Marinobacter hydrocarbonoclasticus is an oil-eating bacterium that possesses a large adhesion protein (MhLap) with the potential to bind extracellular ligands. One of these ligand-binding modules is the ~20-kDa PA14 domain (MhPA14) that has affinity for glucose-based carbohydrates. Previous studies showed this sugar-binding domain is retained on dextran-based size-exclusion resins during chromatography, requiring the introduction of glucose or EDTA to remove the protein from the column. Given the ready availability of such size-exclusion resins in biochemistry laboratories, this study explores the use of MhPA14 as an affinity tag for recombinant protein purification. Two different fusion proteins were tested: 1) Green fluorescent protein (GFP) linked to the N-terminus of the MhPA14 tag; and 2) the ice-binding domain from the Marinomonas primoryensis ice-binding protein (MpIBD) linked to the MhPA14 C-terminus by a TEV cut site. The GFP_MhPA14 fusion visibly bound to Superdex, Sephadex, and Sephacryl resins, but did not bind to Sepharose. Using Superdex resin, dextran-affinity purification proved to be an effective one-step purification strategy for both proteins, superior to even nickel-affinity chromatography. Dextran-affinity chromatography was also the most effective method of separating the MhPA14 tag from MpIBD following TEV proteolysis, as compared to both nickel-affinity and ice-affinity methods. These results indicate that MhPA14 has potential for widespread use in recombinant protein purification.

中文翻译：

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细菌糖结合蛋白作为含葡聚糖的树脂的一步亲和纯化标签。

碳氢弯曲杆菌是一种食油细菌，它具有较大的粘附蛋白（MhLap），具有与细胞外配体结合的潜力。这些配体结合模块之一是〜20-kDa PA14结构域（MhPA14），该结构域对基于葡萄糖的碳水化合物具有亲和力。先前的研究表明，这种糖结合域在色谱过程中保留在基于葡聚糖的尺寸排阻树脂上，需要引入葡萄糖或EDTA才能从色谱柱上除去蛋白质。鉴于此类排阻树脂已在生物化学实验室中提供，本研究探索了MhPA14作为亲和标签用于重组蛋白纯化的用途。测试了两种不同的融合蛋白：1）与MhPA14标签N端连接的绿色荧光蛋白（GFP）；2）通过TEV切割位点与MhPA14 C-末端连接的来自滨海边马的冰结合蛋白（MpIBD）的冰结合结构域。GFP_MhPA14融合物可见地与Superdex，Sephadex和Sephacryl树脂结合，但不与Sepharose结合。使用Superdex树脂，葡聚糖亲和纯化被证明是两种蛋白质的有效一步纯化策略，甚至优于镍亲和层析。与镍亲和法和冰亲和法相比，葡聚糖亲和层析也是在TEV蛋白水解后从MpIBD分离MhPA14标签的最有效方法。这些结果表明，MhPA14具有广泛用于重组蛋白纯化的潜力。GFP_MhPA14融合物可见地与Superdex，Sephadex和Sephacryl树脂结合，但不与Sepharose结合。使用Superdex树脂，葡聚糖亲和纯化被证明是两种蛋白质的有效一步纯化策略，甚至优于镍亲和层析。与镍亲和法和冰亲和法相比，葡聚糖亲和层析也是在TEV蛋白水解后从MpIBD分离MhPA14标签的最有效方法。这些结果表明，MhPA14具有广泛用于重组蛋白纯化的潜力。GFP_MhPA14融合物可见地与Superdex，Sephadex和Sephacryl树脂结合，但不与Sepharose结合。使用Superdex树脂，葡聚糖亲和纯化被证明是两种蛋白质的有效一步纯化策略，甚至优于镍亲和色谱。与镍亲和法和冰亲和法相比，葡聚糖亲和层析也是在TEV蛋白水解后从MpIBD分离MhPA14标签的最有效方法。这些结果表明，MhPA14具有广泛用于重组蛋白纯化的潜力。与镍亲和法和冰亲和法相比，葡聚糖亲和层析也是在TEV蛋白水解后从MpIBD分离MhPA14标签的最有效方法。这些结果表明，MhPA14具有广泛用于重组蛋白纯化的潜力。与镍亲和法和冰亲和法相比，葡聚糖亲和层析也是在TEV蛋白水解后从MpIBD分离MhPA14标签的最有效方法。这些结果表明，MhPA14具有广泛用于重组蛋白纯化的潜力。