Genetically modified bacteria producing three dna repair enzymes and method for the evaluation of dna repair activity
AssigneesUniversidad de la República, Ministerio De Defensa Nacional—Instituto Antartico Uruguayo
InventorsJuan José MARIZCURRENA, Susana CASTRO SOWINSKI
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Abstract
Three recombinant E. coli strains produce the enzymes CPD-photolyase, 6,4-bifunctional photolyase and 6,4-photolyase, from bacterial Antarctic isolates of the genus Hymenobacter the first one and Sphingomonas the others. It is also disclosed a process of production and purification of the recombinant enzymes with high performance, high degree of purity and high catalytic repair activity, having applications in, but it is not limited to, cosmetics and pharmaceutical industry. A fast, cheap and qualitative method is provided for the determination of the CPD photolyase activity.
Claims17
A method for the production of at least one recombinant photolyase selected from CPD-photolyases, 6,4-bifunctional photolyases and 6,4-photolyases that comprises the transformation of Escherichia coli bacteria with the host vector pET28a (+) comprising one optimized gene sequence selected from bacterial Antarctic isolates of the genus Hymenobacter for the CPD photolyase and Sphingomonas for the 6,4-photolyase and the 6,4-bifunctional photolyase, culturing for their expression and purification.
An isolated nucleic acid molecule encoding photolyases consisting of the amino acid sequence of SEQ ID No2, SEQ ID No4 or SEQ ID No6.
A method for the determination of qualitative CPD-photolyase activity consisting in:
- UVC-irradiating the plasmid puc18-2 with 1000 J m−2 using a Spectroline lamp;
- evaluating the full radiation by MseI digestion and 1% agarose-gel electrophoresis at room temperature, for 1 h at 60V, using TAE as running buffer;
- visualizing DNA-restriction fragments by staining with ethidium bromide;
- and
- determining the DNA-damage by the absence of the 780 kbp DNA fragment corresponding to the miniTn5 cassette when the irradiated plasmid pUC18-2 is 25 previously incubated with CPD-photolyases.
Dependencies are inferred from the claim text.