| Determination of Recombinant Mannitol-1-phosphate Dehydrogenase Activity from Ectocarpus sp.
|
|
Author:
Date:
2016-11-05
[Abstract] Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase (M1PDH, EC 1.1.1.17) and a mannitol-1-phosphatase (M1Pase, EC 3.1.3.22). This protocol describes the biochemical characterization of the recombinant catalytic domain of one ...
[摘要] 褐藻属于与绿色植物和动物遥远相关的系统发生谱系，并且主要发现于但不限于潮间带，一种苛刻且频繁变化的环境。由于它们独特的进化史和它们的栖息地，褐藻具有其代谢中的一些特性。其中之一是甘露醇循环，其在其生理学中发挥中心作用，因为甘露糖醇充当碳储存，渗透保护剂和抗氧化剂。该多元醇通过甘露醇-1-磷酸酯脱氢酶（M1PDH，EC 1.1.1.17）和甘露醇-1-磷酸酶（M1Pase，EC 3.1.3.22）的作用直接从光生酸酯果糖-6-磷酸衍生。该协议描述了在Ectocarpus中鉴定的三种M1PDH之一的重组催化结构域的生物化学表征。该重组催化结构域（下文称为M1PDHcat）使用NAD（H）作为辅因子催化果糖-6-磷酸（F6P）向甘露醇-1-磷酸（M1P）的可逆转化。 M1PDHcat活性在两个方向上测定，即，F6P还原和M1P氧化（图1）。
![src =]()
图1.甘露醇-1-磷酸脱氢酶的可逆反应
|
|
|
| Determination of Recombinant Mannitol-1-phosphatase Activity from Ectocarpus sp.
|
|
Author:
Date:
2016-08-20
[Abstract] Brown algae belong to a phylogenetic lineage distantly related to green plants and animals, and are found predominantly, but not exclusively, in the intertidal zone, a harsh and frequently changing environment. Because of their unique evolutionary history and of their habitat, brown algae feature several peculiarities in their metabolism. One of these is the mannitol cycle, which plays a central role in their physiology, as mannitol acts as carbon storage, osmoprotectant, and antioxidant. This polyol is derived directly from the photoassimilate fructose-6-phosphate via the action of a mannitol-1-phosphate dehydrogenase (M1PDH, EC 1.1.1.17) and a mannitol-1-phosphatase (M1Pase, EC 3.1.3.22). This protocol describes the biochemical characterization of a recombinant M1Pase of Ectocarpus ...
[摘要] 褐藻属于与绿色植物和动物遥远相关的系统发生谱系，并且主要发现于但不限于潮间带，一种苛刻且频繁变化的环境。由于它们独特的进化史和它们的栖息地，褐藻具有其代谢中的一些特性。其中之一是甘露醇循环，其在其生理学中发挥中心作用，因为甘露糖醇充当碳储存，渗透保护剂和抗氧化剂。该多元醇通过甘露醇-1-磷酸酯脱氢酶（M1PDH，EC 1.1.1.17）和甘露醇-1-磷酸酶（M1Pase，EC 3.1.3.22）的作用直接从光生酸酯果糖-6-磷酸衍生。该方案描述了Ectocarpus的重组M1P酶的生物化学表征。 M1P酶催化甘露醇-1-磷酸转化为甘露醇（图1）。
图1.甘露醇-1-磷酸酶催化的反应
|
|
|
| Generation of IgG-Fc Glycovariants Using Recombinant Glycosidases and Glycosyltransferases
|
|
Author:
Date:
2016-08-05
[Abstract] The immunoglobulin G (IgG) fragment crystallizable (Fc) domain contains a single, highly conserved asparagine 297 (N297) glycosylation site in the CH2 domain, which is buried within the hydrophobic core of each of the two heavy chains. The biantennary core glycan structure, composed of 2 N-acetylglucosamine (GlcNAc) and 3 mannose residues, can be further decorated with fucose, bisecting GlcNAc and terminal GlcNAc, galactose, and sialic acid. Presence or absence of distinct residues can alter IgG effector functions such as antibody-dependent cell-mediated cytotoxicity (ADCC) or complement-dependent cytotoxicity (CDC). Here, we provide a protocol for the generation of IgG-Fc de-galactosylated, galactosylated, de-sialylated and sialylated IgG antibodies using recombinant glycosidases and ...
[摘要] 免疫球蛋白G（IgG）片段可结晶（Fc）结构域在CH2结构域中包含单个，高度保守的天冬酰胺297（N297）糖基化位点，其掩埋在两条重链的每一条的疏水核内。由2个N-乙酰葡萄糖胺（GlcNAc）和3个甘露糖残基组成的双触角核心聚糖结构可以进一步用岩藻糖，二等分GlcNAc和末端GlcNAc，半乳糖和唾液酸装饰。不同残基的存在或不存在可以改变IgG效应子功能，例如抗体依赖性细胞介导的细胞毒性（ADCC）或补体依赖性细胞毒性（CDC）。在这里，我们提供使用重组糖苷酶和糖基转移酶产生IgG-Fc去半乳糖基化，半乳糖基化，去唾液酸化和唾液酸化IgG抗体的方案。
[背景] 糖基转移酶用于抗体聚糖修饰的用途允许将糖底物连接到预先存在的聚糖残基上。免疫球蛋白G在其每个CH2结构域中携带单个高度保守的N-糖基化位点（Arnold等人，2007）（图1），允许用糖基转移酶进行位点特异性聚糖修饰。如果抗体的Fab结构域含有Asn-X-Ser/Thr（X≠Pro）序列（Mellquist等人，1998），则抗体可携带额外的N-聚糖。因此，仔细选择缺少Fab糖基化的单克隆抗体对于Fc特异性聚糖修饰是重要的。本文所述的方案是基于以下出版物开发的（Kingston，2003; Kaneko等人，2006; Anthony等人，2008; Barb等人。，2009; Quast ...
|
|
|