| 摘要: |
| 【目的】由于LBD基因编码一类植物特有的空间结构尚未解析的转录因子，拟建立2种LBD家族成员（TtRa2和AtLBD37）及其DNA结合结构域的高效表达与纯化体系，为该家族转录因子的结晶研究奠定基础。【方法】以pET 21a表达载体为基架，设计并构建了pHMT载体，该载体在目标基因上游依次融合了His-tag、MBP和TEV蛋白酶酶切位点。将TtRa2和AtLBD37基因的完整编码区及其DNA结合结构域编码区分别插入pHMT载体，获得重组质粒pHMT-AtLBD37、pHMT-AtLBD37-BD、pHMT-TtRa2、pHMT-TtRa2-BD。将上述质粒导入E.coli表达菌株2566诱导表达，先使用amylose亲和柱纯化获得融合蛋白，再用融合His-tag的TEV蛋白酶切除融合蛋白TtRa2-BD中的His6 MBP双标签，使用Ni-NTA亲和层析柱去除TEV蛋白酶和His6-MBP标签，最后获得目标蛋白。【结果】TtRa2和AtLBD37及其DNA结合结构域均获得了高效可溶性融合表达，其表达量占细胞总蛋白的50%～70%；TtRa2-BD经2步亲和纯化后，每升菌液可获得15 mg纯度大于98%的目标蛋白。【结论】成功建立了2种LBD转录因子及其DNA结合结构域的高效表达和纯化体系。 |
| 关键词: LBD家族转录因子 pHMT载体 表达纯化 |
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| 基金项目:中央高校基本科研业务费专项（QN2009070）；西北农林科技大学“千人计划”引进人才项目（Z101021102） |
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| Expression and purification of LBD family transcription factors TtRa2 and AtLBD37 |
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| Abstract: |
| 【Objective】LBD genes define a novel class of plant-specific transcription factors with unresolved spatial structures.This study set up an efficient expression and purification system for two representative LBD members (TtRa2 and AtLBD37) and their DNA-binding domains to improve the crystallization of LBD transcription factors.【Method】Based on expression vector pET-21a,we designed and constructed a pHMT vector and a His-tag,a MBP and a TEV protease sites were successively fused in its upstream.After inserting TtRa2,AtLBD37 and their DNA-binding domains into pHMT vector,four recombinant plasmids,pHMT-AtLBD37,pHMT-AtLBD37-BD,pHMT-TtRa2 and pHMT-TtRa2 BD,were constructed.Recombinant proteins were expressed in E.coli strain and fusion proteins were purified by amylose affinity chromatography.Then,His6-MBP double tags were cut off by TEV protease and the target protein TtRa2-BD was purified by Ni-NTA affinity chromatography.【Result】TtRa2,AtLBD37 and their DNA-binding domains were expressed efficiently in soluble form,account for 50%-70% of total protein.After two steps of affinity purification,15 mg TtRa2-BD with 98% purity were obtained from 1 L culture.【Conclusion】We established an efficient expression and purification process for LBD family members and their DNA-binding domains. |
| Key words: transcription factors of LBD family pHMT vector expression and purification |
