Clavin, a type-1 ribosome-inactivating protein from Aspergillus clavatus IFO 8605. cDNA isolation, heterologous expression, biochemical and biological characterization of the recombinant protein.
We describe the cloning and expression of a new cDNA from the filamentous fungus Aspergillus clavatus IFO 8605. This cDNA contains an open reading frame (ORF) that predicts a putative ribonuclease precursor with 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 designated clavin was purified and characterized. Clavin shows typical type-1 RIP properties: specific cleavage of ribosomal and synthetic RNA and inhibition of protein synthesis in cell-free and cellular systems. When selectively targeted to a tumour cell antigen by coupling to a monoclonal antibody (mAb) clavin was able to inhibit protein synthesis at nanomolar concentration. Pharmacokinetics analysis in mice indicated an elimination half-life (t1/2 beta) of 7.4 h with no particular accumulation in major organs. Liver toxicity was very limited and transient while no alteration of kidney function was observed. Clavin induced a late and very low antibody response in mice. The in vitro and in vivo biological characteristics of clavin, together with its availability in large amounts, suggest the usefulness of this toxin in the production of toxic chemical conjugates.
Parente
D
D
Dipartimento di Biotecnologie, Menarini Ricerche Sud, Pomezia, Italy.
Raucci
G
G
Celano
B
B
Pacilli
A
A
Zanoni
L
L
Canevari
S
S
Adobati
E
E
Colnaghi
M I
MI
Dosio
F
F
Arpicco
S
S
Cattel
L
L
Mele
A
A
De Santis
R
R
D000595:Amino Acid Sequence; D000818:Animals; D001230:Aspergillus / Q000378:metabolism*; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D017931:DNA Primers; D018076:DNA, Complementary / Q000302:isolation & purification / Q000378:metabolism; D004722:Endoribonucleases; D005260:Female; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D006801:Humans; D000922:Immunotoxins / Q000493:pharmacokinetics / Q000633:toxicity; D008657:Metabolic Clearance Rate; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis / Q000187:drug effects; D011500:Protein Synthesis Inhibitors; D011817:Rabbits; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D012156:Reticulocytes / Q000378:metabolism; D012260:Ribonucleases; D012270:Ribosomes / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
D017931:DNA Primers; D018076:DNA, Complementary; D005656:Fungal Proteins; D000922:Immunotoxins; D011500:Protein Synthesis Inhibitors; D011994:Recombinant Proteins; C014801:alpha-sarcin; D004722:Endoribonucleases; D012260:Ribonucleases; C100767:CLA protein, Aspergillus clavatus