Ribosome-inactivating proteins, inactive precursor forms thereof, a process for making and a method of using
AssigneeDowElanco
InventorsTerence A. Walsh, Timothy D. Hey, Alice E. R. Morgan
Life of this patent
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Abstract
The present invention is directed to a ribosome inactivating proteins. The proteins are characterized by being in a single chain proRIP inactive form that can be converted into an active form by cleavage with proteases.
Claims7
A substantially pure protein having the amino acid sequence of SEQ ID NO:2, termed pro-Ribosome Inactivating Protein (proRIP), wherein the proRip has a selectively removable, internal peptide linker sequence that has from about 9 to about 25 amino acids which correspond to residues 162 through 186 of SEQ ID NO:2 or a 70% homologous sequence that will display biological inhibitor activity and is incapable of substantially inactivating eukaryotic ribosomes, but which can be converted by removal of the linker into a protein having .alpha. and .beta. fragments and being capable of substantially inactivating eukaryotic ribosomes, wherein the .alpha. fragment has the amino acid sequence of residues 17 to 161 of SEQ ID NO:2 and the .beta. fragment has the amino acid sequence of residues 187 to 287 of FIG. 1, SEQ ID NO:2.
A substantially pure protein, termed a pro-Ribosome inactivating Protein (proRIP), comprising a selectively removable, internal peptide linker sequence and is incapable of substantially inactivating eukaryotic ribosomes, but it can be converted by removal of the linker into a protein having .alpha. and .beta. fragments, termed a RIP, that is capable of substantially inactivating eukaryotic ribosomes, said proRIP wherein, (1) the RIP is selected from the group consisting of Barley Translation Inhibitor (SEQ ID NO:26), Ricin A-chain RIP (SEQ ID NO:27), Abrin-A A-chain (SEQ ID NO:71), Saporin (SEQ ID NO:72), SLT-1 RIP (SEQ ID NO:73), Trichosanthin (SEQ ID NO:74), Luffin-A (SEQ ID NO:75), MAP (SEQ ID NO:76), Ricinus communis agglutinin (SEQ ID NO:77), Momordin (SEQ ID NO:78), PAP-S (SEQ ID NO:79), Luffin-B (SEQ-ID NO:80), and Dianthin 30 (SEQ ID NO:81, (2) a removable, internal peptide linker sequence that inhibits RIP activity and is 70% homologous to residues 162-186 of SEQ ID NO:2, (3) the proRIP is generated by inserting the linker peptide of (2) into a selected sequence of (1), and is inserted into said selected sequence of (1) in between any two amino acid residues within the following sequences:
- amino acid residues 148-158 of Barley Translation Inhibit or (SEQ ID NO:26), amino acid residues 152-162 of Ricin A-chain RIP (SEQ. ID NO:27), amino acid residues 138-148 of Abrin-A A-chain (SEQ ID NO:71), amino acid residues 153-163 of Saporin (SEQ ID NO:72), amino acid residues of 145-155 of SLT-1 RIP (SEQ ID NO:73), amino acid residues 139-149 of Trichosanthin (SEQ ID NO:74), amino acid residues 138-148 of Luffin-A (SEQ ID NO:75), amino acid residues 145-155 of MAP (SEQ ID NO:76), amino acid residues 152-162 of Ricinus communis agglutinin (SEQ ID NO:77), amino acid residues 138-148 of Momordin (SEQ ID NO:78), amino acid residues 151-161 of PAP-S (SEQ ID NO:79), amino acid residues 139-149 of Luffin-B (SEQ ID NO:80), or amino acid residues 174-184 of Dianthin 30 (SEQ ID NO:81).
A substantially pure protein, termed a Ribosome Inactivating Protein (RIP), having .alpha. and .beta. fragments and being capable of substantially inactivating eukaryotic ribosomes, wherein the .alpha. fragment has the amino acid sequence of residues 17 to 161 of SEQ ID NO:2 and the .beta. fragment has the amino acid sequence of residues 187 to 287 of SEQ ID NO:2.
A fusion protein capable of substantially inactivating eukaryotic ribosomes, said protein comprising an amino acid sequence selected from the group consisting of SEQ ID NOS:7, 9, 11, 13, 15, 17, 19, 21, 23, and 25.
A method for converting a pro-Ribosome Inactivating Protein (proRIP) into a RIP, said method comprising the following steps:
- a) providing a homogeneous protein, termed a proRIP, wherein the proRIP has a selectively removable, internal peptide linker sequence and is incapable of substantially inactivating eukaryotic ribosomes, but which can be converted by removal of the linker into a protein having .alpha. and .beta. fragments, termed a RIP, that is capable of substantially inactivating eukaryotic ribosomes;
- and
- b) contacting the proRIP with a cleaving agent capable of deleting the linker to form a protein having .alpha. and .beta. fragments, termed a RIP, that is capable of substantially inactivating eukaryotic ribosomes.
Dependencies are inferred from the claim text.