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首页> 外文期刊>Protein Expression and Purification >Purification and characterization of a recombinant human testican-2 expressed in baculovirus-infected Sf9 insect cells
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Purification and characterization of a recombinant human testican-2 expressed in baculovirus-infected Sf9 insect cells

机译：在杆状病毒感染的Sf9昆虫细胞中表达的重组人testican-2的纯化和鉴定

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摘要

Testican-2 is a member of the, testican group of brain extracellular proteoglycans where a 45 kDa modular protein core is composed of a follistatin-like domain, a calcium-binding domain, a thyroglobulin type-1 (Tg1) domain and an acid C-terminal region with glycosaminoglycan attachment sites. The modular structure suggests that it could participate in various interactions. The aim of the present study was to express and characterize a recombinant human testican-2 in quantities sufficient for structural and functional studies. Human cDNA coding for a 422 amino acid testican-2 protein was cloned into the pFastBac1 vector and expressed in the Spodoptera frugiperda (Sf9) insect cell expression system. The protein was purified to homogeneity by three chromatographic steps using the His(6) tag in the first two steps and ion exchange chromatography as last one. The final yield of purified recombinant testican-2 was up to 3.5 mg/L culture medium and its molecular mass determined by SDS-PAGE was similar to 55 kDa. Analysis by enzymatic deglycosylation revealed presence of N-linked sugars with a total mass of 4 kDa. In contrast to the Tg1 domain of testican-1, which acts as an inhibitor of the lysosomal cysteine peptidase cathepsin L, the Tg1 domain of testican-2 did not inhibit. cathepsins L, 13, K and S. We identified the Clq subcomponent of complement component Cl as a potential interacting partner of testican-2. The Clq subcomponent is a recognition molecule which acts in concert with other Cl subcomponents to activate the classical pathway of complement activation. The reported new interaction could be of importance in various complement-mediated inflammatory and other immune processes. (c) 2007 Elsevier Inc. All rights reserved.
机译：Testican-2是大脑细胞外蛋白聚糖testican组的成员，其中45 kDa的模块蛋白核心由卵泡抑素样结构域，钙结合结构域，甲状腺球蛋白1型（Tg1）结构域和酸C组成。 -具有糖胺聚糖附着位点的末端区域。模块化结构表明它可以参与各种交互。本研究的目的是表达和表征重组人testican-2的数量，足以进行结构和功能研究。将编码422个氨基酸的testican-2蛋白的人cDNA克隆到pFastBac1载体中，并在Spodoptera frugiperda（Sf9）昆虫细胞表达系统中表达。通过在前两个步骤中使用His（6）标签并在最后一个步骤中使用离子交换色谱法的三个色谱步骤，将蛋白质纯化至均质。纯化的重组testican-2的最终产量高达3.5 mg / L培养基，通过SDS-PAGE测定的分子量约为55 kDa。通过酶促去糖基化的分析显示存在总质量为4kDa的N-连接的糖。与充当溶酶体半胱氨酸肽酶组织蛋白酶L抑制剂的testican-1的Tg1结构域相反，testican-2的Tg1结构域没有抑制作用。组织蛋白酶L，13，K和S。我们确定补体成分Cl的Clq子成分为testican-2的潜在相互作用伴侣。 Clq亚成分是识别分子，其与其他C1亚成分协同作用以激活补体激活的经典途径。报道的新相互作用可能在各种补体介导的炎症和其他免疫过程中具有重要意义。 （c）2007 Elsevier Inc.保留所有权利。

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