Cost-effective and uniform C-13- and N-15-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp PCC 7120

Citation
D. Desplancq et al., Cost-effective and uniform C-13- and N-15-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp PCC 7120, PROT EX PUR, 23(1), 2001, pp. 207-217
Citations number
39
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Biochemistry & Biophysics
Journal title
PROTEIN EXPRESSION AND PURIFICATION
ISSN journal
1046-5928 → ACNP
Volume
23
Issue
1
Year of publication
2001
Pages
207 - 217
Database
ISI
SICI code
1046-5928(200110)23:1<207:CAUCAN>2.0.ZU;2-E
Abstract
Structural studies of biomolecules using nuclear magnetic resonance (NMR) r ely on the availability of samples enriched in C-13 and N-15 isotopes. Whil e C-13/N-5-labeled proteins are generally obtained by overexpression in tra nsformed Escherichia coli cells cultured in the presence of an expensive mi xture of labeled precursors, those of the photoautotrophic cyanobacterium A nabaena sp. PCC 7120 can be uniformly labeled by growing them in medium con taining (NaNO3)-N-15 and (NaHCO3)-C-13 as the sole nitrogen and carbon sour ces. We report here a novel vector-host system suitable for the efficient p reparation of uniformly C-13/N-15-labeled proteins in Anabaena sp. PCC 7120 . The 24-kDa N-terminal domain of the E. coli gyrase B subunit, used as a t est protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter. The transformed Anabaena cells were grown in the presenc e of the labeled mineral salts and culture conditions were optimized to obt ain over 90% of 13C and 15N enrichment in the constitutively expressed 24-k Da polypeptide. The yield of purified 24-kDa protein after dual isotope lab eling under anaerobic conditions was similar to that obtained with E. coli cells bearing a comparable expression vector and cultured in parallel in a commercially available labeling medium. Furthermore, as probed by NMR spect roscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in An abaena was identical to the E. coli sample, demonstrating that it was of su fficient quality for 3D-structure determination. Because the Anabaena syste m was far more advantageous taking into consideration the expense for the l abels that were necessary, these results indicate that Anabaena sp. PCC 712 0 is an economic alternative for the C-13/N-15-labeling of soluble recombin ant proteins destined for structural studies. (C) 2001 Academic Press.