Huang Jenq-Kuen, Tsai Shuhui, Huang George H, Gowda Prashanth G, Walzer Andrew M, Wen Lisa
Department of Chemistry, Western Illinois University, One University Circle, Macomb, IL 61455, USA.
Protein Expr Purif. 2004 May;35(1):32-8. doi: 10.1016/j.pep.2003.12.017.
Mature eukaryotic initiation factor 5A (eIF5A) is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (Nepsilon-(4-amino-2(R)-hydroxybutyl)lysine). The synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Deoxyhypusine synthase is the first of the two enzymes that catalyzes the maturation of eIF5A. We have subcloned the cDNA encoding bovine and human deoxyhypusine synthase into a pET-11a expression vector, separately. T7-tagged bovine and human deoxyhypusine synthase have been overexpressed in Escherichia coli and purified to homogeneity using T7 antibody affinity chromatography. Activities of the enzyme from both human and bovine have been measured by their ability to convert the eIF5A precursor protein to the intermediate, deoxyhypusine form of eIF5A. Our results have shown that bovine deoxyhypusine synthase has considerably higher activity than human deoxyhypusine synthase in catalyzing the synthesis of deoxyhypusine.
成熟的真核生物起始因子5A（eIF5A）是真核细胞中唯一已知的含有罕见氨基酸hypusine（Nε-（4-氨基-2（R）-羟基丁基）赖氨酸）的蛋白质。hypusine的合成对于eIF5A在真核细胞增殖和存活中的功能至关重要。脱氧hypusine合酶是催化eIF5A成熟的两种酶中的第一种。我们已将编码牛和人脱氧hypusine合酶的cDNA分别亚克隆到pET-11a表达载体中。带有T7标签的牛和人脱氧hypusine合酶已在大肠杆菌中过表达，并使用T7抗体亲和层析纯化至均一。通过它们将eIF5A前体蛋白转化为eIF5A的中间脱氧hypusine形式的能力来测定人和牛的该酶活性。我们的结果表明，牛脱氧hypusine合酶在催化脱氧hypusine的合成方面比人脱氧hypusine合酶具有更高的活性。
