铜离子对光合颤绿菌蓝铜蛋白结晶的影响  

The Impact of Copper Ions on the Crystallization of Auracyanin from Candidatus Viridilinea Mediisalina

作　　者：鞠金鑫 马林 李琳 董士尚 秦晓春 JU Jinxin;MA Lin;LI Lin;DONG Shishang;QIN Xiaochun(School of Biological Science and Technology,University of Jinan,Jinan 250022,China)

机构地区：[1]济南大学生物科学与技术学院,山东济南250022

出　　处：《聊城大学学报(自然科学版)》2025年第2期299-306,共8页Journal of Liaocheng University:Natural Science Edition

基　　金：国家自然科学基金项目(32070267,32270260);山东省自然科学基金重大基础研究项目(ZR2019ZD48);国家自然科学基金项目(32200206);山东省自然科学基金项目(ZR2020QC057)资助。

摘　　要：蓝铜蛋白(auracyanin,AC)是一种含铜的重要光合电子传递载体。分别获得高质量的光合颤绿菌(Candidatus Viridilinea mediisalina,Cvm)野生型AC蛋白(Cvm-AC)和不结合铜离子的突变体AC蛋白(Cvm-AC-mutant),通过对比两者结晶条件的差异探讨铜离子对Cvm-AC蛋白结晶的影响。首先通过异源表达获得两种目的蛋白,再分别依次通过镍离子亲和层析、阴离子交换柱层析和凝胶过滤层析分离纯化获得高纯度的Cvm-AC和Cvm-AC-mutant蛋白,并使用十二烷基磺酸钠-聚丙烯酰胺凝胶电泳对蛋白的均一性和纯度进行检测;通过使用坐滴法和悬滴法对Cvm-AC蛋白的结晶条件进行筛选和优化,最终获得了质量较好的Cvm-AC蛋白晶体。然而,使用Cvm-AC蛋白的结晶条件无法获得Cvm-AC-mutant的蛋白质晶体,表明铜离子的结合与否可以直接影响Cvm-AC的结晶。The auracyanin(AC)is an important copper-containing photosynthetic electron transport carrier.The high quality of the(Candidatus Viridilinea mediisalina,Cvm)AC protein(Cvm-AC)and mutant AC protein(Cvm-AC-mutant)were obtained,and the effect of copper ion on the crystallization of Cvm-AC protein was further explored by comparing the differences in crystallization conditions between the two.Firstly,we obtained two target proteins by heterologous,and then isolated and purified the high-purity Cvm-AC and Cvm-AC-mutant proteins by nickel-ion affinity chromatography,anion-exchange column chromatography,and gel filtration chromatography,respectively,and examined the homogeneity and purity of the proteins by using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The crystallization conditions for Cvm-AC protein were screened and optimized using the sitting drop method and the hanging drop method,ultimately yielding high-quality Cvm-AC protein crystals.However,the crystallization conditions for Cvm-AC protein could not yield protein crystals for Cvm-AC-mutant,indicating that the binding of copper ions can directly affect the crystallization of Cvm-AC.

关 键 词：光合颤绿菌 Cvm-AC蛋白 分离 纯化 结晶 

分 类 号：Q936[生物学—微生物学]