Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II.
Uniform 13C isotope labeling of proteins with sodium acetate for NMR studies: application to human carbonic anhydrase II.
复制标题
用乙酸钠对蛋白质进行统一 13C 同位素标记用于 NMR 研究：在人碳酸酐酶 II 上的应用。
DOI：
10.1021/bi00232a017
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发表时间：
1991
期刊：
影响因子：
2.9
通讯作者：
Fierke,CA
中科院分区：
文献类型：
--
作者：
Venters,RA;Calderone,TL;Spicer,LD;Fierke,CA
杜克大学医学中心生物化学系和放射学系，达勒姆，北卡罗来纳州27710，1990年12月4日接收; 1991年1月8日接收的修订版Mandarin pt摘要：用于NMR研究的蛋白质的统一双标记可能非常昂贵，即使使用有效的表达和纯化方案，这主要是由于[13 C6，99%]葡萄糖的高成本。我们在这里证明，均匀（> 95%）的13 C和15 N双标记的蛋白质可以通过在含有[1，2- 13 C2，99%]乙酸钠作为唯一碳源和[15 N，99%]氯化铵作为唯一氮源的限定培养基中培养细胞来制备用于NMR结构/功能研究。此外，我们证明，这种标记方案可以扩展到包括均匀的碳同位素标记到任何所需的水平（低于50%），通过利用培养基含有等量的[1- 13 C，99%]乙酸钠和[2- 13 C，99%]乙酸钠与未标记的乙酸钠。这种技术比使用富含同位素的藻类水解物标记更简单，劳动强度更低。这些标记方案已被用于成功地制备同位素富集的人碳酸酐酶II的NMR量。通过该技术标记的蛋白质的活性和1H NMR谱与从含有标记的葡萄糖的培养基产生的蛋白质获得的活性和1H NMR谱相同;然而，[1，2- 13 C2，99%]乙酸钠生长培养基的成本显著低于[13 C6，99%]葡萄糖生长培养基的成本。我们在这里报告的第一个公布的13 C和15 N NMR光谱的人碳酸酐酶II作为一个重要的步骤，导致分配这29-kDa的锌金属酶。
Department of Biochemistry and Department of Radiology, Duke University Medical Center, Durham, North Carolina 27710 Received December 4, 1990; Revised Manuscript Received January 8, 1991 abstract: Uniform double labeling of proteins for NMR studies can be prohibitively expensive, even with an efficient expression and purification scheme, due largely to the high cost of [13C6, 99%] glucose. We demonstrate here that uniformly (> 95%) 13C and 15N double-labeled proteins can be prepared for NMR structure/function studies by growing cells in defined media containing sodium [1, 2-13C2, 99%] acetate as the sole carbon source and [15N, 99%] ammonium chloride as the sole nitrogen source. In addition, we demonstrate that this labeling scheme can be extended to includeuniform carbon isotope labeling to any desired level (below 50%) by utilizing media containing equal amounts of sodium [1-13C, 99%] acetate and sodium [2-13C, 99%] acetate in conjunction with unlabeled sodium acetate. This technique is less labor intensive and more straightforward than labelingusing isotope-enriched algal hydrolysates. These labeling schemes have been used to successfully prepare NMR quantities of isotopically enriched human carbonic anhydrase II. The activity and the'H NMR spectra of the protein labeled by this technique are the same as those obtained from the protein produced from media containing labeled glucose; however, the cost of the sodium [1, 2-13C2, 99%] acetate growth media is considerably less than the cost of the [13C6, 99%] glucose growth media. We report here the first published 13C and 15N NMR spectra of human carbonic anhydrase II as an important step leading to the assignment of this 29-kDa zinc metalloenzyme.