Recombinant expression of homodimeric 660 kDa human thyroglobulin in soybean seeds: an alternative source of human thyroglobulin.
Recombinant expression of homodimeric 660 kDa human thyroglobulin in soybean seeds: an alternative source of human thyroglobulin.
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大豆种子中同源二聚体 660 kDa 人甲状腺球蛋白的重组表达：人甲状腺球蛋白的替代来源。
DOI：
10.1007/s00299-011-1044-8
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发表时间：
2011
影响因子：
6.2
通讯作者：
Piller,KennethJ
中科院分区：
文献类型：
--
作者：
Powell,Rebecca;Hudson,LauraC;Lambirth,KevinC;Luth,Diane;Wang,Kan;Bost,KennethL;Piller,KennethJ
大豆种子具有许多特性，使它们成为生产重组蛋白的理想靶标。然而，一个经常被忽视的品质是它们储存大量复杂储存蛋白质的能力。由于这一特点，我们假设大豆种子将支持重组表达目前难以或不可能使用传统植物和非植物寄主系统表达的大而复杂的蛋白质。为了验证这一假设，我们用编码人甲状腺球蛋白(HTG)的合成基因转化大豆。HTG是一种660 kDa的同源二聚体蛋白，广泛用于诊断行业对甲状腺疾病的筛查和检测。在缺乏能够产生重组HTG的重组系统的情况下，研究和诊断级HTG继续从身体和手术切除的甲状腺组织中提纯。这些不太理想的组织来源缺乏统一的糖基化和碘化，因此在将纯化的HTG用于敏感的ELISA筛查时引入了变异性。在本研究中，我们报道了重组hTG在大豆种子中的成功表达。使用专门为检测患者血清中HTG而开发的商业ELISA试剂盒证明了大豆衍生蛋白的真实性。Western分析和凝胶过滤层析表明，重组HTG和甲状腺纯化的HTG在大小、质量、电荷和亚基相互作用方面具有相似的生物学特性。重组蛋白经3代稳定，约占种子总可溶性蛋白的1.5%。这些结果支持了我们的假设，即大豆是表达大型复杂蛋白质的传统寄主系统的实用替代品。
Soybean seeds possess many qualities that make them ideal targets for the production of recombinant proteins. However, one quality often overlooked is their ability to stockpile large amounts of complex storage proteins. Because of this characteristic, we hypothesized that soybean seeds would support recombinant expression of large and complex proteins that are currently difficult or impossible to express using traditional plant and non-plant-based host systems. To test this hypothesis, we transformed soybeans with a synthetic gene encoding human thyroglobulin (hTG)—a 660 kDa homodimeric protein that is widely used in the diagnostic industry for screening and detection of thyroid disease. In the absence of a recombinant system that can produce recombinant hTG, research and diagnostic grade hTG continues to be purified from cadaver and surgically removed thyroid tissue. These less-than-ideal tissue sources lack uniform glycosylation and iodination and therefore introduce variability when purified hTG is used in sensitive ELISA screens. In this study, we report the successful expression of recombinant hTG in soybean seeds. Authenticity of the soy-derived protein was demonstrated using commercial ELISA kits developed specifically for the detection of hTG in patient sera. Western analyses and gel filtration chromatography demonstrated that recombinant hTG and thyroid-purified hTG are biologically similar with respect to size, mass, charge and subunit interaction. The recombinant protein was stable over three generations and accumulated to ~1.5% of total soluble seed protein. These results support our hypothesis that soybeans represent a practical alternative to traditional host systems for the expression of large and complex proteins.