Desplancq D, Kieffer B, Schmidt K, Posten C, Forster A, Oudet P, Strub J M, Van Dorsselaer A, Weiss E
Laboratoire de Biotechnologie des Interactions Macromoléculaires, FRE-CNRS 2370, France.
Protein Expr Purif. 2001 Oct;23(1):207-17. doi: 10.1006/prep.2001.1496.
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.
利用核磁共振（NMR）对生物分子进行结构研究依赖于富含（13）C和（15）N同位素的样品。虽然（13）C/（15）N标记的蛋白质通常是通过在含有昂贵标记前体混合物的条件下培养的转化大肠杆菌细胞中过表达获得的，但光合自养蓝藻鱼腥藻Anabaena sp. PCC 7120的蛋白质可以通过在含有Na（15）NO（3）和NaH（13）CO（3）作为唯一氮源和碳源的培养基中培养而实现均匀标记。我们在此报告一种新型载体 - 宿主系统，适用于在鱼腥藻Anabaena sp. PCC 7120中高效制备均匀（13）C/（15）N标记的蛋白质。作为测试蛋白的大肠杆菌gyrase B亚基的24 kDa N端结构域被克隆到受tac启动子控制的pRL25C穿梭载体中。将转化后的鱼腥藻细胞在标记的矿物盐存在下培养，并优化培养条件，以使组成型表达的24 kDa多肽中的（13）C和（15）N富集超过90％。在厌氧条件下进行双同位素标记后，纯化的24 kDa蛋白的产量与在市售标记培养基中平行培养的携带可比表达载体的大肠杆菌细胞所获得的产量相似。此外，通过NMR光谱和质谱检测，在鱼腥藻中表达的24 kDa N端结构域与大肠杆菌样品相同，表明其质量足以用于三维结构测定。考虑到所需标记物的成本，由于鱼腥藻系统具有更大的优势，这些结果表明鱼腥藻Anabaena sp. PCC 7120是用于结构研究的可溶性重组蛋白（13）C/（15）N标记的经济替代方案。
