SikCDPK1基因EF-hand的定点突变、原核表达及蛋白纯化  

Site-directed Mutagenesis,Prokaryotic Expression and Protein Purification of SikCDPK1 Gene EF-hand

作　　者：郭家秀 朱新霞[1] Guo Jiaxiu;Zhu Xinxia(School of Life Sciences,Shihezi University,Shihezi,832003)

机构地区：[1]石河子大学生命科学学院,石河子832003

出　　处：《分子植物育种》2023年第2期503-509,共7页Molecular Plant Breeding

基　　金：国家自然科学基金项目(31760066)资助。

摘　　要：钙依赖性蛋白激酶(calcium dependent protein kinase,CDPK)通过与Ca^(2+)相互作用,在植物响应各种逆境胁迫的过程中发挥重要作用,其C端调控区的EF-hand基序是使CDPK的激活依赖于Ca^(2+)的关键结构。为进一步研究具有极强耐寒性的天山雪莲对逆境的响应机制,本研究采用PCR定点突变技术,将SikCDPK1基因EF-hand基序的第435、第471、第507和第540位氨基酸分别由E(谷氨酸)突变为Q(谷氨酰胺),构建定点突变原核表达载体,转化表达菌株E.coli BL21(DE3),使用IPTG诱导蛋白表达,利用亲和层析系统对重组蛋白进行纯化。结果显示,重组菌株能够成功表达出目的重组蛋白,重组蛋白可溶性表达量较高的诱导培养条件是18℃1 mmol/L IPTG诱导16 h,最后成功纯化出符合预期大小的可溶性蛋白,为进一步探究SikCDPK1中不同EF-hand基序的生物学功能提供了实验依据。Calcium dependent protein kinase(CDPK)plays an important role in plant response to various stresses by interacting with Ca^(2+).Its EF-hand motif in the C-terminal regulatory region is a key structure that makes the activation of CDPK dependent on Ca^(2+).In order to further study the response mechanism of Sasussured involucrata Kar.et Kir.with strong cold tolerance to adversity,this study used PCR site-directed mutagenesis technology to mutate the 435,471,507,and 540 amino acids of the EF-hand motif of SikCDPK1 gene from E(glutamate)to Q(glutamine).A site-directed mutant prokaryotic expression vector was constructed and transformed into the expression strain E.coli BL21(DE3).The recombinant protein was induced by IPTG and purified by an affinity chromatography system.The results showed that the recombinant strain could successfully express the target recombinant protein.The high soluble expression of recombinant protein was induced by 1 mmol/L IPTG at 18℃for 16 h.Finally,the expected size of the soluble protein was successfully purified.This study provide experimental basis for further exploring the biological functions of different EF-hand motif in SikCDPK1.

关 键 词：SikCDPK1 EF-HAND 定点突变 原核表达 蛋白纯化 

分 类 号：Q78[生物学—分子生物学]