Heterologous expression of plant glycosyltransferases for biochemistry and structural biology.
Heterologous expression of plant glycosyltransferases for biochemistry and structural biology.
复制标题
DOI：
10.1016/bs.mcb.2020.05.002
复制
发表时间：
2020
影响因子：
--
通讯作者：
Urbanowicz BR
中科院分区：
文献类型：
--
作者：
Prabhakar PK;Wang HT;Smith PJ;Yang JY;Barnes WJ;Peña MJ;Moremen KW;Urbanowicz BR
光合作用捕获的大部分碳被转化为构成植物细胞壁的多糖。这些复杂的宏观结构由纤维素、半纤维素和果胶以及少量的结构蛋白、矿物质和在许多情况下的木质素组成。壁组件彼此组装和相互作用，以产生具有许多功能的动态结构，包括为植物结构提供机械支撑以及确定植物细胞的形状和大小。尽管它们的丰度，在我们的知识的这些聚合物的结构单元的合成的主要差距仍然存在，主要是由于在体外生化分析的活性合成酶的表达和纯化的无效的方法。半纤维素多糖木葡聚糖占植物初生细胞壁干重的25%。大多数关于参与木葡聚糖生物合成途径的糖基转移酶（GT）的知识来自于敲除突变体的鉴定和碳水化合物分析，提供的关于木葡聚糖在植物中如何催化生物合成的信息很少。在本章中，我们描述了使用HEK293表达平台异源表达植物GT的方法。作为该平台实用性的证明，评估了来自三个不同CAZy家族的九种木葡聚糖相关GT，并且描述了用于表达、纯化和构建体优化的方法以用于生物化学和结构表征。
Much of the carbon captured by photosynthesis is converted into the polysaccharides that constitute plant cell walls. These complex macrostructures are composed of cellulose, hemicellulose, and pectins, together with small amounts of structural proteins, minerals, and in many cases lignin. Wall components assemble and interact with one another to produce dynamic structures with many capabilities, including providing mechanical support to plant structures and determining plant cell shape and size. Despite their abundance, major gaps in our knowledge of the synthesis of the building blocks of these polymers remain, largely due to ineffective methods for expression and purification of active synthetic enzymes for in vitro biochemical analyses. The hemicellulosic polysaccharide, xyloglucan, comprises up to 25% of the dry weight of primary cell walls in plants. Most of the knowledge about the glycosyltransferases (GTs) involved in the xyloglucan biosynthetic pathway has been derived from the identification and carbohydrate analysis of knockout mutants, lending little information on how the catalytic biosynthesis of xyloglucan occurs in planta. In this chapter we describe methods for the heterologous expression of plant GTs using the HEK293 expression platform. As a demonstration of the utility of this platform, nine xyloglucan-relevant GTs from three different CAZy families were evaluated, and methods for expression, purification, and construct optimization are described for biochemical and structural characterization.
登录
查看更多内容
影响因子：
21.1
作者：
He, Yuan;Wang, Kan;Yan, Nieng
通讯作者：
Yan, Nieng
DOI：
10.1007/978-1-4939-2760-9_10
发表时间：
2015-01-01
期刊：
GLYCO-ENGINEERING: METHODS AND PROTOCOLS
影响因子：
--
作者：
Geisler, Christoph;Mabashi-Asazuma, Hideaki;Jarvis, Donald L.
通讯作者：
Jarvis, Donald L.
影响因子：
27.5
作者：
Jensen, Jacob K.;Schultink, Alex;Pauly, Markus
通讯作者：
Pauly, Markus
影响因子：
4.8
作者：
Faik, A;Bar-Peled, M;Keegstra, K
通讯作者：
Keegstra, K
DOI：
10.1073/pnas.1804428115
发表时间：
2018-11-06
影响因子：
11.1
作者：
Muchero W;Sondreli KL;Chen JG;Urbanowicz BR;Zhang J;Singan V;Yang Y;Brueggeman RS;Franco-Coronado J;Abraham N;Yang JY;Moremen KW;Weisberg AJ;Chang JH;Lindquist E;Barry K;Ranjan P;Jawdy S;Schmutz J;Tuskan GA;LeBoldus JM
通讯作者：
LeBoldus JM