Sechiumin, a ribosome-inactivating protein from the edible gourd, Sechium edule Swartz - Purification, characterization, molecular cloning and expression
Journal
European Journal of Biochemistry
Journal Volume
255
Journal Issue
2
Pages
400-408
Date Issued
1998
Author(s)
Abstract
A new ribosome-inactivating protein (RIP), sechiumin, was purified from the seeds of edible gourd, Sechium edule Swartz by gel-filtration and ion-exchange chromatography, with an apparent relative molecular mass of 27 kDa. It inhibits the protein synthesis of rabbit reticulocyte lysate strongly with a concentration causing 50% inhibition (IC50) of 0.7 nM, but has a much lower inhibitory effect on intact HeLa cells, with an IC50 of 5000 nM. Sechiumin has a highly specific RNA N-glycosidase activity towards 28S rRNA, as does the A-chain of abrin. It suggests that sechiumin is one of the type-I ribosome-inactivating proteins. The cDNA of sechiumin has been cloned and expressed using a pET expression system in Escherichia coli. The sechiumin cDNA has 951 nucleotides, encoding a protein with 285 amino acids. The amino acid sequence deduced from the cDNA reveals that the first 21 N-terminal amino acid residues constitutes a signal peptide. Sechiumin has nearly 60% similarity to several type-I RIPs purified from the Cucurbitaceae family, such as luffin-a (62.5%) and trichosanthin (64.8%), but less similarity to other type-I RIPs. Two amino acid residues, Glu160 and Arg163, at the putative active site of sechiumin, are known to be catalytically active in ricin and abrin. The N-terminal amino acid sequence of sechiumin is very Similar to that of trichosanthin. The recombinant sechiumin was obtained as an insoluble protein, and the preparation of the active soluble form was achieved by renaturing the denatured protein. These studies suggest that the recombinant sechiumin could be used for the preparation of immunotoxin as a potential cancer chemotherapeutic agent.
Subjects
Cytotoxicity; Inhibition of protein synthesis; Ribosome-inactivating protein; Sechium edule Swartz; Sechiumin
SDGs
Other Subjects
glycosidase; recombinant protein; ribosome inactivating protein; amino acid sequence; article; enzyme activity; Escherichia coli; gel filtration; in vitro study; ion exchange chromatography; molecular cloning; molecular weight; nonhuman; phytochemistry; priority journal; protein analysis; protein purification; protein synthesis inhibition; reticulocyte lysate; Sechium edule
Publisher
Blackwell Publishing Ltd
Type
journal article