Enzyme based system for production of messenger RNA with increased transfection efficiency
A method for the production of proteins used in the in vitro transcription (IVT) of messenger RNA (mRNA), wherein the proteins are evaluated for purity and efficacy by the efficiency with which mRNA synthetically derived therefrom, subsequently transfects cells and produces encoded proteins.
1. A method for in vitro synthesis of mRNA, the method comprising:
transfecting a first plurality of competent bacterial cells with a first plasmid vector comprising an arabinose promoter linked to a nucleic acid sequence encoding T7 RNA polymerase, wherein the first plasmid vector comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 19;
transfecting a second plurality of competent bacterial cells with a second plasmid vector comprising at least one arabinose promoter linked to a nucleic acid sequence encoding vaccinia virus capping enzyme (VVCE), wherein the second plasmid vector comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 20;
transfecting a third plurality of competent bacterial cells with a third plasmid vector comprising a UUG start codon, an arabinose promoter linked to a nucleic acid sequence encoding polyadenosine (poly (A)) polymerase, wherein the third plasmid vector comprises a nucleotide sequence that is at least 95% identical to SEQ ID NO: 21;
expressing and purifying T7 RNA polymerase from the first plurality of competent bacterial cells;
expressing and purifying VVCE from the second plurality of competent bacterial cells;
expressing and purifying poly (A) polymerase from the third plurality of competent bacterial cells;
adding linearized plasmid DNA to a first composition comprising the purified T7 RNA polymerase to produce RNA transcripts;
adding the RNA transcripts to a second composition comprising the purified VVCE to produce capped RNA transcripts;
adding the capped RNA transcripts to a third composition comprising the purified poly (A) polymerase to produce polyadenylated capped mRNA transcripts; and
purifying mRNA from the third composition.
2. The method of claim 1 , wherein the first plasmid vector comprises a nucleotide sequence that comprises SEQ ID NO: 19.
3. The method of claim 1 , wherein the second plasmid vector comprises a nucleotide sequence that comprises SEQ ID NO: 20.
4. The method of claim 1 , wherein the third plasmid vector comprises a nucleotide sequence that comprises SEQ ID NO: 21.
5. The method of claim 1 , wherein the bacterial competent cells are E. Coli BL21 (DE3) competent cells.
6. The method of claim 1 , wherein VVCE is expressed at 18° C.
7. The method of claim 1 , wherein VVCE is expressed over an 18 hour fermentation period.