| 摘要: |
| 【目的】对莱姆病螺旋体（Borrelia burgdorferi）端粒解离酶（telomere resolvase，ResT）进行原核可溶性表达纯化，并筛选高衍射度的ResT-DNA复合物晶体，为探究ResT DNA复合物的结构和功能奠定基础。【方法】合成pET28-b-ResT表达质粒，构建原核表达载体pSUMO-ResT，分析ResT可溶性蛋白在BL21(DE3)、BL21(DE3) pLysS、BL21 Gold(DE3) pLysS、BL21 Codon Plus(DE3)、Rosetta(DE3)和Rosetta(DE3) pLysS中的表达量，获得最优的ResT原核表达菌株。基于AlphaFold预测的ResT蛋白模型，利用定点突变及ResT可溶性蛋白表达分析，获得高效可溶性表达ResT突变体。经Ni-NTA亲和层析、HiTrap Heparin HP亲和层析及分子筛层析对ResT突变体 ResT（W94H）进行蛋白纯化。利用EMAS检测ResT（W94H）与DNA的结合能力。使用蛋白晶体接种（seeding）和交叉接种（cross-seeding）方法，利用多种商业化结晶试剂盒筛选并优化ResT-DNA复合物的结晶条件，对复合物晶体进行X射线衍射分析。【结果】BL21 Gold(DE3) pLysS菌株是ResT最佳的高效可溶性表达菌株，W94H突变体比野生型具有更强的可溶性表达。获得了高纯度（95%）且高质量浓度（15 mg/mL）的ResT（W94H），其能够与底物DNA形成稳定的ResT DNA复合物。在18 ℃下，ResT DNA最佳结晶条件是：PEG3350 150 g/L、二甲苯二酸钠0.1 mol/L（pH 6.6）、NaCl 0.15 mol/L，ResT-DNA晶体分辨率最高为3.52 ?，晶体空间群为P4。【结论】得到了高纯度ResT (W94H）蛋白，获得了ResT-DNA复合物晶体。 |
| 关键词: 莱姆病螺旋体 端粒解离酶 原核表达和纯化 蛋白结晶 X-射线衍射 |
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| 基金项目:国家自然科学基金项目（31900876）；河南省教育厅自然科学项目（22A180013）；河南工业大学高层次人才项目（2019BS020） |
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| Expression,purification and preliminary X-ray diffraction of telomere resolvase ResT from Borrelia burgdorferi |
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HU Yuansen,WANG Yuan,LÜ Yangyong,et al
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| Abstract: |
| 【Objective】This study conducted prokaryotic expression and purification of telomere resolvase ResT from Borrelia burgdorferi,screened crystallization conditions of ResT-DNA complex and analyzed complex crystals to provide basis for further research on its structure and biological function.【Method】The pET28-b-ResT was synthesized and plasmid pSUMO-ResT was constructed,and the best expression host for ResT in prokaryote expression system was selected through comparing the expressions of ResT in BL21(DE3),BL21(DE3) pLysS,BL21 Gold(DE3) pLysS,BL21 Codon Plus(DE3),Rosetta(DE3) and Rosetta(DE3) pLysS.Based on the AlphaFold model of ResT prediction,ResT mutations were constructed by sitedirected mutagenesis and ResT soluble expression was investigated.Then,ResT (W94H) was purified via Ni-NTA chromatography,HiTrap Heparin HP chromatography and size exclusion chromatography,and the interaction of ResT(W94H) with the sequence specific DNA was checked by EMAS.At last,the crystallization screening was performed via a variety of crystals screening kits by seeding and crossseeding methods,and the x-ray diffraction of ResT-DNA complex crystal was conducted.【Result】BL21 Gold(DE3) pLysS was the best strain for soluble expression.The purified ResT(W94H) protein with high purity (95%) and high concentration (15 mg/mL) was obtained and it formed stable ResT-DNA complex.The best crystallization conditions at 18 ℃ were PEG3350 150 g/L,sodium cacodylate 0.1 mol/L (pH 6.6) and NaCl 0.15 mol/L,and the products had the highest resolution of 3.52 and the space group of P4. 【Conclusion】High-purity protein of ResT was obtained and ResT-DNA complex crystal was screened and analyzed. |
| Key words: Borrelia burgdorferi telomere resolvase prokaryotic expression and purification protein crystallization X-ray diffraction |
