Krishzyme mRNA Cap 2 -O-methyltransferase was derived from a recombinant E. coli strain that carries the gene for the vaccinia mRNA Cap 2 -O-Methyltransferase. This enzyme adds a methyl group at the 2 -O position of the first nucleotide adjacent to the cap structure at the 5 end of the RNA. The enzyme utilizes S-adenosylmethionine (SAM) as a methyl donor to methylate capped RNA (cap-0) resulting in a cap-1 structure.
The Cap 1 structure can increase the translation efficiency, improving the expression of mRNA in transfection and microinjection experiments. This enzyme specifically requires RNA with an m7GpppN cap as substrate. It cannot utilize RNA with pN, ppN, pppN or GpppN at the 5 end. Capped RNA may be prepared via in vitro transcription using cap analog or through enzymatic capping using the Vaccinia Capping Enzyme.
Storage buffer 20 mM Tris-HC lpH 8.0 25 ), 100 mM NaCl, 1 mM DTT, 0.1 mM EDTA, 0.1% Triton X-100 50% glycerol.
Unit Definition One unit is defined as the amount of enzyme required to methylate 10 pmoles of 80 nt capped RNA transcript in 1 hour at 37 C.
Specifications:
Exonuclease: 50 U of mRNA Cap 2 -O-Methyltransferase with 1 ug gamaa-Hind III digest DNA at 37 C for 16 hours yields no degradation as determined by agarose gel electrophoresis.
Endonuclease: 50 U of mRNA Cap 2 -O-Methyltransferase with 1 ug gamaa DNA at 37 C for 16 hours yields no degradation as determined by agarose gel electrophoresis.
Nickase: 50 U of mRNA Cap 2 -O-Methyltransferase with 1 ug pBR322 at 37 C for 16 hours yields no degradation as determined by agarose gel electrophoresis.
RNase: 50 U of mRNA Cap 2 -O-Methyltransferase with 1.6 ug MS2 RNA for 4 hours at 37 C yields no degradation as determined by agarose gel electrophoresis E.coli DNA: 50 U of mRNA Cap 2 -O-Methyltransferase is screened for the presence of E. coli genomic DNA using TaqMan qPCR with primers specific for the E. coli 16S rRNA locus. The E. coli genomic DNA contamination is 0.1 pg/50 U.
Bacterial Endotoxin: 10 EU/mg.
Application:
To improve mRNA expression during microinjection and transfection experiments.