The expression of saporin, a ribosome-inactivating protein from the plant Saponaria officinalis, in Escherichia coli.
We have isolated and sequenced genomic clones from the DNA of Saponaria officinalis using a cDNA probe that encodes proteins with high homology to saporin-6, one of the most potent of the ribosome-inactivating proteins that is currently used for the construction of immunotoxins and mitotoxins. Sequence differences in the clones suggest a multigene family of proteins. These data agree with observations of several different proteins with ribosome-inactivating protein activity and similar structure. Two of the genomic clones encode proteins that have identical sequences to two of the four isoforms of saporin-6. We have inserted the DNA from one genomic clone into an Escherichia coli expression system that encodes a signal sequence for export to the bacterial periplasmic space. Exportation is observed and the isolated gene product has ribosome-inactivation activity similar to the native protein. Sequence analysis shows differential processing of the remaining plant signal sequence. The majority of the expressed protein remains intracellular and this material also shows high specific activity and differential processing. Saporin as an immunotoxin in clinical trial and as a mitotoxin in experimental models has been extremely efficacious. These data indicate the ability to produce fusion proteins with saporin and cell-binding ligands for production of new reagents for further clinical and experimental use.
Barthelemy
I
I
Department of Molecular and Cellular Growth Biology, Whittier Institute for Diabetes and Endocrinology, La Jolla, California 92037.
Martineau
D
D
Ong
M
M
Matsunami
R
R
Ling
N
N
Benatti
L
L
Cavallaro
U
U
Soria
M
M
Lappi
D A
DA
D000595:Amino Acid Sequence; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli; D015870:Gene Expression; D000922:Immunotoxins; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010944:Plants; D016133:Polymerase Chain Reaction; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000378:metabolism*; D000076985:Saporins; D017386:Sequence Homology, Amino Acid
D000922:Immunotoxins; D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D004247:DNA; D009699:N-Glycosyl Hydrolases; D000076985:Saporins