Better M, Bernhard S L, Lei S P, Fishwild D M, Carroll S F
XOMA Corporation, Santa Monica, California 90404.
J Biol Chem. 1992 Aug 15;267(23):16712-8.
A synthetic gene for the Aspergillus protein toxin mitogillin has been synthesized and expressed in Escherichia coli. The recombinant mitogillin is a potent inhibitor of protein synthesis in vitro with an IC50 of 9.7 pM. Immunoconjugates of recombinant mitogillin derivatized with S-acetylmercaptosuccinic anhydride and 5-methyl-2-iminothiolane modified H65 antibody kill T cell lines and peripheral blood mononuclear cells expressing the human CD5 surface antigen. Native mitogillin contains 4 cysteine residues which form two disulfide pairs (Fernandez-Luna, J. L., Lopez-Otin, C., Soriano, F., and Mendez, E. (1985) Biochemistry 24, 861-867). Three derivatives of mitogillin have been assembled which substitute alanine residues for cysteine residues 5, 147, or 5 and 147. Each of these molecules retains the ability to inhibit protein synthesis in vitro with at most a 2-fold reduction in activity. The derivative mitogillinC147A can be conjugated to 5-methyl-2-iminothiolane- modified H65 antibody directly without pretreatment with S-acetylmercaptosuccinic anhydride, and the immunoconjugate is active against HSB2 cells. Genetic manipulation of toxin genes to expose an accessible cysteine residue into a recombinant product can thus be used to generate immunotoxins without initial derivatization by nonspecific cross-linking reagents.
已合成了曲霉属蛋白毒素米托吉林的合成基因，并在大肠杆菌中进行了表达。重组米托吉林是一种体外蛋白质合成的有效抑制剂，IC50为9.7 pM。用S - 乙酰巯基琥珀酸酐衍生化的重组米托吉林与5 - 甲基 - 2 - 亚氨基硫杂环戊烷修饰的H65抗体的免疫缀合物可杀死表达人CD5表面抗原的T细胞系和外周血单个核细胞。天然米托吉林含有4个半胱氨酸残基，形成两对二硫键（Fernandez - Luna, J. L., Lopez - Otin, C., Soriano, F., and Mendez, E. (1985) Biochemistry 24, 861 - 867）。已构建了三种米托吉林衍生物，用丙氨酸残基取代半胱氨酸残基5、147或5和147。这些分子中的每一个都保留了体外抑制蛋白质合成的能力，活性最多降低2倍。衍生物米托吉林C147A可以直接与5 - 甲基 - 2 - 亚氨基硫杂环戊烷修饰的H65抗体偶联，无需用S - 乙酰巯基琥珀酸酐预处理，并且该免疫缀合物对HSB2细胞具有活性。因此，对毒素基因进行基因操作，使重组产物中暴露一个可接近的半胱氨酸残基，可用于生成无需用非特异性交联试剂进行初始衍生化的免疫毒素。
