Buffer solution for enhancing activity of TET enzyme

[0027] Example 1: Effects of four buffer systems on TET enzyme activity.

[0028] In this example, we verified the 3-(N-morpholino)propanesulfonic acid sodium salt-sodium hydroxide buffer system (Mops-NaOH), tris-hydrochloric acid buffer system (Tris-HCl) , bis-hydroxyethyltrismethylolmethylamine-sodium hydroxide buffer system (Bis-Tris-NaOH) or 4-hydroxyethylpiperazine sulfuric acid-potassium hydroxide buffer system (Hepes-KOH) four reactions The impact of the system on each TET enzyme, the specific implementation is as follows:

[0029] TET enzyme reaction buffer includes 10-100 mM buffer system (pH 7.5), 10-300 mM sodium chloride, 0.1-10 mM ascorbic acid (L-AA), 0.1-10 mM s adenosine triphosphate (ATP), 0.1-10 mM α - Ketoglutarate (α-KG), 30-300 uM ferrous ammonium sulfate, 0.1-5 mM dithiothreitol.

[0030] Epigentek's Epigenase 5mC-hydroxylase TET activity / inhibition assay kit (fluorescence method) was used to measure NgTET1, mTET1, mTET2, hTET1, hTET2, and hTET3 accordin

[0031] Example 2: Effect of small molecule additives on TET enzyme activity.

[0032] In this example, we verified the effects of different small molecule additives on TET enzyme activity. Small molecule additives are 1 mM hydroquinone, 1 mM tetrafluoro-p-benzoquinone, 1 mM tetrachloro-p-benzoquinone, 1 mM tetrabromo-p-benzoquinone, 1 mM tetraiodobenzoquinone, 1 mM L-cysteine , 1 mM L-proline, 1 mM potassium L-glutamate, 1 mM tris(2-carboxyethyl)phosphine, 1 mM dithiothreitol, 1 mM thioethylene glycol. Using Epigentek's Epigenase 5mC-hydroxylase TET activity / inhibition assay kit (fluorescence method), add the above-mentioned small molecule additives respectively, and measure the enzyme activity of TET according to the procedure in the manual. The enzyme activity assay results were as figure 2 , phenol and quinone compounds (hydroquinone, tetrafluoro-p-benzoquinone, tetrachloro-p-benzoquinone, tetrabromo-p-benzoquinone and tetraiodobenzoquinone), amino acid analogs (L-cystein

[0033] Example 3: Effects of concentrations of hydroquinone, chloro-p-benzoquinone, L-cysteine ​​and tris(2-carboxyethyl)phosphine on TET enzyme activity.

[0034] In this example, we verified the effects of hydroquinone, chloro-p-benzoquinone, L-cysteine ​​and tris (2-carboxyethyl) phosphine on the enzyme activity of NgTET1 in the TET enzyme reaction buffer. Refer to Examples 1 and 2 for the flow process. The result is as image 3 As indicated, 1-5 mM hydroquinone, 0.5-1 mM chloranil, 2-10 mM L-cysteine, and 0.1-0.5 mM tris(2-carboxyethyl)phosphine have the effect on TETase Facilitation works best.