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大肠杆菌holo-ACP突变体的表达纯化及相应acyl-ACP的性质初探

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大肠杆菌holo-ACP突变体的表达纯化及相应acyl-ACP的性质初探
Expression and Purification of holo-ACP Mutant and Property Study on the Corresponding acyl-ACP from E`scherichia Coli
投稿时间：2019-04-12
DOI：10.15918/j.tbit1001-0645.2019.123
中文关键词:acyl-ACP单点突变体高效液相色谱性质差异
English Keywords:acyl-ACPsingle-site mutanthigh-performance liquid phase chromatographyproperty difference
基金项目:国家自然科学基金资助项目（21877110，21708040，21506025）
作者单位E-mail
王文青大连理工大学 生物工程学院, 辽宁, 大连 116024
杨淼中国科学院 大连化学物理研究所, 辽宁, 大连 116023
冯延宾中国科学院 大连化学物理研究所, 辽宁, 大连 116023
姬芳玲大连理工大学 生物工程学院, 辽宁, 大连 116024fanglingji@dlut.edu.cn
薛松中国科学院 大连化学物理研究所, 辽宁, 大连 116023xuesong@dicp.ac.cn
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中文摘要:
酰基-酰基载体蛋白（acyl-acyl carrier protein，acyl-ACP）作为酰基供体，在脂肪酸、聚酮等天然产物合成途径中具有重要作用.目前，常以酰基-辅酶A（acyl-CoA）来代替尚未商品化的acyl-ACP进行相关酶的体外活性研究.由于底物蛋白acyl-ACP的ACP部分与相关酶发生相互作用，影响酶的催化特性，这种替代无法真实认识相关酶的酰基转移特性.本文以大肠杆菌pET-28a（+）-ACP为模板，构建12株单点突变体，表达纯化出相应holo-ACP，并进一步合成C16：0-ACP和C18：1-ACP.高效液相色谱的结果表明，ACP单点突变会影响acyl-ACP整体的性质，其中T40残基的改变对acyl-ACP的疏水性、紫外吸收响应和稳定性均产生了显著影响.ACP突变体的性质表征为acyl-ACP与相关酶的互作机制研究奠定了基础.
English Summary:
As an acyl donor, the acyl-acyl carrier protein (acyl-ACP) plays pivotal roles in biosynthesis of various natural products, including fatty acids, polyketones and so on. At present the acyl-CoA has been used as a substitute for uncommercialized acyl-ACP to perform in vitro study of the relevant enzyme activity. By using acyl-CoA, the catalytic specificity of the enzymes can't be revealed correctly because the substrate protein acyl-ACP could interact with the relevant enzymes through the ACP. Based on the plasmid pET-28a(+) -ACP derived from Escherichia coli, 12 single-site ACP mutants were designed, and the corresponding holo-ACPs were expressed and purified. These holo-ACPs were subsequently used as substrates to synthesize C16:0-ACP and C18:1-ACP. The HPLC results show that, ACP mutant with change in a single amino acid can effect on the acyl-ACP feature. In particular, the ACP mutant with change in T40 residue has notable impacts on hydrophobicity, ultraviolet absorption response and stability of acyl-ACP as well.
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相关话题/辽宁 中国科学院 大连化学物理研究所 大连理工大学 生物工程

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