CLAVIN A TYPE-1 RIBOSOME-INACTIVATING PROTEIN FROM ASPERGILLUS-CLAVATUS IFO-8605 - CDNA ISOLATION, HETEROLOGOUS EXPRESSION, BIOCHEMICAL ANDBIOLOGICAL CHARACTERIZATION OF THE RECOMBINANT PROTEIN

We describe the cloning and expression of a new cDNA from the filament ous fungus Aspergillus clavatus IFO 8605. This cDNA contains an open r eading frame (ORF) that predicts a putative ribonuclease precursor wit h high similarity to the alpha-sarcin family of ribosome-inactivating proteins (RIPs). The cDNA encoding the mature protein was expressed in Escherichia coli, and the recombinant protein, a 17-kDa polypeptide d esignated clavin was purified and characterized. Clavin shows typical type-1 RIP properties: specific cleavage of ribosomal and synthetic RN A and inhibition of protein synthesis in cell-free and cellular system s. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able ro inhibit protein synthesi s at nanomolar concentration. Pharmacokinetics analysis in mice indica ted an elimination half-life ((t1/2 beta)) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and tra nsient while no alteration of kidney function was observed. Clavin ind uced a late and very low antibody response in mice. The in vitro and i n vivo biological characteristics of clavin, together with its availab ility in large amounts, suggest the usefulness of this toxin in the pr oduction of toxic chemical conjugates.