Department of Applied Chemistry, Kogakuin University, Hachioji, Tokyo, Japan.
PLoS One. 2013 Nov 11;8(11):e78669. doi: 10.1371/journal.pone.0078669. eCollection 2013.
Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)6 tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N'-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N'-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.
酸性哺乳动物几丁质酶（AMCase）已被证明与小鼠模型中的哮喘、过敏炎症和食物加工有关。在这里，我们描述了一种大肠杆菌表达系统，该系统允许在周质中产生与蛋白 A 融合的活性 AMCase，该蛋白 A 融合在 N 端和 V5 表位和 C 端（His）6 标签（V5-His）（蛋白 A-AMCase-V5-His）在大肠杆菌中。将小鼠 AMCase cDNA 克隆到载体 pEZZ18 中，该载体是一种表达载体，包含葡萄球菌蛋白 A 启动子，带有信号序列和蛋白 A 的截断形式，用于大肠杆菌的细胞外表达。大多数蛋白 A-AMCase-V5-His 存在于周质空间中，具有几丁质酶活性，这是通过使用显色底物 4-硝基苯基 N,N'-二乙酰基-β-D-壳二糖苷来测量的。蛋白 A-AMCase-V5-His 从周质级分中通过 IgG Sepharose 柱随后通过 Ni Sepharose 色谱法进行纯化。重组蛋白显示出强烈的活性峰，最大观察到的活性在 pH 2.0 时，最佳温度为 54°C。当该蛋白在 pH 1.0 和 pH 11.0 之间在冰上预孵育 1 小时时，保留了完整的几丁质酶活性。该蛋白在 pH 2.0 和 7.0 时在 54°C 下也具有热稳定性。重组 AMCase 对 4-硝基苯基 N,N'-二乙酰基-β-D-壳二糖苷的几丁质酶活性与 CHO 表达的 AMCase 相当。此外，重组 AMCase 与壳聚糖珠结合，切割胶体壳聚糖并释放主要是 N,N'-二乙酰壳二糖片段。因此，大肠杆菌表达的蛋白 A-小鼠 AMCase-V5-His 融合蛋白具有与 CHO 表达的 AMCase 相当的几丁质酶功能。这种重组蛋白可用于阐明小鼠 AMCase 的详细生物医学功能。
