Cost-effective and uniform (13)C- and (15)N-labeling of the 24-kDa N-terminal domain of the Escherichia coli gyrase B by overexpression in the photoautotrophic cyanobacterium Anabaena sp. PCC 7120.
Structural studies of biomolecules using nuclear magnetic resonance (NMR) rely on the availability of samples enriched in (13)C and (15)N isotopes. While (13)C/(15)N-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 Na(15)NO(3) and NaH(13)CO(3) as the sole nitrogen and carbon sources. We report here a novel vector-host system suitable for the efficient preparation of uniformly (13)C/(15)N-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 (13)C and (15)N 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 (13)C/(15)N-labeling of soluble recombinant proteins destined for structural studies.
Desplancq
D
D
Laboratoire de Biotechnologie des Interactions Macromoléculaires, FRE-CNRS 2370, France. desplanc@esbs.u-strasbg.fr
Kieffer
B
B
Schmidt
K
K
Posten
C
C
Forster
A
A
Oudet
P
P
Strub
J M
JM
Van Dorsselaer
A
A
Weiss
E
E
D002247:Carbon Isotopes / Q000191:economics / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods*; D000458:Cyanobacteria / Q000235:genetics*; D027081:DNA Gyrase / Q000096:biosynthesis* / Q000737:chemistry; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes / Q000191:economics / Q000378:metabolism; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D014169:Transformation, Bacterial
D002247:Carbon Isotopes; D029968:Escherichia coli Proteins; D009587:Nitrogen Isotopes; D011994:Recombinant Proteins; D027081:DNA Gyrase