Objective To predict the characteristics and function of Cthe_2401 protein,and perform the cloning,expression and purification of Escherichia coli in vitro.Methods The sequence and evolutionary characteristics of Cthe_2401 protein were analyzed by the National Center for Biotechnology Information database(NCBI)and protein Conserved Domain database(CDD),and SWISS-MODEL online software was used to conduct homology modeling and predict the structure and function of the protein.Cthe_2401 gene was amplified by polymerase chain reaction(PCR),and the pET28a-Cthe_2401 expression plasmid was constructed with the pET28a vector.Cthe_2401 protein expression was induced by using isopropylthioate galactose(IPTG)after transformation of E.coli.Cthe_2401 protein was purified using 50％ammonium sulfate precipitation,Ni-NTA column as well as SephadexG75 dextran gel column.Results Bioinformatic predictions suggest that the Cthe_2401 protein belongs to the Veg protein family and may be related to biofilm occurrence and sporulation.Cthe_2401 protein was successfully cloned and expressed in E.coli and purified in vitro to obtain protein bands of about 10 ku in size,consistent with the expected results.Conclusion The function of Cthe_2401 protein is successfully predicted,and the cloning,expression and purification of Cthe_2401 protein in E.coli are successfully obtained.
关键词
热纤梭菌/Cthe_2401/Veg蛋白/生物信息学/蛋白表达与纯化/大肠杆菌
Key words
Clostridium thermocellum/Cthe_2401/Veg protein/bioinformatics/porotein expression and purification/Escherichia coli
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