To investigate the enzymatic properties of histone lysine demethylase, the demethylases originated from zebrafish(Lsd1) and coffee(Jmjc) were cloned and heterologously expressed in E. coli BL21(DE3), respectively. The fermentation process was further optimized, and the enzymatic properties were studied after the purification of the expressed products via affinity chromatography on a nickel column. Based on the results, two E. coli recombinants (BL21 (DE3)/pET28a-lsD1 and BL21(DE3)/pET28a-jmjC) were constructed successfully. The soluble target protein could be obtained under 25 ℃ and 0.1 mmol/L isopropyl-β-D-thiogalactopyranoside(IPTG) induction. These two enzymes could be further purified by eluting with 100 mmol/L and 200 mmol/L imidazole. In terms of reaction activity, the corresponding optimum temperature and pH for Lsd1 were 35 ℃ and 7.0, while for Jmjc were 20 ℃ and 8.0. Comparing Lsd1 with Jmjc in stability, Lsd1 showed better thermostability, but Jmjc was found to have better tolerance to the change of pH. The enzyme activity of these two demethylases was promoted by the addition of Mn2+. In contrast, Co2+, Mg2+, Ca2+, Zn2+ and Ni2+ would limit the activity on both enzymes to varies extent. According to the kinetic study, the catalytic coefficient of these two enzymes was relatively low since the overall reaction time was long. The kcat/Km of Lsd1 and Jmjc were 8.23×10-6 L/(mol·s) and 1.06×10-5 L/(mol·s), respectively. The current results could provide meaningful information for the application of histone lysine demethylase in the field of the degradation of non-histone N—CH3 compound as well as the research about the development of inhibitors in vitro.
LI Jinrong
,
XIN Yu
,
SHI Guiyang
,
GU Zhenghua
,
ZHANG Liang
. Recombinant expression and characterization of histone lysine specific demethylase in Escherichia coli[J]. Food and Fermentation Industries, 2021
, 47(24)
: 71
-76
.
DOI: 10.13995/j.cnki.11-1802/ts.027435
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