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Preparation, optimization and characterization of a polyphenylalanine synthetizing system from Agrobacterium
C Alegre1, R Iglesias, J M Ferreras
1Departamento de Bioquimíca, Biología Molecular y Fisiología, Facultad de Ciencias, Universidad de Valladolid, Spain.
Abstract:
A very active cell-free translation system was prepared from Agrobacterium tumefaciens, a bacterium broadly used to transfect plant cells to introduce foreign genes and one that produces tumours in plants. Once optimized for Mg2+, NH4+, high speed supernatant S 370, purified ribosomes and time, the system translates polyuridylic acid very efficiently. A. tumefaciens purified ribosomes were inhibited in vitro by several well-known translational inhibitors including some ribosome-inactivating proteins. Treatment of A. tumefaciens purified ribosomes with type 1-RIP crotin 2 lead to the depurination of the 23S rRNA which, upon treatment with acid aniline, released a diagnostic RNA fragment of about 235 nucleotides.