Acute lymphoblastic leukaemia (ALL) affects lymphoblastic cells and is the most common neoplasm during childhood. Among the pharmaceuticals used in the treatment protocols for ALL, Asparaginase (ASNase) from Escherichia coli (EcAII) is an essential biodrug. Meanwhile, the use of EcAII in neoplastic treatments causes several side effects, such as immunological reactions, hepatotoxicity, neurotoxicity, depression, and coagulation abnormalities. Commercial EcAII is expressed as a recombinant protein, similar to novel enzymes from different organisms; in fact, EcAII is a tetrameric enzyme with high molecular weight (140 kDa), and its overexpression in recombinant systems often results in bacterial cell death or the production of aggregated or inactive EcAII protein, which is related to the formation of inclusion bodies. On the other hand, several commercial expression strains have been developed to overcome these expression issues, but no studies on a systematic evaluation of the E. coli strains aiming to express recombinant asparaginases have been performed to date. In this study, we evaluated eleven expression strains at a low temperature (16 °C) with different characteristics to determine which is the most appropriate for asparaginase expression; recombinant wild-type EcAII (rEcAII) was used as a prototype enzyme and the secondary structure content, oligomeric state, aggregation and specific activity of the enzymes were assessed. Structural analysis suggested that a correctly folded tetrameric rEcAII was obtained using ArcticExpress (DE3), a strain that co-express chaperonins, while all other strains produced poorly folded proteins. Additionally, the enzymatic assays showed high specific activity of proteins expressed by ArcticExpress (DE3) when compared to the other strains used in this work.

中文翻译：

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不同大肠杆菌菌株低温表达的抗白血病酶L-天冬酰胺酶II的功能和结构评价

急性淋巴细胞白血病 (ALL) 影响淋巴细胞，是儿童时期最常见的肿瘤。在 ALL 治疗方案中使用的药物中，来自大肠杆菌 (EcAII) 的天冬酰胺酶 (ASNase) 是一种必不可少的生物药物。同时，在肿瘤治疗中使用 EcAII 会引起多种副作用，例如免疫反应、肝毒性、神经毒性、抑郁和凝血异常。商业 EcAII 表达为重组蛋白，类似于来自不同生物体的新型酶；事实上，EcAII是一种高分子量（140 kDa）的四聚体酶，其在重组系统中的过度表达往往导致细菌细胞死亡或产生聚集或失活的EcAII蛋白，这与包涵体的形成有关。另一方面，已经开发了几种商业表达菌株来克服这些表达问题，但迄今为止尚未对旨在表达重组天冬酰胺酶的大肠杆菌菌株进行系统评估。在本研究中，我们在低温 (16 °C) 下评估了 11 种具有不同特性的表达菌株，以确定哪种表达菌株最适合表达天冬酰胺酶；以重组野生型 EcAII (rEcAII) 为原型酶，评估酶的二级结构含量、寡聚状态、聚集和比活性。结构分析表明，使用ArcticExpress (DE3) 获得了正确折叠的四聚体rEcAII，这是一种共表达伴侣蛋白的菌株，而所有其他菌株均产生折叠不良的蛋白质。此外，