Individual Expression of Recombinant α- and β-Tubulin from Haemonchus contortus: Polymerization and Drug Effects

Oxberry, M.E.; Geary, Timothy G.; Winterrowd, C.A.; Prichard, Roger K.

doi:10.1006/prep.2000.1347

Cited by 30 publications

(21 citation statements)

References 33 publications

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“…Initially, these oryzalin analogs were evaluated in vitro for their ability to inhibit the polymerization of tubulin purified from L. amazonensis (Werbovetz et al, 1999), and ongoing drug discovery efforts against this promising target will continue to require in vitro evaluation of novel compounds against purified leishmanial tubulin. Reports detailing the expression of tubulin from parasites have appeared (MacDonald et al, 2003;Fennell et al, 2006), some of which have demonstrated the assembly of recombinant αβ tubulin (Oxberry et al, 2001;MacDonald et al, 2004). No such expression system currently exists for the leishmanial protein, however, and antileishmanial drug discovery work will continue to rely on the purification of tubulin directly from the parasite in the near future.…”

Section: Introductionmentioning

confidence: 99%

Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

Yakovich

Ragone

Alfonzo

et al. 2006

Experimental Parasitology

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Previously, tubulin has been purified from Leishmania amazonensis and used to identify novel molecules with selective antimitotic activity. However, Leishmania amazonensis is pathogenic and requires a relatively expensive medium for large-scale cultivation. Herein, the purification and characterization of tubulin from the non-pathogenic Leishmania tarentolae is reported, together with the sequence of α-and β-tubulin from this organism. This protein was purified by sonication, diethylaminoethyl-Sepharose chromatography, and one assembly-disassembly cycle in 1% overall recovery based on total cellular protein. L. tarentolae tubulin was indistinguishable from the corresponding L. amazonensis protein in terms of binding affinity for dinitroaniline sulfanilamides and sensitivity to assembly inhibition by these compounds. The amino acid sequences derived from the L. tarentolae α-and β-tubulin genes were 99.6% and 99.4% identical to the corresponding amino acid sequences from the L. major Friedlin strain. These results indicate that tubulin from L. tarentolae is suitable for use in drug screening.

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Section: Introductionmentioning

confidence: 99%

Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

Yakovich

Ragone

Alfonzo

et al. 2006

Experimental Parasitology

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“…Such -and -homodimers have been reported in recombinant nematode tubulin. 34) Polymerization occurred at both GTP concentrations tested and in the absence of GTP (Fig. 6).…”

Section: Polymerization Of Tubulinmentioning

confidence: 85%

“…34) Optimal formation of typical long microtubule-like structures occurred at ! 1 mg/ml of each tubulin concentration (Fig.…”

Section: Polymerization Of Tubulinmentioning

confidence: 99%

Cloning, Purification, and Polymerization ofCapsicum annuumRecombinant α and β Tubulin

Jang

Kim

Kalme

et al. 2008

Bioscience, Biotechnology, and Biochemistry

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“…Furthermore, ABZ interaction with β-tubulin is well established in a variety of fungi, protozoa and helminths (Fetterer 1986;Lacey and Prichard 1986;Lubega and Prichard 1990;Lubega and Prichard 1991;Jimenez-Gonzalez et al 1991;Cruz and Edlind 1997;Oxberry et al, 2001;MacDonald et al 2004;Henriquez et al 2008). The interaction of ABZ, but not TCBZ, with crude liver fluke tubulin preparations has been demonstrated experimentally by displacement of radio-labelled colchicine (Fetterer 1986).…”

Section: Introductionmentioning

confidence: 94%

Liver fluke β-tubulin isotype 2 binds albendazole and is thus a probable target of this drug

et al. 2010

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Albendazole is a benzimidazole drug which can be used to treat liver fluke (Fasciola hepatica) infections. Its mode of action is believed to be the inhibition of microtubule formation through binding to β-tubulin. However, F. hepatica expresses at least six different isotypes of β-tubulin, and this has confused, rather than clarified, understanding of the molecular mechanisms of benzimidazole drugs in this organism. Recombinant F. hepatica β-tubulin proteins were expressed in, and purified from, Escherichia coli. These proteins were then used in pull-down assays in which albendazole was covalently linked to Sepharose. β-Tubulin isotype 2 was pulled down in this assay, and this interaction could be reduced by adding competing albendazole. Molecular modelling of β-tubulin isotypes suggests that changes in the side change conformations of residue 200 in the putative albendazole binding site may be important in determining whether, or not, a particular isotype will bind to the drug. These results, together with previous work demonstrating that albendazole causes disruption of microtubules in the liver fluke, strongly suggest that β-tubulin isotype 2 is one of the targets of this drug.

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Individual Expression of Recombinant α- and β-Tubulin from Haemonchus contortus: Polymerization and Drug Effects

Cited by 30 publications

References 33 publications

Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

Leishmania tarentolae: Purification and characterization of tubulin and its suitability for antileishmanial drug screening

Cloning, Purification, and Polymerization ofCapsicum annuumRecombinant α and β Tubulin

Liver fluke β-tubulin isotype 2 binds albendazole and is thus a probable target of this drug

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