PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.
PDZ affinity chromatography: a general method for affinity purification of proteins based on PDZ domains and their ligands.
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DOI：
10.1016/j.pep.2014.02.015
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发表时间：
2014-06
影响因子：
1.6
通讯作者：
Kennedy MB
中科院分区：
文献类型：
--
作者：
Walkup WG 4th;Kennedy MB
PDZ（PSD-95，DiscsLarge，ZO 1）结构域在自然界中作为支架和膜相关蛋白内的蛋白结合结构域起作用。它们包含约90个残基，并与互补C-末端肽序列、其他PDZ结构域和磷脂发生特异性、高亲和力相互作用。我们假设PDZ结构域与其配体的特异性强相互作用将使其非常适合用于亲和色谱。在这里，我们描述了一种新的亲和层析方法，适用于含有PDZ结构域结合配体的蛋白质的纯化，无论是天然的还是通过基因工程引入的。我们创建了一系列亲和树脂，其由来自支架蛋白PSD-95或来自神经元一氧化氮合酶（nNOS）的PDZ结构域组成，与固体支持物偶联。我们使用它们来纯化异源表达的神经元蛋白或含有内源性PDZ结构域配体的蛋白结构域，用游离PDZ结构域肽配体洗脱蛋白质。我们表明，缺乏内源性PDZ结构域配体的感兴趣的蛋白质（POI）可以被工程化为含有C-末端PDZ结构域配体肽或内部、N-或C-末端PDZ结构域的融合产物，然后可以通过相同的方法纯化。使用该方法，我们回收了与活性形式的PDZ结构域配体融合的重组GFP，如通过荧光产率所验证的。类似地，如通过酶测定法评估的，以活性形式纯化与C-末端PDZ结构域配体或N-末端PDZ结构域融合的氯霉素乙酰转移酶（CAT）和β-半乳糖苷酶（LacZ）。一般而言，对于该方法，源自PSD-95的PDZ结构域和配体上级源自nNOS的那些。PDZ结构域亲和层析有望成为纯化多种天然和重组蛋白的通用和有效的方法。
PDZ (PSD-95, DiscsLarge, ZO1) domains function in nature as protein binding domains within scaffold and membrane-associated proteins. They comprise ~ 90 residues and make specific, high affinity interactions with complementary C-terminal peptide sequences, with other PDZ domains, and with phospholipids. We hypothesized that the specific, strong interactions of PDZ domains with their ligands would make them well suited for use in affinity chromatography. Here we describe a novel affinity chromatography method applicable for the purification of proteins that contain PDZ domain-binding ligands, either naturally or introduced by genetic engineering. We created a series of affinity resins comprised of PDZ domains from the scaffold protein PSD-95, or from neuronal nitric oxide synthase (nNOS), coupled to solid supports. We used them to purify heterologously expressed neuronal proteins or protein domains containing endogenous PDZ domain ligands, eluting the proteins with free PDZ domain peptide ligands. We show that Proteins of Interest (POIs) lacking endogenous PDZ domain ligands can be engineered as fusion products containing C-terminal PDZ domain ligand peptides or internal, N- or C-terminal PDZ domains and then can be purified by the same method. Using this method, we recovered recombinant GFP fused to a PDZ-domain ligand in active form as verified by fluorescence yield. Similarly, chloramphenicol acetyltransferase (CAT) and β-Galactosidase (LacZ) fused to a C-terminal PDZ domain ligand or an N-terminal PDZ domain were purified in active form as assessed by enzymatic assay. In general, PDZ domains and ligands derived from PSD-95 were superior to those from nNOS for this method. PDZ Domain Affinity Chromatography promises to be a versatile and effective method for purification of a wide variety of natural and recombinant proteins.
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影响因子：
3.4
作者：
Follenius-Wund, A;Bourotte, M;Pigault, C
通讯作者：
Pigault, C
影响因子：
16.2
作者：
ADAMS, ME;BUTLER, MH;FROEHNER, SC
通讯作者：
FROEHNER, SC
DOI：
10.1073/pnas.87.2.682
发表时间：
1990-01-01
影响因子：
11.1
作者：
BREDT, DS;SNYDER, SH
通讯作者：
SNYDER, SH
影响因子：
16.2
作者：
Firestein, BL;Brenman, JE;Bredt, DS
通讯作者：
Bredt, DS
影响因子：
46.9
作者：
Guignet, EG;Hovius, R;Vogel, H
通讯作者：
Vogel, H