J. Lacadena et al., OVERPRODUCTION AND PURIFICATION OF BIOLOGICALLY-ACTIVE NATIVE FUNGAL ALPHA-SARCIN IN ESCHERICHIA-COLI, Gene, 142(1), 1994, pp. 147-151
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.