Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells
Schistosoma mansoni cathepsin D1: Biochemical and biophysical characterization of the recombinant enzyme expressed in HEK293T cells
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DOI：
10.1016/j.pep.2019.105532
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发表时间：
2020-03-01
影响因子：
1.6
通讯作者：
Silva-Jr, F. P.
中科院分区：
文献类型：
--
作者：
Araujo-Montoya, B. O.;Senger, M. R.;Silva-Jr, F. P.
血吸虫表达多种在蠕虫病理生理学中具有不同作用的乙酰基蛋白酶（AP），其中宿主血红蛋白的降解是关键，因为它是这些寄生虫的主要氨基酸来源。来自曼氏血吸虫（Schistosoma mansoni）的组织蛋白酶D样AP（SmCD 1）已被用作在染色体中进行疫苗和药物开发研究的模型酶，但尚未报道用于容易地以高产量生产重组酶的可靠表达系统。为了有助于进一步推进关于这种有价值的抗人β-内酰胺酶靶点的知识，我们在HEK 293 T哺乳动物细胞中开发了一种瞬时表达系统，并对重组酶（rSmCD 1）进行了生物化学和生物物理表征。这是可能的，表达的重组SmCD 1的C-末端截短形式（rSmCD 1 Δ CT），并从培养上清中以高产量（16 mg/L）纯化它。当通过尺寸排阻色谱和多角度激光散射分析时，rSmCD 1 Delta CT在中性pH下表现为二聚体，这对于组织蛋白酶D是不寻常的，在介质酸化后变成单体。通过分析超速离心，证实了二聚体在溶液中的游离rSmCD 1 Delta CT以及与胃酶抑制剂相互作用期间单体的稳定性。在这里使用的哺乳动物细胞表达系统能够产生rSmCD 1 Delta CT的高产率，允许第一次的重要动力学参数的表征，以及其生物物理特性的初步描述。
Schistosomes express a variety of aspartyl proteases (APs) with distinct roles in the helminth pathophysiology, among which degradation of host haemoglobin is key, since it is the main amino acid source for these parasites. A cathepsin D-like AP from Schistosoma mansoni (SmCD1) has been used as a model enzyme for vaccine and drug development studies in schistosomes and yet a reliable expression system for readily producing the recombinant enzyme in high yield has not been reported. To contribute to further advancing the knowledge about this valuable antischistosomal target, we developed a transient expression system in HEK 293T mammalian cells and performed a biochemical and biophysical characterization of the recombinant enzyme (rSmCD1). It was possible to express a recombinant C-terminal truncated form of SmCD1 (rSmCD1 Delta CT) and purify it with high yield (16 mg/L) from the culture supernatant. When analysed by Size-Exclusion Chromatography and multi-angle laser light scattering, rSmCD1 Delta CT behaved as a dimer at neutral pH, which is unusual for cathepsins D, turning into a monomer after acidification of the medium. Through analytical ultrancentrifugation, the dimer was confirmed for free rSmCD1 Delta CT in solution as well as stabilization of the monomer during interaction with pepstatin. The mammalian cell expression system used here was able to produce rSmCD1 Delta CT with high yields allowing for the first time the characterization of important kinetic parameters as well as initial description of its biophysical properties.