蛋白激酶MARK激酶结构域的分子克隆及蛋白纯化专业论文推荐.docVIP

PAGE 蛋白激酶MARK激酶结构域的 分子克隆及蛋白纯化 摘要：微管亲和性调节激酶（microtubule affinity regulating kinases, MARKs）是一类哺乳动物丝氨酸/苏氨酸激酶，属于Ca2+钙调蛋白依赖性蛋白激酶(Ca2+-calmodulin-dependent protein kinases, CaMK)家族腺苷单磷酸活化蛋白激酶（adenosine monophosphate-activated protein kinases, AMPK）亚族。MARKs在细胞极性决定、胞内物质运输和细胞信号传导等多种细胞过程中发挥重要作用。MARKs能够磷酸化微管结合蛋白（microtubule-associated proteins, MAPs）的微管结合结构域，导致它们从微管上脱离，从而增加微管的运动性。人类MARKs由四个基因编码，产生四种异构体：MARK1，MARK2，MARK3，MARK4。该论文主要涉及人类MARK1-3激酶结构域的分子克隆及蛋白纯化。我们将编码MARK1-3激酶结构域野生型和无活性突变体的基因插入到pET28a-His-TEV原核表达载体上，通过Ni2+亲和层析和凝胶过滤层析进行目的蛋白的纯化。 关键词：MARKs MAPs PAR-1 MST1 MST2 分子克隆 蛋白纯化 Cloning and purification of catalytic domains of protein kinase MARKs Abstract: Microtubule affinity regulating kinases (MARKs), as a kind of mammalian serine/threonine kinases, are members of the adenosine monophosphate-activated protein kinases (AMPK) subfamily, which belongs to the Ca2+-calmodulin-dependent protein kinases (CaMK) group of kinases. MARKs have vital roles in a variety of cell processes, such as cellular polarity determination, intracellular transport and cell signaling. They can phosphorylate the tubulin-binding domain of microtubule-associated proteins (MAPs), which causes their detachment from microtubules, and then increases microtubule dynamics. Human MARKs are encoded by four genes to get four isoforms MARK1, MARK2, MARK3 and MARK4. In this research, we focus on the cloning and purification of MARK1-3 catalytic domains. The gene encoding wild type and inactive mutants of human. MARK1-3 catalytic domains was constrcuted to the expression vector pET28a-His-TEV, and then transformed into E.coli condon plus cells. The recombinant proteins are purified by Ni2+ affinity HYPERLINK app:ds:chromatography \t chromatography and gel filtration chromatography. Key words: MARKs MAPs PAR-1 MST1 MST2 molecular cloning purification 目录 TOC \o 1-3 \h \z \u HYPERLINK \l _Toc328219723 1. 引言 PAGEREF _Toc328219723 \h 1 HYPERLINK \l _Toc328219724 1.1. 微管、微管结合蛋白和MARKs PAGEREF _To