Leahy P, Carmichael G G, Rossomando E F
Department of BioStructure and Function, University of Connecticut Health Center, Farmington 06030, USA.
Bioconjug Chem. 1996 Sep-Oct;7(5):545-51. doi: 10.1021/bc960044q.
A new method for coupling proteins to plasmid expression vectors is presented. Biotin was covalently attached to a plasmid expression vector containing a chloramphenicol acetyltransferase (CAT) gene. The specific label was one biotin per 100 bp. An electrophoretic mobility shift assay showed that the plasmid was capable of binding multiple streptavidin molecules. When transfected into mouse fibroblasts, the biotinylated plasmid retained 40% of the native plasmid's biological activity, as determined by CAT assay, and was not affected by the binding of streptavidin. The method allows for attachment of any protein to plasmid DNA expression vector while retaining biological function. Hybrid plasmids in which the transcription cassettes were kept free of biotin label were constructed by digesting biotinylated and unbiotinylated plasmids at sites outside the transcription cassette and re-ligating the digestion products. Electron microscopy studies show that the ligation products formed large tangled assemblages of plasmid DNA. When equimolar (with respect to gene number) amounts of these large hybrid biotinylated plasmids were transfected into mouse fibroblasts by means of calcium phosphate precipitation, an increase in CAT expression 25-fold greater than that of original biotinylated plasmid was observed. Slot-blot analysis of total DNA extracted from transfected cells shows that this enhanced activity was not due to increased transfection efficiency. Receptor-mediated delivery could not be shown when a complex comprising biotinylated asialoglycoprotein/streptavidin/biotinylated CAT expression vector was placed in media containing Hep G2 cells.
本文介绍了一种将蛋白质与质粒表达载体偶联的新方法。将生物素共价连接到含有氯霉素乙酰转移酶（CAT）基因的质粒表达载体上。特异性标记为每100 bp一个生物素。电泳迁移率变动分析表明该质粒能够结合多个链霉亲和素分子。当转染到小鼠成纤维细胞中时，通过CAT分析测定，生物素化的质粒保留了天然质粒40%的生物学活性，且不受链霉亲和素结合的影响。该方法允许在保留生物学功能的同时将任何蛋白质连接到质粒DNA表达载体上。通过在转录盒外部的位点消化生物素化和未生物素化的质粒并重新连接消化产物，构建了转录盒不含生物素标记的杂交质粒。电子显微镜研究表明，连接产物形成了质粒DNA的大的缠结聚集体。当通过磷酸钙沉淀将等摩尔量（相对于基因数量）的这些大的杂交生物素化质粒转染到小鼠成纤维细胞中时，观察到CAT表达比原始生物素化质粒增加了25倍。对从转染细胞中提取的总DNA进行狭缝印迹分析表明，这种增强的活性不是由于转染效率的提高。当将包含生物素化去唾液酸糖蛋白/链霉亲和素/生物素化CAT表达载体的复合物置于含有Hep G2细胞的培养基中时，未显示出受体介导的递送。
