Method for extracting 2′,3′-cyclic nucleoside monophosphates

A method for preparing 2′, 3′-cNMPs comprising steps of:

• (1) extracting genomic DNA from Escherichia coli K12, and amplifying translation initiation factor If3 gene which encodes translation initiation factor IF3 protein by polymerase chain reaction (PCR),
• wherein the
• F-GGAATTCCATATGATTAAAGGCGGAAAACG (SEQ ID NO:
• 1) and R-CCGCTCGAGCTGTTTCTTCTTAGG (SEQ ID NO:
• 2);
• (2) digesting the gene If3 from step (1) with specific restriction enzymes, ligating the digested If3 gene into vector pET-22b (+) that has been digested with the same restriction enzymes to generate an expression vector, transforming the generated expression vector into E. coli BL21 (DE3) to obtain a recombinant strain;
• c culturing the obtained recombinant strain and collecting the fermentation broth;
• wherein the restriction enzymes are Nde I and Xho I, and digesting is performed with:
• buffer
• 2 μl
• expression plasmid pET-22b/gene If3
• 8 μl
• restriction enzyme Nde I
• 1 μl
• restriction enzyme Xho I
• 1 μl
• sterilized distilled water
• 8 μl
• and under the following condition:
• water bath at 37° C. for 30 min;
• (3) collecting the cells from the fermentation broth of step (2) by centrifugation, breaking the collected cells and purifying the recombinant protein IF3 therein by Ni2+-nitrilotriacetic acid resin to obtain recombinant protein IF3 solution,
• incubating the recombinant protein IF3 solution at 0° C. for 2.5-3.5 days to release 2′, 3′-cNMPs from the recombinant protein IF3, centrifuging the recombinant protein IF3 solution and collecting the supernatant containing 2′, 3′-cNMPs;
• (4) ultra-filtrating the supernatant from step (3) to remove proteins and obtain filtrate, and preparing four kinds of 2′, 3′-cNMPs from the filtrate by high-performance liquid chromatographic (HPLC) on a C18 reversed-phase column.