Abstract

L-asparaginase (ASNase) is an efficient inhibitor of tumor development, used in chemotherapy sessions against acute lymphoblastic leukemia (ALL) tumor cells; its use results in 80% complete remission of the disease in treated patients. Saccharomyces cerevisiae’s L-asparaginase II (ScASNaseII) has a high potential to substitute bacteria ASNase in patients that developed hypersensitivity, but the endogenous production of it results in hypermannosylated immunogenic enzyme. Here we describe the genetic process to acquire the ScASNaseII expressed in the extracellular medium. Our strategy involved a fusion of mature sequence of protein codified by ASP3 (amino acids 26–362) with the secretion signal sequence of Pichia pastoris acid phosphatase enzyme; in addition, this DNA construction was integrated in P. pastoris Glycoswitch^® strain genome, which has the cellular machinery to express and secrete high quantity of enzymes with humanized glycosylation. Our data show that the DNA construction and strain employed can express extracellular asparaginase with specific activity of 218.2 IU mg⁻¹. The resultant enzyme is 40% more stable than commercially available Escherichia coli’s ASNase (EcASNaseII) when incubated with human serum. In addition, ScASNaseII presents 50% lower cross-reaction with anti-ASNase antibody produced against EcASNaseII when compared with ASNase from Dickeya chrysanthemi.

摘要

L-天冬酰胺酶 (ASNase) 是一种有效的肿瘤发展抑制剂，用于针对急性淋巴细胞白血病 (ALL) 肿瘤细胞的化疗；它的使用使接受治疗的患者的疾病完全缓解了 80%。酿酒酵母的 L-天冬酰胺酶 II (ScASNaseII) 在发生超敏反应的患者中具有很高的替代细菌 ASNase 的潜力，但它的内源性产生会导致高甘露糖化免疫原性酶。在这里，我们描述了获得在细胞外培养基中表达的 ScASNaseII 的遗传过程。我们的策略涉及将ASP3编码的成熟蛋白质序列（氨基酸 26-362）与毕赤酵母的分泌信号序列融合酸性磷酸酶；此外，这种 DNA 构建被整合到P. pastoris Glycoswitch ^®菌株基因组中，该基因组具有表达和分泌大量具有人源化糖基化的酶的细胞机制。我们的数据表明，所用的 DNA 构建和菌株可以表达比活性为 218.2 IU mg ^-1的胞外天冬酰胺酶。当与人血清孵育时，所得酶比市售的大肠杆菌ASNase (EcASNaseII)稳定 40% 。此外，与来自Dickeya chrysanthemi的 ASNase 相比，ScASNaseII 与针对 EcASNaseII 产生的抗 ASNase 抗体的交叉反应降低了 50% 。