Expression of recombinant human acid sphingomyelinase in insect Sf21 cells:: purification, processing and enzymatic characterization
Expression of recombinant human acid sphingomyelinase in insect Sf21 cells:: purification, processing and enzymatic characterization
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DOI：
10.1016/s0168-1656(98)00070-4
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发表时间：
1998-07-30
影响因子：
4.1
通讯作者：
Sandhoff, K
中科院分区：
文献类型：
--
作者：
Bartelsen, O;Lansmann, S;Sandhoff, K
人酸性鞘磷脂酶（haSMase）的生物化学和结构研究依赖于均质生物活性酶的获得。由于人体组织中天然haSMase （n-haSMase）的丰度较低，传统的纯化策略不适合分离该酶的制备量。我们描述了一种利用杆状病毒表达载体系统进行haSMase功能表达和纯化的新方法。用编码haSMase的重组杆状病毒感染frugiperda Spodoptera 21细胞，导致糖基化的75 kDa前体蛋白的表达，该蛋白随后被加工成酶活性分泌的72 kDa haSMase。n -糖基化和蛋白水解成熟的差异解释了成熟重组酶（72 kDa）和人胎盘haSMase （75 kDa）之间分子质量的差异。重组haSMase （r-haSMase）的n端氨基酸测序显示，与胎盘酶相比，重组haSMase的n端延长了23个残基。在胶束测定体系中，r-haSMase降解鞘磷脂的表观K-m和V-max值分别为32 μ M和0.56 mmol h(-1) mg（-1）。总之，建立的杆状病毒表达载体体系为haSMase的表达和功能表征提供了有效的工具。(C) 1998 Elsevier Science B.V.版权所有
Biochemical and structural studies on human acid sphingomyelinase (haSMase) depend on the access to homogeneous biologically active enzyme. Due to the low abundance of native haSMase (n-haSMase) in human tissue, conventional purification strategies are not suitable for the isolation of preparative amounts of the enzyme. We describe a novel approach to the functional expression and purification of haSMase employing the baculovirus expression vector system. Infection of Spodoptera frugiperda 21 cells with recombinant baculovirus encoding haSMase leads to the expression of a glycosylated 75 kDa precursor protein, which is subsequently processed to an enzymatically active secreted 72 kDa haSMase. Variations in N-glycosylation and proteolytic maturation account for the difference in molecular mass between mature recombinant (72 kDa) and human placental haSMase (75 kDa). N-terminal amino acid sequencing of recombinant haSMase (r-haSMase) reveals a 23-residue N-terminal extension compared to the placental enzyme. The apparent K-m and V-max values for sphingomyelin degradation by r-haSMase in a micellar assay system are 32 mu M and 0.56 mmol h(-1) mg(-1), respectively. In conclusion, the established baculovirus expression vector system provides an efficient tool for the expression and functional characterization of haSMase. (C) 1998 Elsevier Science B.V. All rights reserved.