Cost-Effective and Uniform ¹³C- and ¹⁵N-Labeling of the 24-kDa N-Terminal Domain of the Escherichia coli Gyrase B by Overexpression in the Photoautotrophic Cyanobacterium Anabaena sp. PCC 7120

Authors: Desplancq D.¹; Kieffer B.²; Schmidt K.³; Posten C.³; Forster A.⁴; Oudet P.⁴; Strub J-M.⁵; Van Dorsselaer A.⁵; Weiss E.¹

Source: Protein Expression and Purification, Volume 23, Number 1, October 2001 , pp. 207-217(11)

Publisher: Academic Press

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Abstract:

Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in ¹³C and ¹⁵N isotopes. While ¹³C/¹⁵N-labeled proteins are generally obtained by overexpression in transformed Escherichia coli cells cultured in the presence of an expensive mixture of labeled precursors, those of the photoautotrophic cyanobacterium Anabaena sp. PCC 7120 can be uniformly labeled by growing them in medium containing Na¹⁵NO₃ and NaH¹³CO₃ as the sole nitrogen and carbon sources. We report here a novel vector-host system suitable for the efficient preparation of uniformly ¹³C/¹⁵N-labeled proteins in Anabaena sp. PCC 7120. The 24-kDa N-terminal domain of the E. coli gyrase B subunit, used as a test protein, was cloned into the pRL25C shuttle vector under the control of the tac promoter. The transformed Anabaena cells were grown in the presence of the labeled mineral salts and culture conditions were optimized to obtain over 90% of ¹³C and ¹⁵N enrichment in the constitutively expressed 24-kDa polypeptide. The yield of purified 24-kDa protein after dual isotope labeling 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 spectroscopy and mass spectrometry, the 24-kDa N-terminal domain expressed in Anabaena was identical to the E. coli sample, demonstrating that it was of sufficient quality for 3D-structure determination. Because the Anabaena system was far more advantageous taking into consideration the expense for the labels that were necessary, these results indicate that Anabaena sp. PCC 7120 is an economic alternative for the ¹³C/¹⁵N-labeling of soluble recombinant proteins destined for structural studies. Copyright 2001 Academic Press.

Keywords: cyanobacteria; Anabaena sp. PCC 7120; ¹³C/¹⁵N-labeling; overexpression; N-terminal domain of E. coli gyrase B; NMR.

Language: English

Document Type: Research article

Affiliations: 1: Laboratoire de Biotechnologie des Interactions Macromoléculaires, FRE-CNRS 2370 2: Laboratoire de RMN, Ecole Supérieure de Biotechnologie de Strasbourg, boulevard Sébastien Brandt, Illkirch, 67400, France 3: Universität Karlsruhe, Karlsruhe, 76128, Germany 4: Institut de Génétique et de Biologie Moléculaire et Cellulaire, 1 rue Laurent Fries, Illkirch, 67400, France 5: Laboratoire de Spectrométrie de Masse Bio-organique, 1 rue Blaise Pascal, Strasbourg, 67000, France

Publication date: 2001-10-01