Purification of Active Cysteine Proteases by Affinity Chromatography with Attached E-64 Inhibitor

Govrin, Eri M; Levine, Alex

doi:10.1006/prep.1999.1033

Cited by 12 publications

(6 citation statements)

References 18 publications

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“…To test this hypothesis, the infecting dose of MGAS5005Δ srv was suspended in 333 µM of E64 (0.1 mL). E64 is a commercially available inhibitor of cysteine proteases [43], [44]. Mice were infected as before and monitored for 8 days ( n = 10).…”

Section: Resultsmentioning

confidence: 99%

“…We utilized the specific inhibitor of cysteine proteases E64, which irreversibly binds the active thiol group of SpeB [43], [44], [66]. E64 is commonly used as a cysteine protease inhibitor during in vitro assays, but this is the first study to our knowledge to demonstrate the effects of E64 treatment on GAS infection in vivo [11], [16], [65], [67], [68].…”

Section: Discussionmentioning

confidence: 99%

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Dispersal of Group A Streptococcal Biofilms by the Cysteine Protease SpeB Leads to Increased Disease Severity in a Murine Model

et al. 2011

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Group A Streptococcus (GAS) is a Gram-positive human pathogen best known for causing pharyngeal and mild skin infections. However, in the 1980's there was an increase in severe GAS infections including cellulitis and deeper tissue infections like necrotizing fasciitis. Particularly striking about this elevation in the incidence of severe disease was that those most often affected were previously healthy individuals. Several groups have shown that changes in gene content or regulation, as with proteases, may contribute to severe disease; yet strains harboring these proteases continue to cause mild disease as well. We and others have shown that group A streptococci (MGAS5005) reside within biofilms both in vitro and in vivo. That is to say that the organism colonizes a host surface and forms a 3-dimensional community encased in a protective matrix of extracellular protein, DNA and polysaccharide(s). However, the mechanism of assembly or dispersal of these structures is unclear, as is the relationship of these structures to disease outcome. Recently we reported that allelic replacement of the streptococcal regulator srv resulted in constitutive production of the streptococcal cysteine protease SpeB. We further showed that the constitutive production of SpeB significantly decreased MGAS5005Δsrv biofilm formation in vitro. Here we show that mice infected with MGAS5005Δsrv had significantly larger lesion development than wild-type infected animals. Histopathology, Gram-staining and immunofluorescence link the increased lesion development with lack of disease containment, lack of biofilm formation, and readily detectable levels of SpeB in the tissue. Treatment of MGAS5005Δsrv infected lesions with a chemical inhibitor of SpeB significantly reduced lesion formation and disease spread to wild-type levels. Furthermore, inactivation of speB in the MGAS5005Δsrv background reduced lesion formation to wild-type levels. Taken together, these data suggest a mechanism by which GAS disease may transition from mild to severe through the Srv mediated dispersal of GAS biofilms.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Dispersal of Group A Streptococcal Biofilms by the Cysteine Protease SpeB Leads to Increased Disease Severity in a Murine Model

et al. 2011

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“…Comparison with kinetic constants determined for other Original Paper cysteine proteases is difficult and will not be tried here because of the different methods and substrates used in the respective studies. Many cysteine proteases are strongly and irreversibly inhibited by trans-(epoxysuccinyl)-l-leucylamino-4-guanidinobutane (E64) from Aspergillus japonicus [18]. The irreversible inhibition is due to the reaction of the epoxy group at position C-2 of the inhibitor with the cysteine residue at the catalytic site of the protease.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterisation of a Cysteine Protease (Phytolacain G), fromPhytolacca americanaRoots

Sussner

Abel²,

Schulte³

et al. 2004

Planta med

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Protein extracts obtained from dried and fresh roots of Phytolacca americana L. (Phytolaccaceae) were examined in order to identify and characterise individual proteins. The extracts were compared with a commercial pokeweed mitogen standard using SDS polyacrylamide gel electrophoresis (SDS-PAGE). A dominant protein, present in both the extracts and the pokeweed mitogen standard, was isolated by subsequent ammonium sulphate fractionation, anion exchange chromatography, gel filtration and SDS-PAGE. In this way it was purified 140-fold with about 20 % yield and 70-fold with about 13 % yield from dried and fresh roots, respectively. Its molecular mass as determined by gel filtration and SDS-PAGE was estimated to be about 25 kDa. Subsequent isoelectric focussing revealed one single protein band at pH 6.0. LysC digestion of the 25 kDa protein yielded several peptides which were subjected to micro-sequencing. Comparison with published sequences revealed that the protein isolated was phytolacain G, a cysteine protease previously isolated from unripe fruits of P. americana L. The enzyme showed a high affinity towards the oxidised insulin B-chain and was completely inhibited by trans-epoxysuccinyl- L-leucylamido(4-guanidino)-butane (E64). The purified phytolacain G showed "lectin-like" activities such as haemagglutination and mitogenic effects towards human lymphocytes.

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“…This reaction prevents the reaction of E-64 with the cysteine residue at the protease catalytic center. However, this does not affect the bond between the inhibitor and cysteine-protease; instead, it only results in inhibition of the proteolytic activity (Govrin, 1999).…”

Section: Protease Inhibitors Affinity-based Separation Of Parasite Prmentioning

confidence: 99%

Affinity-Based Methods for the Separation of Parasite Proteins

Alves¹,

Silva²,

Oliveira³

et al. 2012

Affinity Chromatography

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Purification of Active Cysteine Proteases by Affinity Chromatography with Attached E-64 Inhibitor

Cited by 12 publications

References 18 publications

Dispersal of Group A Streptococcal Biofilms by the Cysteine Protease SpeB Leads to Increased Disease Severity in a Murine Model

Dispersal of Group A Streptococcal Biofilms by the Cysteine Protease SpeB Leads to Increased Disease Severity in a Murine Model

Isolation and Characterisation of a Cysteine Protease (Phytolacain G), fromPhytolacca americanaRoots

Affinity-Based Methods for the Separation of Parasite Proteins

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