火球菌复制蛋白A的表达纯化和活性研究

Expression, Purification and Biochemical Characterization of Replication Protein A from Pyrococcus Furiosus

摘要: 将火球菌(Pyrococcus furiosus)的3个复制蛋白A相关基因pfuRPA41(PF2020),pfuRPA32(PF2018),pfuRPA14(PF2019)克隆到pDEST17质粒,并在E.coli Rosetta(DE3)表达菌株中成功表达,最终通过固定化镍离子亲和层析分别得到纯度较高的三个复制蛋白A相关蛋白,对它们的单链DNA结合活性进行了详细测试.其中单独的pfuRPA41能结合单链DNA,而单独的pfuRPA32以及pfuRPA14不能结合单链DNA;然而pfuRPA32能够促进pfuRPA41的单链DNA结合能力.同时单链DNA长度对pfuRPA41结合单链DNA的能力有显著影响;在一定长度范围内,pfuRPA41结合单链DNA能力与单链DNA长度成正比

Abstract: The genes encoding replication protein A of Pyrococcus furiosus, including pfuRPA41(PF2020), pfuRPA32(PF2018) and pfuRPA14(PF2019), were cloned into plasmid pDest17. The proteins were successfully expressed in E. coli Rosetta (DE3). Purified recombinant proteins were obtained by using Ni²⁺affinity chromatography. Biochemical characterizations showed that pfuRPA41 alone had the single strand DNA binding activity, while pfuRPA32 or pfuRPA14 had little ssDNA binding activity. PfuRPA32 could greatly enhance the ssDNA binding activity of pfuRPA41. Meanwhile, the length of ssDNA could affect the ssDNA binding activity of pfuRPA41