Lacadena J, Martínez del Pozo A, Barbero J L, Mancheño J M, Gasset M, Oñaderra M, López-Otín C, Ortega S, García J, Gavilanes J G
Departamento de Bioquímica y Biología Molecular I, Universidad Complutense de Madrid, Spain.
Gene. 1994 May 3;142(1):147-51. doi: 10.1016/0378-1119(94)90370-0.
An efficient system was developed to produce, in Escherichia coli, large amounts of native alpha-sarcin (alpha Sar), a cytotoxin from the mold Aspergillus giganteus. The protein has been purified to homogeneity with a yield of 1.5 micrograms/ml of original culture. The constructed expression vector (pINPG alpha S) is based on the synthesis of a fusion protein between alpha Sar and a modified version of the OmpA signal peptide. This peptide seems to favour the postranslational processing of the fusion protein. The purified recombinant alpha-sarcin (re-alpha Sar) is structurally identical to the mature fungal protein according to the following criteria: N-terminal amino acid (aa) sequence, aa composition, electrophoretic mobility, chromatographic behaviour, immunoreactivity and spectroscopic features. Indeed, the recombinant protein recovered is completely functional, since it cleaves, in vitro, eukaryotic rRNA and it is able to interact with phospholipid vesicles with the same specificity as the native fungal alpha Sar.
已开发出一种高效系统，用于在大肠杆菌中大量生产天然α-杀稻瘟菌素（α Sar），这是一种来自巨大曲霉的细胞毒素。该蛋白质已被纯化至同质，产量为每毫升原始培养物1.5微克。构建的表达载体（pINPGαS）基于α Sar与OmpA信号肽修饰版本之间融合蛋白的合成。该肽似乎有利于融合蛋白的翻译后加工。根据以下标准，纯化的重组α-杀稻瘟菌素（re-α Sar）在结构上与成熟真菌蛋白相同：N端氨基酸（aa）序列、aa组成、电泳迁移率、色谱行为、免疫反应性和光谱特征。实际上，回收的重组蛋白具有完全功能，因为它在体外能切割真核生物rRNA，并且能够以与天然真菌α Sar相同的特异性与磷脂囊泡相互作用。
