Expression of human αI-proteinase inhibitor in Aspergillus niger
Expression of human αI-proteinase inhibitor in Aspergillus niger
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DOI：
10.1186/1475-2859-6-34
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发表时间：
2007-10-29
影响因子：
6.4
通讯作者：
Shiloach, Joseph
中科院分区：
文献类型：
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作者：
Karnaukhova, Elena;Ophir, Yakir;Shiloach, Joseph
背景：人α(I)-蛋白水解酶抑制物(α(I)-PI)又称抗胰蛋白酶，是血浆中含量最丰富的丝氨酸蛋白酶抑制物。它的缺乏与进行性的、最终致命的肺气肿的发展有关。目前在美国，阿尔法(I)-PI作为FDA许可的血浆衍生(PD)产品可用于替代治疗。然而，血浆来源本身是有限的；此外，即使在血浆产品的制造中采用了有效的病毒灭活步骤，新出现的病毒污染的风险仍然存在。因此，重组α(I)-PI(r-α(I)-PI)可以提供一种有吸引力的替代方案。结果：我们探索了在丝状真菌黑曲霉(A.niger)中表达人α(I)-PI基因的可能性，该系统与常用的酵母宿主相比，能够为分泌蛋白提供更多的“哺乳动物样”糖基化模式。我们的表达策略是基于α(I)-PI与一个强表达的、分泌的前导蛋白(糖淀粉酶G2)的融合，通过提供体内切割的二碱性加工位点(N-V-I-S-K-R)分离。通过十二烷基硫酸钠-PAGE、Western印迹、ELISA法和α(I)-PI活性分析，我们筛选到了分泌生物活性糖基化r-α(I)-PI的转化子(S)，产率高达12 mg/L。对黑曲霉的r-α(I)-PI进行了体外稳定性测试，并与PD-α(I)-PI参照物和未糖化的人r-α(I)-PI进行了比较。结论：我们研究了丝状真菌A.niger表达人α(I)-PI基因的适宜性。成功地在黑曲霉中实现了人α(I)-PI基因的异源表达，并在黑曲霉中获得了具有生物活性的糖基化蛋白，摇瓶发酵产量高达12 mg/L。
Background: Human alpha(I)-proteinase inhibitor (alpha(I)-PI), also known as antitrypsin, is the most abundant serine protease inhibitor (serpin) in plasma. Its deficiency is associated with development of progressive, ultimately fatal emphysema. Currently in the United States, alpha(I)-PI is available for replacement therapy as an FDA licensed plasma-derived (pd) product. However, the plasma source itself is limited; moreover, even with efficient viral inactivation steps used in manufacture of plasma products, the risk of contamination from emerging viruses may still exist. Therefore, recombinant alpha(I)-PI (r-alpha(I)-PI) could provide an attractive alternative. Although r-alpha(I)-PI has been produced in several hosts, protein stability in vitro and rapid clearance from the circulation have been major issues, primarily due to absent or altered glycosylation.Results: We have explored the possibility of expressing the gene for human alpha(I)-PI in the filamentous fungus Aspergillus niger (A. niger), a system reported to be capable of providing more "mammalian-like" glycosylation patterns to secretable proteins than commonly used yeast hosts. Our expression strategy was based on fusion of alpha(I)-PI with a strongly expressed, secreted leader protein (glucoamylase G2), separated by dibasic processing site (N-V-I-S-K-R) that provides in vivo cleavage. SDS-PAGE, Western blot, ELISA, and alpha(I)-PI activity assays enabled us to select the transformant(s) secreting a biologically active glycosylated r-alpha(I)-PI with yields of up to 12 mg/L. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis further confirmed that molecular mass of the r-alpha(I)-PI was similar to that of the pd-alpha(I)-PI. In vitro stability of the r-alpha(I)-PI from A. niger was tested in comparison with pd-alpha(I)-PI reference and non-glycosylated human r-alpha(I)-PI from E. coli.Conclusion: We examined the suitability of the filamentous fungus A. niger for the expression of the human gene for alpha(I)-PI, a medium size glycoprotein of high therapeutic value. The heterologous expression of the human gene for alpha(I)-PI in A. niger was successfully achieved to produce the secreted mature human r-alpha(I)-PI in A. niger as a biologically active glycosylated protein with improved stability and with yields of up to 12 mg/L in shake-flask growth.