Silent mutations at the 5'-end of the cDNA of actinoporins from the sea anemone Stichodactyla helianthus allow their heterologous overproduction in Escherichia coli.
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.
Alegre-Cebollada
Jorge
J
Departamento de Bioquímica y Biología Molecular I, Facultad de Química, Universidad Complutense, 28040 Madrid, Spain.
Clementi
Giorgia
G
Cunietti
Michela
M
Porres
Christian
C
Oñaderra
Mercedes
M
Gavilanes
José G
JG
Pozo
Alvaro Martínez Del
AM
D000818:Animals; D001483:Base Sequence; D002942:Circular Dichroism; D018076:DNA, Complementary / Q000235:genetics*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D011489:Protein Denaturation; D020033:Protein Isoforms / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D012615:Sea Anemones / Q000235:genetics*
D018076:DNA, Complementary; D050505:Mutant Proteins; D020033:Protein Isoforms; D011994:Recombinant Proteins