Characterization of a saporin isoform with lower ribosome-inhibiting activity

Abstract

We have expressed in Escherichia coli five isoforms of saporin, a single-chain ribosome-inactivating protein (RIP). Translation inhibition activities of the purified recombinant polypeptides in itro were compared with those of recombinant dianthin 30, a less potent and closely related RIP, and of ricin A chain. Dianthin 30, and a saporin isoform encoded by a cDNA from leaf tissue (SAP-C), both had about one order of magnitude lower activity in translation inhibition assays than all other isoforms of saporin tested. We recently demonstrated that saporin extracted from seeds of Saponaria officinalis binds to α2-macroglobulin receptor (α2MR ; also termed low density lipoprotein-receptor-related-protein), indicating a general mechanism of interaction of plant RIPs with the α2MR system [Cavallaro, Nykjaer, Nielsen and Soria (1995) Eur. J. Biochem.

Saporin as a novel suicide gene in anticancer gene therapy

“…RTA has a very low lysine content and a slightly acidic pI whereas 10% of the amino acids in SAP are lysine residues, which confer also an extremely high pI (almost 10) to this polypeptide. In addition, SAP can be internalized through members of the low-density lipoprotein-related receptor family 15,16 and has a much higher cell-free RIP activity, compared to RTA. 15,16 Acting catalytically, SAP has very high intrinsic activity as it has been established that only a few molecules of toxin reaching the cytosolic compartment are sufficient to inhibit protein synthesis and cause cell death.…”

“…In addition, SAP can be internalized through members of the low-density lipoprotein-related receptor family 15,16 and has a much higher cell-free RIP activity, compared to RTA. 15,16 Acting catalytically, SAP has very high intrinsic activity as it has been established that only a few molecules of toxin reaching the cytosolic compartment are sufficient to inhibit protein synthesis and cause cell death. 17 Seed-extracted SAP has been widely used to develop mouse models selectively impaired in central nervous system functions.…”

Expression and purification of cysteine introduced recombinant saporin

“…In our case, the important factors for the choice of the mutation site were: 1) the modification should not affect ribosome binding (i.e., should not be in the catalytic site of the saporin), 2) the engineered site should be easily accessible for conjugation, and 3) the new protein should be stable. Previous studies show that all residues in the catalytic active site cleft of RIP proteins are conserved 15 and mutations at these catalytic active site cleft residues cause a drastic loss of activity 33 . In addition, our observations indicate that mutations in the N terminus produce unstable proteins (unpublished).…”

“…The new product was named as Cys255sap-3. NcoI and EcoRI enzyme restriction sites were preserved at the ends of the recombinant saporin gene, allowing cloning into pET11d plasmid using the same cloning strategy as Fabbrini et al (1997) 15 . This eliminated the T7 terminator on the plasmid; but did not have an appreciable effect on protein expression yield.…”

“…Saporin, a type I RIP with a molecular weight of 30 kDa, is extracted from the seeds of Saponaria officinalis L. Saporin destroys both eukaryotic and prokaryotic ribosomes 15,16,17 . There are five isoforms of saporin isolated from Saponaria officinalis L., four are from the seed of the plant (isoforms 1, 3, 4 and 6) 18 and one is isolated from the leaf of the plant 19,20 .…”