Huang J, Sutliff T D, Wu L, Nandi S, Benge K, Terashima M, Ralston A H, Drohan W, Huang N, Rodriguez R L
Applied Phytologics, Inc., 4110 North Freeway Boulevard, Sacramento, CA 95834, Department of Chemical Engineering, Osaka Prefecture University, 1-1 Gakuen-Cho, Sakai 599-8531, Japan.
Biotechnol Prog. 2001 Jan-Feb;17(1):126-33. doi: 10.1021/bp0001516.
Human alpha-1-antitrypsin (AAT), the most abundant protease inhibitor found in the blood, was expressed in rice embryonic tissue suspension cell culture. This was accomplished by cloning the codon-optimized AAT gene into a vector containing the rice RAmy3D promoter and its signal sequence. The synthetic gene incorporates codons synonymous with those found in highly expressed rice genes. Approximately 1000 stable transformed calli were produced by particle bombardment mediated transformation and were screened for high AAT expression using a porcine elastase inhibitory activity assay. The band shift assay also confirmed that rice-derived AAT is functional regarding its binding capability to the elastase substrate. Time course studies were conducted to determine the optimum, postinduction expression levels from cell culture. AAT expression equivalent to 20% of the total secreted proteins was achieved, and a purification scheme was developed that yielded active AAT with purity greater than 95%. The potential applications of purified plant-derived AAT for treatments of various AAT-deficient diseases are discussed.
人α-1-抗胰蛋白酶（AAT）是血液中含量最丰富的蛋白酶抑制剂，在水稻胚性组织悬浮细胞培养物中得以表达。这是通过将密码子优化的AAT基因克隆到一个含有水稻RAmy3D启动子及其信号序列的载体中来实现的。该合成基因包含与高表达水稻基因中发现的密码子同义的密码子。通过粒子轰击介导的转化产生了约1000个稳定转化的愈伤组织，并使用猪弹性蛋白酶抑制活性测定法筛选高AAT表达。泳带迁移分析也证实了水稻来源的AAT在其与弹性蛋白酶底物的结合能力方面具有功能。进行了时间进程研究以确定细胞培养诱导后最佳的表达水平。实现了相当于总分泌蛋白20%的AAT表达，并开发了一种纯化方案，得到了纯度大于95%的活性AAT。讨论了纯化的植物源AAT在治疗各种AAT缺乏疾病方面的潜在应用。
