Overproduction and purification of biologically active native fungal α-sarcin in Escherichia coli

Lacadena, Javier; Pozo, Álvaro Martı́nez del; Barbero, José Luís; Mancheño, José M.; Gasset, Marı́a; Oñaderra, Mercedes; López-Otı́n, Carlos; Ortega, Sagrario; Garcı́a, José Luis; Gavilanes, José G.

doi:10.1016/0378-1119(94)90370-0

Cited by 63 publications

(112 citation statements)

References 28 publications

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“…E. coli BL21 (DE3) cells, previously cotransformed with a thioredoxin-producing plasmid (pT-Trx) and the corresponding plasmid (pINPGαSH137Q), were used to produce the catalytically inactive -sarcin H137Q mutant, also as previously described (García-Ortega et al, 2000;Lacadena et al, 1994Lacadena et al, , 1995Lacadena et al, , 1999. This mutant retains the structural features of the wild-type protein, as well as its ability to interact with membranes, but lacks the characteristic ribonucleolytic activity of ribotoxins (Lacadena et al, 1995(Lacadena et al, , 1999.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

“…This mutant retains the structural features of the wild-type protein, as well as its ability to interact with membranes, but lacks the characteristic ribonucleolytic activity of ribotoxins (Lacadena et al, 1995(Lacadena et al, , 1999. SDS-PAGE of proteins, Western blots, protein hydrolysis, amino acid analysis, and spectroscopic characterization were performed according to standardized procedures described before (García-Ortega et al, 2000, 2002Lacadena et al, 1994;Martínez-Ruiz et al, 2001). According to all these criteria, the three proteins used in this study were purified to homogeneity and retained their structural and functional properties.…”

Section: Protein Production and Purificationmentioning

confidence: 99%

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Fungal extracellular ribotoxins as insecticidal agents

Olombrada¹,

Herrero-Galán²,

Tello³

et al. 2013

Insect Biochemistry and Molecular Biology

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Fungal ribotoxins were discovered almost 50 years ago as extracellular ribonucleases (RNases) with antitumoral properties. However, the biological function of these toxic proteins has remained elusive. The discovery of the ribotoxin HtA, produced by the invertebrates pathogen H. thompsonii, revived the old proposal that insecticidal activity would be their long searched function. Unfortunately, HtA is rather singular among all ribotoxins known in terms of sequence and structure similarities. Thus, it was intriguing to answer the question of whether HtA is just an exception or, on the contrary, the paradigmatic example of the ribotoxins function. The work presented uses HtA and -sarcin, the most representative member of the ribotoxins family, to show their strong toxic action against insect larvae and cells.

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Section: Protein Production and Purificationmentioning

confidence: 99%

Section: Protein Production and Purificationmentioning

confidence: 99%

Fungal extracellular ribotoxins as insecticidal agents

Olombrada¹,

Herrero-Galán²,

Tello³

et al. 2013

Insect Biochemistry and Molecular Biology

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“…Earlier gene and cDNA coding for restrictocin have been expressed in Aspergillus nidulans and Saccharomyces cerevisiae, respectively, to study the role of leader peptide on the survival of the host [7,8]. Ribotoxins, ~-sarcin and mitogillin have been expressed earlier using ompA and pelB signal sequences but the protein levels obtained were low [9][10][11][12][13]. Asp f L a homologue of mitogillin has also been expressed in E. coli but the recombinant protein was 10-fold less active than the native toxin [14].…”

Section: Introductionmentioning

confidence: 99%

Expression of ribonucleolytic toxin restrictocin in Escherichia coli: purification and characterization

1996

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Restrictocin is a toxin produced by the fungusAspergillus restrictus. The DNA coding for restrictocin was isolated from the host by polymerase chain reaction and cloned into a T7 promoter-based expression vector. The protein was overproduced in Escherichia coli and remained insoluble in the cell in the form of inclusion bodies. Recombinant restrictocin was purified in large amounts, by a simple denaturation-renaturation protocol involving a redox system, with typical yields of 45 mg/l of original culture. Restrictocin could be secreted into the bacterial medium using ompA, pelB and LTB signal sequences.Among the three signal sequences, ompA was found to be the most efficient in secreting the recombinant protein. The protein secreted into the extracellular medium was properly processed as evident by the amino-terminal sequencing. Recombinant restrictucin was readily purified to homogeneity from either the medium or inclusion bodies by simple chromatographic techniques and was found to be functionally as active as the native fungal protein in inhibiting the eukaryotic translation.

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“…Cloning procedures and DNA manipulations were carried out according to standard methods (Lacadena et al, 1994;Maassen, 1999;Schotte et al, 6 2000;Sambrook and Russell, 2001). Suitable deoxyoligonucleotides were used as primers for PCR amplification, using a series of plasmids constructed before as templates (Lacadena et al, 1994(Lacadena et al, , 1995García-Ortega et al, 2002, containing the cDNA corresponding to the five proteins studied: Asp f 1, α-sarcin, Asp f 1 Δ(7-22), and α-sarcins Δ(7-22) and H137Q.…”

Section: Cloning Proceduresmentioning

confidence: 99%

“…Suitable deoxyoligonucleotides were used as primers for PCR amplification, using a series of plasmids constructed before as templates (Lacadena et al, 1994(Lacadena et al, , 1995García-Ortega et al, 2002, containing the cDNA corresponding to the five proteins studied: Asp f 1, α-sarcin, Asp f 1 Δ(7-22), and α-sarcins Δ(7-22) and H137Q. These amplified DNA fragments were flanked by NgoMIV and BamHI restriction sites that were used to clone the proteins into the corresponding cloning sites of the plasmid pT1NX, in frame with the secretion signal leader of the lactococcal usp45 gene placed under the control of the constitutive promoter P1 (Steidler et al, 1995.…”

Section: Cloning Proceduresmentioning

confidence: 99%