产量(工程)
亮佩平
溶解
化学
蛋白酶
圆周率
色谱法
特里斯
蛋白酶抑制剂(药理学)
核化学
生物化学
酶
材料科学
有机化学
生物
冶金
人类免疫缺陷病毒(HIV)
抗逆转录病毒疗法
病毒载量
免疫学
出处
期刊:Vox Sanguinis
[Wiley]
日期:2006-11-01
卷期号:91 (4): 309-315
被引量:5
标识
DOI:10.1111/j.1423-0410.2006.00841.x
摘要
The aim of this study was to increase the yield of active alpha(1)-proteinase inhibitor (alpha(1)-PI) from Cohn fraction IV-1 paste during the manufacture of this therapeutic protein and to investigate the molecular mechanism for this yield increase.Dissolution experiments with IV-1 paste investigated the impact of different variables on the yield of alpha(1)-PI activity. Solutions of IV-1 paste prepared under different conditions were assayed for evidence of protease activity by Western blots of alpha(1)-PI following SDS-PAGE, by azocaseinolytic and amidolytic (S-2288) assays, and by zymography, and for the extent of alpha(1)-PI oligomerization by Western blots following nondenaturing PAGE.Minor modification of the manufacturing process by combining dissolution of IV-1 paste with the subsequent pH adjustment (to 9.25-9.50 with NaOH), achieved by addition of a standard concentration of NaOH to the 10-mm Tris base dissolvent for IV-1 paste, was found to give a highly reproducible 9.4 +/- 0.9% increase in yield of active alpha(1)-PI. Solutions of IV-1 paste prepared with this combined dissolvent contained reduced amounts of low molecular weight fragments of alpha(1)-PI, reduced protease activity, and reduced amounts of oligomers of alpha(1)-PI. Addition of the protease inhibitor leupeptin to the 10-mm Tris base dissolvent for IV-1 paste also caused an increase in the yield of alpha(1)-PI activity.Dissolution of IV-1 paste in a more alkaline medium gave a significant increase in the yield of active alpha(1)-PI. This yield increase was attributed to a reduction both in protease activity and in the extent of oligomerization of alpha(1)-PI.
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