TcPINK1蛋白的表达、纯化及活性检测

Expression, Purification, and Activity Determination of TcPINK1

摘要

PINK1是一种定位在线粒体外膜上的丝氨酸/苏氨酸激酶,可感受线粒体损伤,通过招募PARKIN启动线粒体自噬,其功能性突变与家族性帕金森氏症(PD)密切相关．为探索PINK1家族蛋白N端对激酶催化功能的影响,本研究将赤拟谷盗PINK1(TcPINK1)3种不同长度N端截短体的基因克隆至pET15b表达载体,然后转化至大肠埃希氏菌BL21(DE3)中,并对诱导剂IPTG的浓度、诱导温度等进行优化．目标蛋白经Ni-NTA亲和层析、阴离子交换层析和分子筛纯化后,利用FITC标记的泛素作为底物进行激酶活性检测．实验显示,在OD₆₀₀值为0.6～0.8、表达温度为16 ℃、IPTG浓度为0.4 mmol/L、诱导表达18 h时,TcPINK1不同截短体蛋白可溶性表达量均较高,并显示出磷酸化泛素的活性．分子排阻色谱和酶活性检测结果显示,TcPINK1的N端αA螺旋对激酶的聚集状态及活性具有调节作用．TcPINK1和人源PINK1(hPINK1)的序列和结构对比均显示激酶区具有较高同源性,对TcPINK1酶活性调节机制的研究与探究可为PINK1蛋白家族的生物学功能提供重要信息．

Abstract

PTEN-induced putative kinase 1(PINK1)is a serine/threonine kinase located on the outer membrane of mitochondria. It detects mitochondrial damage and initiates mitochondrial autophagy by recruiting the PARKIN protein. Its functional mutation is closely related to familial Parkinson’s disease(PD). To explore the influence of the N-terminal of PINK1 family proteins on the catalytic function of kinases, three N-terminal truncated genes of Tribolium castaneum PINK1(TcPINK1) with different lengths were cloned into the pET15b expression vector and transformed into Escherichia coli BL21(DE3). The concentration of the inducer IPTG and induction temperature were optimized. The target protein was purified by Ni-NTA affinity chromatography, anion exchange chromatography, and molecular sieve chromatography, and FITC-labeled ubiquitin was used as the substrate for kinase activity detection. The findings indicated that the soluble expression levels of various truncated TcPINK1 proteins were consistently high under the conditions of 0.4 mmol/L IPTG, an induction period of 18 h, OD₆₀₀ values of 0.6—0.8, and a temperature of 16 ℃. All variants exhibited phosphorylation activity toward ubiquitin. In addition, results from molecular sieving combined with enzyme activity assays demonstrated that the N-αA helix of TcPINK1 played a regulatory role in the aggregation state and enzymatic activity of the kinase. Comparative analyses of the sequence and structure of TcPINK1 and human PINK1(hPINK1)demonstrated a high degree of homology within the kinase domain. This study of the regulatory mechanism of TcPINK1 enzymatic activity provides important information for exploring the biological function of the PINK1 protein family.

关键词

帕金森氏症 / TcPINK1 / AlphaFold 3 / 原核表达

Key words

Parkinson’s disease(PD) / Tribolium castaneum PINK1 / AlphaFold 3 / prokaryotic expression

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基金资助

天津市自然科学基金资助项目(21JCQNJC01660)