Baculovirus-mediated expression and purification of human serum paraoxonase 1A.
Baculovirus-mediated expression and purification of human serum paraoxonase 1A.
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发表时间：
2001-06
影响因子：
6.5
通讯作者：
R. Brushia;T. Forte;M. Oda;B. La Du;J. Bielicki
中科院分区：
文献类型：
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作者：
R. Brushia;T. Forte;M. Oda;B. La Du;J. Bielicki
人对氧磷酶1(HPON1)是一种在高密度脂蛋白上转运的脂质相关酶。人们对hPON1很感兴趣，因为它被认为具有抗氧化/抗动脉粥样硬化的特性。我们建立了一种重组杆状病毒(BV)，以大量产生hPON1A用于结构和功能研究，并在此描述了该酶的生产和分离方法。Hi-5昆虫细胞(40 mg/L)产生高水平的重组hPON1A型(RPON1a)，一部分(约10 mg/L)分泌到细胞培养上清液中，但大部分(约30 mg/L)仍与寄主昆虫细胞结合。通过洗涤剂提取(Tergitol NP-10)和三个简单的层析步骤(DEAE-Sepharose、Sephacryl S-200和刀豆蛋白A)纯化细胞相关的rPON1a。纯化后的酶与刀豆蛋白A结合，经内切糖苷酶H消化后转变为低分子量，表明rPON1a含有高甘露糖N-糖链。伴随着酶的脱糖作用，芳香酯酶的活性显著降低(>；99%)。RPON1A芳酯酶活性依赖于Ca(2+)，其动力学参数与天然hPON1A相似(K(M)=3.8+/-2.1对3.7+/-2.0 mm，V(Max)=1,305+/-668对1,361+/-591 U/mg蛋白，rPON1A和hPON1A)。RPON1a和hPON1a均能有效抑制脂氧合酶介导的磷脂过氧化。与内糖苷酶H处理敏感的芳香酯酶活性不同，酶促脱糖不会抑制rPON1A的抗氧化活性。总之，我们的BV介导的PON1A表达系统似乎非常适合于生产相对大量的rPON1A用于结构功能研究。
Human paraoxonase 1 (hPON1) is a lipid-associated enzyme transported on HDL. There is considerable interest in hPON1 because of its putative antioxidative/antiatherogenic properties. We have created a recombinant baculovirus (BV) to generate hPON1A in large quantities for structure-function studies and here describe the method for production and isolation of the enzyme. A high level of recombinant hPON1 type A (rPON1A) was produced by Hi-5 insect cells (40 mg/l); a fraction ( approximately 10 mg/l) was secreted into the cell culture medium, but the majority ( approximately 30 mg/l) remained associated with the host insect cells. Cell-associated rPON1A was purified by detergent extraction (Tergitol NP-10) followed by three simple chromatography steps (DEAE-Sepharose, Sephacryl S-200, and concanavalin A). The purified enzyme bound to concanavalin A and was converted to a lower molecular mass by endoglycosidase H digestion, suggesting that rPON1A contained high-mannose N-glycan chains. There was a significant decrease in arylesterase activity (>99%) concomitant with enzymatic deglycosylation. rPON1A was dependent on Ca(2+) for arylesterase activity, exhibiting kinetic parameters similar to native hPON1A (K(m) = 3.8 +/- 2.1 vs. 3.7 +/- 2.0 mM and V(max) = 1,305 +/- 668 vs. 1,361 +/- 591 U/mg protein, rPON1A and hPON1A, respectively). Both rPON1A and hPON1A efficiently inhibited lipoxygenase-mediated peroxidation of phospholipid. In contrast to the arylesterase activity, which was sensitive to endoglycosidase H treatment, enzymatic deglycosylation did not inhibit the antioxidant activity of rPON1A. In conclusion, our BV-mediated PON1A expression system appears ideally suited for the production of relatively large quantities of rPON1A for structure-function studies.