Cloning, Expression and Characterization of a Heat-Labile Uracil-DNA lycosylase from <i>Scophthalmus maximus</i>

doi:10.13523/j.cb.20191005

China Biotechnology

2019, Vol. 39

Issue (10): 34-43 DOI: 10.13523/j.cb.20191005

Cloning, Expression and Characterization of a Heat-Labile Uracil-DNA lycosylase from Scophthalmus maximus

HAN Ting-han¹,ONG Xue-mei¹,ING Ya-fang²,U Chen¹,ZHANG Kun-xiao¹,AO song¹,U Heng-hao¹

1 Jiangsu Key Laboratory of Marine Bioresources and Environment, Jiangsu Key Laboratory of Marine harmaceutical Compound Screening,Co?Innovation Center of Jiangsu Marine Bio-ndustry Technology,Huaihai Institute of Technology, Lianyungang　222005, China
2 Wuhan Institute for Food and Cosmetic Control, Wuhan　430012, China

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Abstract

Objective: Uracil-DNA glycosylase (UDGase) is a tool enzyme widely used in qPCR, NGS and other related fields. Because of its application characters, only heat-labile UDGase has good application potential. There are only 2 species origins of heat-labile UDGase as developed tool enzymes, which are patent protected and expensive. Therefore developing new species origins of heat-labile UDGase is of urgent need.Methods: Based on previous studies and sequence analysis, it is speculated that Scophthalmus maximus has a heat-labile UDGase. It was confirmed that the liver homogenate exhibited UDGase activity. The gene of UDGase of Scophthalmus maximus, SmUDGase, was cloned from the liver homogenate. Recombinant expression of SmUDGase was achieved in E.coli, and the enzyme was purified and characterized.Results: equence alignments showed that the cloning of SmUDGase was successful. Gone through recombinant expression, purification with affinity and ion-exchange chromatography, the purified enzyme achieved a purity of 95%, a productive rate of 1.51mg/L, and a specific activity of 2 295.08U/mg. SmUDGase was heat-labile with a rapid decrease of activity above 40℃. For other enzymatic properties, such as pH range, metal ion dependency and sensitivity to the inhibitor, SmUDGase was consistent with the current commercial UDGase.Conclusion: The study successfully cloned and characterized a new species origin SmUDGase from Scophthalmus maximus. SmUDGase was heat-labile with other enzymatic properties close to current commercial UDGase. Recombinant expression and purification methods of the enzyme also were explored. The purified enzyme has basically reached the commercial production standards. These provided theoretical reference and technical reserve for development of tool enzymes of this kind.

Key words： Uracil-DNA glycosylase Scophthalmus maximus Heat-labile Recombinant expression

Received: 19 March 2019 Published: 12 November 2019

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	Ting-han HAN
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Cite this article:

HAN Ting-han,ONG Xue-mei,ING Ya-fang,U Chen,ZHANG Kun-xiao,AO song,U Heng-hao. Cloning, Expression and Characterization of a Heat-Labile Uracil-DNA lycosylase from Scophthalmus maximus. China Biotechnology, 2019, 39(10): 34-43.

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https://manu60.magtech.com.cn/biotech/10.13523/j.cb.20191005 OR https://manu60.magtech.com.cn/biotech/Y2019/V39/I10/34

Table 1 Primer list

Table 2 Enzyme activity of crude liver extract

Fig.1 Cloning of the Uracil-DNA glycosylase gene from Scophthalmus maximus (a) Sequence alignments of the UDGase protein sequences from E.coli (Ecoli UDG), Gadus morhua (cod UDG) and Scophthalmus maximus (SmUDG) (b) PCR amplified UDGase gene fragment from the cDNA sample

Fig.2 Recombinant expression and purification of SmUDGase (a) Schematic of pET28b-SmUDGase plasmid (b) SDS-PAGE analysis of the purification samples from different purification steps are indicated at the top of respective lanes

Table 3 Purification parameters

Fig.3 Enzyme activity and thermo sensitivity tests of SmUDGase (a) Kinetics of SmUDGase activity under different temperatures (b) Heat inactivation of UDGase from E.coli (Ecoli UDGase), Gadus morhua (Cod UDGase) and Scophthalmus maximus (SmUDGase) ^* P<0.05, ^** P<0.01

Fig.4 Characterization of the adaptation of SmUDGase (a) SmUDGase activity in pH range 6.0-11.0 (b) SmUDGase activity under different concentrations of Na⁺ and K⁺ (c) SmUDGase activity under different concentrations of Mg²⁺ and Ca²⁺ (d) Comparison of inactivation of SmUDGase by heat and Ugi ^* P<0.05, ^** P<0.01


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