Refolding of the Cupressus arizonica major pollen allergen Cup a1.02 overexpressed in Escherichia coli. (Articolo in rivista)

Type
Label
  • Refolding of the Cupressus arizonica major pollen allergen Cup a1.02 overexpressed in Escherichia coli. (Articolo in rivista) (literal)
Anno
  • 2004-01-01T00:00:00+01:00 (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#doi
  • 10.1016/j.pep.2004.06.034 (literal)
Alternative label
  • Rea G., Iacovacci P., Ferrante P., Zelli M., Brunetto B., Lamba D., Boffi A., Pini C., Federico R. (2004)
    Refolding of the Cupressus arizonica major pollen allergen Cup a1.02 overexpressed in Escherichia coli.
    in Protein expression and purification (Print)
    (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#autori
  • Rea G., Iacovacci P., Ferrante P., Zelli M., Brunetto B., Lamba D., Boffi A., Pini C., Federico R. (literal)
Pagina inizio
  • 419 (literal)
Pagina fine
  • 425 (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#url
  • http://www.elsevier.com/locate/yprep (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#numeroVolume
  • 37 (literal)
Rivista
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#pagineTotali
  • 7 (literal)
Note
  • ISI Web of Science (WOS) (literal)
Http://www.cnr.it/ontology/cnr/pubblicazioni.owl#affiliazioni
  • Rea G. -IC-CNR, Roma Iacovacci P. -Istituto Superiore di Sanità, Roma Ferrante P. -Department of Biology, Università degli Studi “Roma Tre”, Zelli M. -Department of Biology, Università degli Studi “Roma Tre”, Brunetto B. -Istituto Superiore di Sanità, Roma Lamba D. -IC-CNR, Trieste Boffi A. -Dep. of Biochemical Sciences “Alessandro Rossi Fanelli”, Università di Roma “La Sapienza” Pini C. -Istituto Superiore di Sanità, Roma Federico R. -Department of Biology, Università degli Studi “Roma Tre” (literal)
Titolo
  • Refolding of the Cupressus arizonica major pollen allergen Cup a1.02 overexpressed in Escherichia coli. (literal)
Abstract
  • The cDNA encoding an isoform of the cypress major pollen allergen, Cup a1.02, has been cloned and expressed in Escherichia coli as a N-terminal 6× His-tagged protein. To increase recovery, Cup a1.02 was expressed at high levels exploiting the T5 strong promoter and led to accumulate as inclusion bodies. The insoluble purified aggregates were solubilized in 6 M guanidine hydrochloride, immobilized using nickel-chelating affinity chromatography, and successfully refolded by controlled removal of the chaotropic reagent. Enhanced protein refolding was observed by reducing the protein concentration at 0.6–0.8 mg/ml. SDS–PAGE and gel filtration chromatography indicated an apparent molecular mass of H40 kDa and the occurrence of the protein as monomers. The reconstituted fusion protein displayed the same immunological properties of the native Cup a1.02 protein as proven by IgE immunoreactivity. Immunoblotting, ELISA, and histamine release test showed that the tag did not preclude the protein functionality hence validating its correct three-dimensional folding. The protein fold was also assessed by CD spectroscopy and deconvolution of the spectrum allowed to estimate the secondary structure as a prevalence of ² structures (higher than 60%) and a small contribution from ± helices (less than 12%). The reported procedure has proven to be useful for the production of multi-milligrams of recombinant Cup a1.02 allergen suitable for structural biology studies and for the molecular and functional characterization of the IgE binding sites. (literal)
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