ABSTRACT
Proteins provided with unique functional groups such as affinity labels or fluorescence moieties offer high potential in many biotechnological or biomedical investigations, e.g., immobilization studies or high throughput screenings. An attractive alternative to known posttranslational methods o f protein modification is the site-directed cotranslational incorpora tion o f unnatural amino acids. Here we point out different aspects o f this method with regard to the synthesis o f protein conjugates bearing different functionalities. Moreover we describe a cell-free system enabling tRNA-based synthesis o f modified proteins even with large functional groups in high yields. This specialized cell-free system contains a substantially decreased level o f release factor 1 (RF1) and originates from an E . coli strain encoding a tagged RF1 variant. We present the efficient amber suppressor tRNA-mediated incorporation o f the unnatural amino acid biocytin (biotinylated lysine) into stoichiometrically defined protein conjugates, containing the biotin label at the desired position. As potential applications we demonstrate the usefiilness o f the system for the immobilization o f biotinylated proteins direcdy from a cell-free system and for interaction studies. Finally we show the cotranslational incorporation o f a large fluorescent amino acid using our system.
