obtained confirmed the successful refolding of the protein and suggested a nucleation-condensation mechanism, whereby the sugar-binding site acts as a nucleus to initiate the refolding process. The refolded monomers, after adopting their native three-dimensional structures, spontaneously assemble to form tetramers.Keywords: Artocarpus incisa lectin; frutalin; lectin refolding; lectin unfolding; protein refolding.Our current understanding of the protein folding mechanism is the result of intense studies using both theoretical and experimental biophysical methods. This complex problem concerning the mechanism by which proteins adopt one specific fold among those possible, has been experimentally investigated recently [1][2][3]. Understanding this mechanism would provide a powerful tool for drug design and for comprehension of cellular organization at the molecular level. The fact that proteins with different sequences adopt the same fold suggests that the number of folding pathways is limited, probably, to a few hundred [4]. The b sheet class of proteins has been poorly represented in folding studies [5], even though this is critical for a complete understanding of the formation of the b sheet that differs from the folding properties of helical and mixed a/b proteins. In recent years, the participation of abnormal b sheet structures in Alzheimer's, Huntington's and prion diseases has been demonstrated [6]. On the other hand, this class of b sheet proteins contains families whose members show high structural homology and sequential identity, although with different levels of specificity and affinity for ligands [7,8]. Some of these b sheet proteins are the lectins, a particular carbohydrate-binding protein class widely distributed in all life forms that can mediate several biological events such as the recognition of molecules present in membranes or in the extracellular matrix [9].We have described and studied s...

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Section: R E S U L T S a N D Discussioncontrasting

confidence: 51%

Unfolding and refolding studies of frutalin, a tetrameric d‐galactose binding lectin

Campana

Moraes

Monteiro-Moreira

et al. 2002

European Journal of Biochemistry

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“…We chose mammalian cells for expression because SVSPs are often glycosylated and have a number of disulfide bonds, which present obstacles to effectively refold the recombinant proteins as active forms from E. coli -expressed insoluble inclusion bodies, even under conditions intended to favour disulfide bond formation [ 35 , 36 ]. Such refolding steps are crucial but are frequently unsuccessful and/or time-consuming to optimise sufficient yield recovery, as reported in several previous snake venom toxin expression studies [ 31 , 32 , 53 , 54 ]. Consequently, here we used mammalian HEK293F cells for the expression of three pathogenically relevant SVSP toxins sourced from geographically diverse medically important viperid snakes, specifically ancrod from C. rhodostoma , batroxobin from B. atrox and RVV-V from D. russelli .…”

Section: Discussionmentioning

confidence: 99%

Exploring the Utility of Recombinant Snake Venom Serine Protease Toxins as Immunogens for Generating Experimental Snakebite Antivenoms

Alomran

Blundell

Alsolaiss

et al. 2022

Toxins

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Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies in order to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms, were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed that they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.

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“…We chose mammalian cells for expression because SVSPs are often glycosylated and have a number of disulfide bonds, which present obstacles to effectively refold the recombinant proteins as active forms from E. coli- expressed insoluble inclusion bodies, even under conditions intended to favour disulfide bond formation (35, 36). Such refolding steps are crucial but are frequently unsuccessful and/or time-consuming to optimise sufficient yield recovery, as reported in several previous snake venom toxin expression studies (31, 32, 54, 55). Consequently, here we used mammalian HEK293F cells for the expression of three pathogenically relevant SVSP toxins sourced from geographically diverse medically important viperid snakes, specifically ancrod from C. rhodostoma , batroxobin from B. atrox and RVV-V from D. russelli .…”

Section: Discussionmentioning

confidence: 99%

Exploring the utility of recombinantly expressed snake venom serine protease toxins as immunogens for generating experimental snakebite antivenoms

Alomran

Blundell

Alsolaiss

et al. 2022

Preprint

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Snakebite is a neglected tropical disease that causes high rates of global mortality and morbidity. Although snakebite can cause a variety of pathologies in victims, haemotoxic effects are particularly common and are typically characterised by haemorrhage and/or venom-induced consumption coagulopathy. Despite polyclonal antibody-based antivenoms being the mainstay life-saving therapy for snakebite, they are associated with limited cross-snake species efficacy, as there is often extensive toxin variation between snake venoms, including those used as immunogens for antivenom production. This restricts the therapeutic utility of any antivenom to certain geographical regions. In this study, we explored the feasibility of using recombinantly expressed toxins as immunogens to stimulate focused, pathology-specific, antibodies to broadly counteract specific toxins associated with snakebite envenoming. Three snake venom serine proteases (SVSP) toxins, sourced from geographically diverse and medically important viper snake venoms were successfully expressed in HEK293F mammalian cells and used for murine immunisation. Analyses of the resulting antibody responses revealed that ancrod and RVV-V stimulated the strongest immune responses, and that experimental antivenoms directed against these recombinant SVSP toxins, and a mixture of the three different immunogens, extensively recognised and exhibited immunological binding towards a variety of native snake venoms. While the experimental antivenoms showed some reduction in abnormal clotting parameters stimulated by the toxin immunogens and crude venom, specifically reducing the depletion of fibrinogen levels and prolongation of prothrombin times, fibrinogen degradation experiments revealed they broadly protected against venom- and toxin-induced fibrinogenolytic functional activities. Overall, our findings further strengthen the case for the use of recombinant venom toxins as supplemental immunogens to stimulate focused and desirable antibody responses capable of neutralising venom-induced pathological effects, and therefore potentially circumventing some of the limitations associated with current snakebite therapies.

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Expression, Refolding, and Activity of a Recombinant Nonhemorrhagic Snake Venom Metalloprotease

Cited by 25 publications

References 34 publications

Unfolding and refolding studies of frutalin, a tetrameric d‐galactose binding lectin

Unfolding and refolding studies of frutalin, a tetrameric d‐galactose binding lectin

Exploring the Utility of Recombinant Snake Venom Serine Protease Toxins as Immunogens for Generating Experimental Snakebite Antivenoms

Exploring the utility of recombinantly expressed snake venom serine protease toxins as immunogens for generating experimental snakebite antivenoms

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