Cloning, Expression, Purification and Refolding of Microtubule Affinity-Regulating Kinase 4 Expressed in Escherichia coli
Cloning, Expression, Purification and Refolding of Microtubule Affinity-Regulating Kinase 4 Expressed in Escherichia coli
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DOI：
10.1007/s12010-014-0733-5
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发表时间：
2014-03-01
影响因子：
3
通讯作者：
Hassan, Md Imtaiyaz
中科院分区：
文献类型：
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作者：
Naz, Farha;Asad, Mohd;Hassan, Md Imtaiyaz
微管相关蛋白/微管亲和力调节激酶4（MARK 4）是Ser/Thr激酶家族的成员，并且参与许多生物学功能，包括微管束形成、神经系统发育、程序性细胞死亡的正调控、细胞周期控制、细胞极性决定、细胞形状改变、细胞分裂等。我们需要足够的蛋白质。在本文中，我们报告了一种新的MARK 4克隆策略。我们在PQE 30载体中克隆了MARK 4催化结构域，包括59个N端功能未知的额外残基和单独的催化结构域。重组MARK 4在M15细胞中以包涵体形式表达。包涵体用1.5%N-月桂酰肌氨酸在碱性缓冲液中有效地溶解，随后用Ni-NTA亲和层析在一个步骤中纯化，具有高纯度和良好的浓度。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳检测蛋白纯度，并通过质谱免疫印迹进行鉴定。利用ATP酶活性测定验证了重组蛋白的复性效果。我们的纯化程序快速，简单，并在有限的步骤中产生足够数量的高纯度蛋白质。
Microtubule-associated protein/microtubule affinity-regulating kinase 4 (MARK4) is a member of the family Ser/Thr kinase and involved in numerous biological functions including microtubule bundle formation, nervous system development, positive regulation of programmed cell death, cell cycle control, cell polarity determination, cell shape alterations, cell division etc. For various biophysical and structural studies, we need this protein in adequate quantity. In this paper, we report a novel cloning strategy for MARK4. We have cloned MARK4 catalytic domain including 59 N-terminal extra residues with unknown function and catalytic domain alone in PQE30 vector. The recombinant MARK4 was expressed in the inclusion bodies in M15 cells. The inclusion bodies were solubilized effectively with 1.5 % N-lauroylsarcosine in alkaline buffer and subsequently purified using Ni-NTA affinity chromatography in a single step with high purity and good concentration. Purity of protein was checked on sodium dodecyl sulphate-polyacrylamide gel electrophoresis and identified by using mass spectrometry immunoblotting. Refolding of the recombinant protein was validated by ATPase assay. Our purification procedure is quick, simple and produces adequate quantity of proteins with high purity in a limited step.