一种来源于大菱鲆的热敏型尿嘧啶DNA糖苷酶的克隆表达及酶学性质鉴定

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

目的尿嘧啶DNA糖苷酶UDGase是一种广泛应用于qPCR、二代测序等领域的工具酶,由于其应用特性,只有热敏性UDGase才具有较大开发利用潜力。目前全球热敏性UDGase工具酶仅有2个物种来源,均有专利保护且价格昂贵,亟待开发新来源且具有优良热敏特性的UDGase。方法根据前人研究及序列分析推测大菱鲆(Scophthalmus maximus)具有热敏性UDGase。经验证,大菱鲆肝脏匀浆呈现UDGase活性。从大菱鲆肝脏匀浆克隆得到大菱鲆UDGase基因SmUDGase,并使用大肠杆菌工程菌株实现重组表达,分离纯化后进行活力表征检测。结果序列比对结果表明,SmUDGase基因克隆成功。重组表达并经亲和层析、离子交换层析分离纯化,获得纯酶纯度约95%,产率1.51mg/L,比活力2295.08U/mg。SmUDGase具有热敏性,在40℃时酶活即开始迅速降低。其他酶学性质,如pH适应范围、金属离子依赖性和对抑制剂的敏感性均与当前商业化UDGase一致。结论成功克隆并鉴定来自大菱鲆的新来源SmUDGase,该酶具有热敏感性,酶学特性接近目前商业化UDGase。并探索该酶的重组表达和纯化工艺,所得纯酶基本达到商业化生产水平,为该类型生物工具酶的开发提供了理论参考和技术储备。

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: Sequence 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. 51 mg/L,and a specific activity of 2 295. 08 U/mg. Sm UDGase 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 Sm UDGase 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.