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n4噬菌体n4ssb蛋白和大肠杆菌rna聚合酶的原核表达、纯化及互作验证
首发时间：2024-07-26
摘要：目的验证n4噬菌体编码的单链dna结合蛋白（n4-coded single-stranded dna-binding protein，n4ssb）和大肠杆菌rna聚合酶（rna polymerase，rnap）之间是否存在相互作用，解析n4ssb调控大肠杆菌rnap转录n4噬菌体晚期基因的分子机制。方法本文构建了his标签位于a亚基c端的rna聚合酶核心酶蛋白表达质粒、未携带标签的σ蛋白表达质粒以及his标签位于n端的n4ssb蛋白表达质粒；并利用大肠杆菌异源表达系统分别表达n4ssb和大肠杆菌rnap；优化纯化过程，获得均一稳定的活性蛋白；最终通过凝胶过滤层析验证这两种蛋白之间是否存在相互作用；若存在相互作用，则继续利用结构生物学、生物化学等方法解析n4ssb蛋白调控rnap转录其后期基因的分子机制。结果成功构建his标签在a亚基c端的rna聚合酶核心酶蛋白表达质粒、未携带标签的σ蛋白表达质粒以及his标签位于n端的n4ssb蛋白表达质粒；利用大肠杆菌异源表达系统表达获得n4ssb蛋白和his标签分别在β\'亚基c端和a亚基c端的rnap；优化蛋白纯化条件，获得均一稳定的目的蛋白；凝胶过滤层析验证发现n4ssb与不同标签位置的大肠杆菌rnap均不存在相互作用。结论凝胶过滤层析无法验证n4ssb和大肠杆菌rnap之间的相互作用，可能需要特殊的启动子dna序列辅助n4ssb结合并调控rnap或者通过其他方法验证。
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prokaryotic expression, purification and interaction verification of the bacteriophage n4-coded single-stranded dna-binding protein and escherichia coli rna polymerase
abstract：objective verify whether there is an interaction between n4 phage-encoded single-stranded dna-binding protein (n4ssb) and escherichia coli (e. coli) rna polymerase (rnap), and explore the mechanism by which n4ssb regulates the transcription of the n4 phage late gene by e. colirnap. method construct protein expression plasmids for rnap core with his tag at the c-terminus of the α-subunit and for untagged σ, as well as protein expression plasmids for n4ssb with his tag at the n-terminus. express n4ssb and e. coli rnap through e. coli heterologous expression system; optimize the purification method to obtain homogeneous and stable active proteins, prokaryotic expression, purification and interaction verification of the n4ssb) and e.coli rnap. ultimately verify the existence of interactions between the two proteins by gel filtration chromatography. .continue to analyze the molecular mechanism of n4ssb regulating the transcription of late n4 gene by escherichia coli rnap by structural biology and biochemistry, if there is an interaction.results successfully construct protein expression plasmids for the rnap core enzyme with his tag at the c-terminal of the a-subunit, a protein expression plasmid for σ that does not carry the tag, and a protein expression plasmid for n4ssb with his tag located at the n-terminal. successfully express n4ssb and rnap with his tags at the c-terminal of β\' subunit and c-terminal of α-subunit, respectively using e. coli heterologous expression system. optimize the protein purification conditions and finally obtain homogeneous and stable target protein. gel filtration chromatography validation revealed that n4ssb did not interact with e.coli rnap with different labeling positions. conclusion gel filtration chromatography was unable to verify the interaction between n4ssb and e. coli rnap, and special promoter dna sequences may be required to assist n4ssb binding and modulate rnap or to validate by other means.
keywords： n4 bacteriophage late gene n4ssb transcription rna polymerase
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n4噬菌体n4ssb蛋白和大肠杆菌rna聚合酶的原核表达、纯化及互作验证
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