Production of recombinant human monocyte neutrophil elastase inhibitor (rM/NEI)
Production of recombinant human monocyte neutrophil elastase inhibitor (rM/NEI)
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DOI：
10.1006/prep.1998.0951
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发表时间：
1998-10-01
影响因子：
1.6
通讯作者：
Mandle, RJ
中科院分区：
文献类型：
--
作者：
Cooley, J;Mathieu, B;Mandle, RJ
用杆状病毒表达系统表达重组人单核细胞/中性粒细胞弹性蛋白酶抑制剂（rM/NEI）。纯化的重组蛋白被证明抑制人中性粒细胞弹性蛋白酶的形成一个稳定的等摩尔复合物，如已被证明为M/NEI从单核细胞衍生的细胞系分离。rM/NEI在pH 6至pH 8的水性缓冲液中非常稳定，但在低于pH 6的缓冲液中不稳定。rM/NEI在冻融循环和低温保存条件下活性稳定，在冻干过程中无明显活性损失。通过阴离子交换和尺寸排阻色谱法获得了1.6 g rM/NEI纯度大于95%的批次，产量为7 - 8 mg/L培养的昆虫细胞。选择与大规模cGMP生产相容的方法和方案，并适用于rM/NEI作为囊性纤维化治疗剂的生化表征和临床前评价。大量纯化rM/NEI的可用性将有助于临床评价rM/NEI预防弹性蛋白酶介导的肺组织破坏与囊性纤维化的发病率和死亡率。(C)北京：科学出版社.
Recombinant human monocyte/neutrophil elastase inhibitor (rM/NEI) was expressed with a baculovirus expression system. The purified recombinant protein was shown to inhibit human neutrophil elastase by the formation of a stable equimolar complex, as had been shown for M/NEI isolated from monocyte-derived cell lines. rM/NEI was remarkably stable in aqueous buffers from pH 6 to pH 8, but not in buffers below pH 6. rM/NEI activity was stable when subjected to freeze-thaw cycles and low temperature storage in Tris or phosphate buffers, rM/NEI could also be lyophilized without significant loss of activity. A 1.6-g batch of greater than 95% purity in rM/NEI was obtained by anion exchange and size exclusion chromatography with yields of 7 to 8 mg per liter of cultured insect cells. Methods and protocols were chosen for compatibility with large-scale cGMP production and were suitable for biochemical characterization and preclinical evaluation of rM/NEI as a therapeutic agent for cystic fibrosis. The availability of large amounts of purified rM/NEI will facilitate clinical evaluation of rM/NEI for prevention of the elastase-mediated destruction of lung tissue associated with the morbidity and mortality of cystic fibrosis. (C) 1998 Academic Press.