OVERPRODUCTION AND PURIFICATION OF BIOLOGICALLY-ACTIVE NATIVE FUNGAL ALPHA-SARCIN IN ESCHERICHIA-COLI

An efficient system was developed to produce, in Escherichia coli, lar ge amounts of native alpha-sarcin (alpha Sar), a cytotoxin from the mo ld Aspergillus giganteus. The protein has been purified to homogeneity with a yield of 1.5 mu g/ml of original culture. The constructed expr ession vector (pINPG alpha S) is based on the synthesis of a fusion pr otein between alpha Sar and a modified version of the OmpA signal pept ide. This peptide seems to favour the postranslational processing of t he 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 compos ition, electrophoretic mobility, chromatographic behaviour, immunoreac tivity and spectroscopic features. Indeed, the recombinant protein rec overed is completely functional, since it cleaves, in vitro, eukaryoti c rRNA and it is able to interact with phospholipid vesicles with the same specificity as the native fungal alpha Sar.