1. Silent mutations at the 5'-end of the cDNA of actinoporins from the sea anemone Stichodactyla helianthus allow their heterologous overproduction in Escherichia coli.
- Author
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Alegre-Cebollada J, Clementi G, Cunietti M, Porres C, Oñaderra M, Gavilanes JG, and Pozo AM
- Subjects
- Animals, Base Sequence, Circular Dichroism, Electrophoresis, Polyacrylamide Gel, Escherichia coli genetics, Molecular Sequence Data, Mutant Proteins biosynthesis, Mutant Proteins genetics, Mutant Proteins isolation & purification, Mutant Proteins metabolism, Nucleic Acid Conformation, Plasmids genetics, Plasmids metabolism, Protein Denaturation, Protein Isoforms biosynthesis, Protein Isoforms chemistry, Protein Isoforms isolation & purification, Protein Isoforms metabolism, Recombinant Proteins isolation & purification, Recombinant Proteins metabolism, DNA, Complementary genetics, Escherichia coli metabolism, Mutation genetics, Recombinant Proteins biosynthesis, Recombinant Proteins genetics, Sea Anemones genetics
- Abstract
Wild-type actinoporins StnI and StnII from the sea anemone Stichodactyla helianthus, as well as their NH(2)-terminal six-His tagged versions, have been overproduced in Escherichia coli. Overproduction of both wild-type proteins was only possible after introducing silent mutations within the 5'-end of their original cDNA sequences. These mutations would prevent the formation of RNA secondary structures blocking the ribosome-binding site and the initiation codon. The four recombinant proteins were purified to homogeneity in milligrams amount and characterized from spectroscopic and functional points of view. All the isolated proteins behaved as the corresponding natural ones although the six-His tagged variants exhibited a decreased lytic activity. The strategy described will be useful to allow the production of mutant variants of these proteins and probably of other actinoporins.
- Published
- 2007
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