期刊信息
- 刊名： 河北师范大学学报（自然科学版）Journal of Hebei Normal University (Natural Science)
- 主办： 河北师范大学
- ISSN： 1000-5854
- CN： 13-1061/N
- 中国科技核心期刊
- 中国期刊方阵入选期刊
- 中国高校优秀科技期刊
- 华北优秀期刊
- 河北省优秀科技期刊
人酪蛋白激酶1家族δ型蛋白的异源表达和纯化及晶体结构研究
- （1.北京工业大学 化学与生命科学学院,北京 100124;2.北京大学 临床医学高等研究院,北京 100083;3.华科精准医疗科技有限公司,北京 100062;4.中国科学院高能物理研究所多学科研究中心,北京 100049）
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DOI：10.13763/j.cnki.jhebnu.nse.202504005
Study on Heterologous Expression Purification and Crystal Structure of Human Casein Kinase 1 Delta Protein
摘要/Abstract
摘要：
酪蛋白激酶1-δ型蛋白（casein kinase 1 isoform delta,CK1δ）是一个高度保守的丝氨酸/苏氨酸蛋白激酶，在众多细胞过程中发挥重要的调节作用.针对CK1δ蛋白的X射线晶体结构研究可以揭示激酶发挥功能的结构基础.本研究构建了人源CK1δ1-293原核表达载体pET28a(+)-His-CK1δ1-293,并完成了重组蛋白的诱导表达，经过镍亲和层析、透析酶切和凝胶过滤层析获得了高纯度、高浓度的CK1δ激酶结构域蛋白.使用多种结晶条件试剂盒和气相扩散坐滴法筛选获得了CK1δ1-293蛋白的单晶体.经X射线衍射测定蛋白质晶体的衍射分辨率为2.5 Å，空间群为P212121，晶胞参数为a=45.8 Å,b=72.7 Å,c=330.3 Å，α,β,γ=90°.该晶胞参数与已知的CK1δ1-293蛋白晶体结构相比均不相同，为一个全新的晶型.结构分析发现新晶型中具有独特结构特征，为CK1δ蛋白抑制剂的设计提供了新的信息.
Abstract：
Casein kinase 1 isoform delta (CK1δ) is a highly conserved serine/threonine protein kinase that plays a crucial regulatory role in many cellular processes.Investigation into the X-ray crystal structure of CK1δ could reveal the structural basis of its kinase function.In this study,a human CK1δ1-293 prokaryotic expression vector,pET28a(+)-His-CK1δ1-293,was constructed.The recombinant protein was expressed,and the high-purity,high-concentration CK1δ kinase domain protein was obtained after purification by nickel-affinity chromatography,dialysis proteolysis,and gel filtration chromatography.Various crystallization condition kits and the vapor diffusion sitting drop method yielded single crystals of CK1δ1-293 protein.X-ray diffraction determined the crystal structure at a 2.5 Å resolution,in the P212121 space group with cell parameters a=45.8 Å,b=72.7 Å,c=330.3 Å,α,β,γ=90°.This structure,differing from known CK1δ1-293 protein crystal structures,represents a novel crystal form.Structural analysis revealed unique structural features in the new crystal form,which provided fresh insights for the design of CK1δ inhibitors.
关键词
Key words：
CK1δ
;
prokaryotic expression
;
protein purification
;
X-ray crystallography analysis
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