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  

Study on Heterologous Expression Purification and Crystal Structure of Human Casein Kinase 1 Delta Protein

作　　者：李怡莹 姜文艳 宋嘉宁 吕博强 曹鹏 龚勇 王娟[1] LI Yiying;JIANG Wenyan;SONG Jianing;LV Boqiang;CAO Peng;GONG Yong;WANG Juan(College of Chemistry and Life Science,Beijing University of Technology,Beijing 100124,China;Biomedical Engineering Department,Peking University,Beijing 100083,China;Sinovation Medical Technology Co.,Ltd.Beijing 100062,China;Multi-disciplinary Research Division,Institute of High Energy Physics of the Chinese Academy of Sciences,Beijing 100049,China)

机构地区：[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

出　　处：《河北师范大学学报(自然科学版)》2025年第3期287-296,共10页Journal of Hebei Normal University:Natural Science

基　　金：国家自然科学基金(32370805,31970044)。

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CK1δ^(1-293)原核表达载体pET28a(+)-His-CK1δ^(1-293),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CK1δ^(1-293)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2.5Å,空间群为P2_(1)2_(1)2_(1),晶胞参数为a=45.8Å,b=72.7Å,c=330.3Å,α,β,γ=90°.该晶胞参数与已知的CK1δ^(1-293)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.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 CK1δcould reveal the structural basis of its kinase function.In this study,a human CK1δ^(1-293)prokaryotic expression vector,pET28a(+)-His-CK1δ^(1-293),was constructed.The recombinant protein was expressed,and the high-purity,high-concentration CK1δkinase 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 CK1δ^(1-293)protein.X-ray diffraction determined the crystal structure at a 2.5Åresolution,in the P2_(1)2_(1)2_(1)space group with cell parameters a=45.8Å,b=72.7Å,c=330.3Å,α,β,γ=90°.This structure,differing from known CK1δ^(1-293)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：CK1δ蛋白 原核表达 蛋白纯化 X射线晶体结构解析 

分 类 号：Q71[生物学—分子生物学]