Immobilized poly(n)polymerase
The present invention relates to an immobilized poly(N)polymerase (PNP), methods of producing said PNP and uses thereof. Further disclosed is an enzyme reactor and kit comprising the PNP for producing polynucleotidylated ribonucleic acid poly(N)RNA)molecules which are useful in gene therapy, immunotherapy, protein replacement therapy and/or vaccination.
1. A method of producing polyadenylated ribonucleic acid (poly(A)RNA) molecules comprising contacting an RNA molecule with a poly(A)polymerase covalently immobilized onto a solid support, said poly(A)polymerase comprising at least 90% sequence identity to SEQ ID NO: 113, wherein the poly(A)polymerase comprises at least one newly introduced cysteine residue compared to a native poly(A)polymerase, wherein the at least one newly introduced cysteine residue is located at a terminus of the protein, wherein the poly(A)polymerase is immobilized via a thiol group of the at least one newly introduced cysteine residue, and wherein the at least one cysteine residue is attached to the poly(A)polymerase via a peptide linker element comprising the sequence of SEQ ID NO: 162 (GlyGlyGlyGly), and,
wherein at least 80% of the produced poly(A)RNA has a poly(A) length of at least 100 nucleotides.
2. The method according to claim 1 , wherein the poly(A)polymerase is immobilized by covalent binding to a thiol-activated solid support.
3. The method according to claim 1 , wherein the covalent binding is a disulfide bridge, thioester bond or a thioether bond.
4. The method according to claim 1 , wherein the solid support comprises a member selected from the group consisting of sepharose, thiopropyl-sepharose, sephadex, agarose, silica, magnetic beads, methacrylate beads, and nanoparticles, preferably the solid support comprises a member selected from the group consisting of sepharose, thiopropyl-sepharose, sephadex, agarose, silica, magnetic beads, and nanoparticles.
5. The method according to claim 1 , wherein the solid support is selected from the group consisting of activated thiol sepharose, thiopropyl-sepharose, thiol-activated sephadex, thiol-activated agarose, silica-based thiol-activated matrix, silica-based thiol-activated magnetic beads, pyridyl disulfide-functionalized nanoparticles, maleimide-activated agarose and mixtures thereof.
6. The method according to claim 1 , wherein the poly(A)polymerase comprises an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:113.
7. The method according to claim 1 , wherein the poly(A)polymerase comprises only one cysteine residue.
8. The method according to claim 1 , wherein the peptide linker element comprises the sequence of SEQ ID NO: 168 (Gly 4 SerGly 4 ).
9. The method according to claim 1 , wherein the poly(A)polymerase comprises a purification tag as depicted in SEQ ID NOs: 181-201.
10. The method of claim 1 , wherein the poly(A)polymerase is immobilized onto the solid support through a single cysteine residue.
11. The method of claim 1 , wherein each of the poly(A)RNA molecules comprises at least 120 adenylates.
12. The method of claim 1 , wherein the poly(A)polymerase covalently immobilized onto a solid support is used for more than one enzymatic polyadenylation cycle.
13. The method of claim 1 , wherein the method comprises at least one ATP feeding step.
14. The method of claim 6 , wherein the poly(A)polymerase comprises only one cysteine residue.
15. The method of claim 6 , wherein the poly(A)polymerase comprises an amino acid sequence having at least 98% sequence identity to the amino acid sequence of SEQ ID NO:113.
16. The method of claim 15 , wherein the poly(A)polymerase comprises the amino acid sequence of SEQ ID NO: 113.
17. The method of claim 1 , wherein the RNA molecule comprises a 5′ Cap.