MODIFICATION OF RNA-RELATED ENZYMES FOR ENHANCED PRODUCTION
The present invention provides, among other things, methods and compositions for large-scale production of capped mRNA using SUMO-Guanylyl Transferase fusion protein.
1 .- 6 . (canceled)
7 . A fusion protein, wherein the fusion protein comprises guanylyl transferase and a small ubiquitin-like molecule (SUMO) protein comprising 90% identity to SEQ ID NO: 8 and SEQ ID NO: 7.
8 . The fusion protein of claim 7 , wherein the guanylyl transferase comprises SEQ ID NO: 8 and SEQ ID NO: 7.
9 .- 10 . (canceled)
11 . The fusion protein of claim 7 , wherein the fusion protein has comparable phosphatase activity, guanylyl transferase activity and methylation activity relative to a wild-type guanylyl transferase protein.
12 . A vector encoding a fusion protein comprising guanylyl transferase protein and a small ubiquitin-like molecule (SUMO) protein.
13 . The vector of claim 12 , wherein the vector comprises SEQ ID NO: 1 and SEQ ID NO: 2.
14 . The vector of claim 12 , wherein the vector comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3.
15 . The vector of claim 12 , wherein the vector comprises SEQ ID NO: 4 and SEQ ID NO: 3.
16 . A method to produce a guanylyl transferase by fermentation, comprising: a) culturing in a fermentation medium a microorganism that is transformed with at least one recombinant nucleic acid molecule comprising a nucleic acid sequence encoding a guanylyl transferase fusion protein that has an amino acid sequence that is at least 90% identical to SEQ ID NO: 8 and SEQ ID NO: 7; and b) collecting a product produced from the step of culturing.
17 . (canceled)
18 . The method of claim 16 , wherein the guanylyl transferase fusion protein has comparable phosphatase activity, guanylyl transferase activity and methylation activity relative to a wild-type guanylyl transferase protein.
19 . The method of claim 16 , wherein the guanylyl transferase fusion protein comprises a small ubiquitin-like molecule (SUMO) protein.
20 . The method of claim 19 , wherein the guanylyl transferase fusion protein comprises SEQ ID NO: 8.
21 . The method of claim 19 , wherein the SUMO protein is bound to the guanylyl transferase by a covalent link.
22 . The method of claim 21 , wherein the covalent link is between the SUMO protein and a large subunit of the guanylyl transferase.
23 . The method of claim 16 , wherein the fermentation medium is selected from the group consisting of Terrific Broth, Cinnabar, 2×YT and LB.
24 . The method of claim 16 , wherein the microorganism is a bacterium.
25 . The method of claim 16 , wherein the nucleic acid sequence encoding the guanylyl transferase is at least 90% identical to SEQ ID NO: 2 and SEQ ID NO: 3.
26 . The method of claim 16 , wherein the recombinant nucleic acid molecule further comprises a nucleic acid sequence encoding a small ubiquitin-like molecule (SUMO) protein.
27 . The method of claim 26 , wherein the nucleic acid sequence encoding a small ubiquitin-like molecule (SUMO) protein is at least 90% identical to SEQ ID NO: 1.
28 . The method of claim 16 , wherein the product is a guanylyl transferase.
29 . The method of claim 28 , wherein the product is a guanylyl transferase fusion protein that further comprises a small ubiquitin-like molecule (SUMO) protein.
30 . (canceled)