Overproduction and purification of biologically active native fungal alpha-sarcin in Escherichia coli.
An efficient system was developed to produce, in Escherichia coli, large amounts of native alpha-sarcin (alpha Sar), a cytotoxin from the mold Aspergillus giganteus. The protein has been purified to homogeneity with a yield of 1.5 micrograms/ml of original culture. The constructed expression vector (pINPG alpha S) is based on the synthesis of a fusion protein between alpha Sar and a modified version of the OmpA signal peptide. This peptide seems to favour the postranslational processing of the fusion protein. The purified recombinant alpha-sarcin (re-alpha Sar) is structurally identical to the mature fungal protein according to the following criteria: N-terminal amino acid (aa) sequence, aa composition, electrophoretic mobility, chromatographic behaviour, immunoreactivity and spectroscopic features. Indeed, the recombinant protein recovered is completely functional, since it cleaves, in vitro, eukaryotic rRNA and it is able to interact with phospholipid vesicles with the same specificity as the native fungal alpha Sar.
Lacadena
J
J
Departamento de Bioquímica y Biología Molecular I, Universidad Complutense de Madrid, Spain.
Martínez del Pozo
A
A
Barbero
J L
JL
Mancheño
J M
JM
Gasset
M
M
Oñaderra
M
M
López-Otín
C
C
Ortega
S
S
García
J
J
Gavilanes
J G
JG
D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000302:isolation & purification*; D001230:Aspergillus / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D004274:DNA, Recombinant; D004722:Endoribonucleases; D004926:Escherichia coli; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008969:Molecular Sequence Data; D010957:Plasmids; D011500:Protein Synthesis Inhibitors / Q000302:isolation & purification*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification
D000970:Antineoplastic Agents; D004274:DNA, Recombinant; D005656:Fungal Proteins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C014801:alpha-sarcin; D004722:Endoribonucleases