Zhong Qixin, Xu Li, Zhang Cheng, Glatz Charles E
Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA.
Appl Microbiol Biotechnol. 2007 Sep;76(3):607-13. doi: 10.1007/s00253-007-1037-2. Epub 2007 Jun 14.
Plants have attracted interest as hosts for protein expression because of the promise of a large production capacity and a low production cost. However, recovery costs remain a challenge as illustrated for recovery of recombinant aprotinin, a trypsin inhibitor, with removal of native corn trypsin inhibitor from transgenic corn (Azzoni et al. in Biotechnol Bioeng 80:268-276, 2002). When expression is targeted to corn grain fractions, dry milling can separate germ and endosperm fractions. Hence, only the product-containing fraction needs to be extracted, reducing the cost of extraction and the impurity level of the extract. Selective extraction conditions can reduce impurity levels to the point that low-cost adsorbents can result in relatively high purity levels. In this work, we attempted to achieve comparable purity with these lower cost methods. We replaced whole grain extraction and purification of recombinant aprotinin with sequential trypsin affinity and IMAC steps with an alternative of germ fraction extraction and purification with ion exchange and hydrophobic interaction chromatography (HIC). Using germ extraction at acidic pH supplemented with heat precipitation to remove additional host proteins resulted in a higher specific activity feed to the chromatographic steps. The cation exchange step provided 7.6x purification with 76.4% yield and no sodium dodecyl sulfate-polyacrylamide gel electrophoresis detectable native corn trypsin inhibitor. After the HIC step (2.7x step purification with 44.0% yield), the final product had a specific activity that was 75.3% of that of the affinity-purified aprotinin.
由于植物具有巨大的生产能力和较低的生产成本，因此作为蛋白质表达的宿主受到了关注。然而，回收成本仍然是一个挑战，如从转基因玉米中回收重组抑肽酶（一种胰蛋白酶抑制剂）并去除天然玉米胰蛋白酶抑制剂所示（Azzoni等人，《生物技术与生物工程》，2002年，第80卷，第268 - 276页）。当表达靶向玉米籽粒组分时，干法研磨可以分离胚芽和胚乳组分。因此^，只需要提取含产物的组分，从而降低提取成本和提取物的杂质水平。选择性提取条件可以将杂质水平降低到低成本吸附剂能够获得相对较高纯度的程度。在这项工作中，我们试图用这些低成本方法实现相当的纯度。我们用胰蛋白酶亲和和IMAC步骤的序列替代了重组抑肽酶的全谷物提取和纯化，采用离子交换和疏水相互作用色谱（HIC）对胚芽组分进行提取和纯化。在酸性pH下进行胚芽提取并辅以热沉淀以去除额外的宿主蛋白，从而为色谱步骤提供了更高比活的进料。阳离子交换步骤实现了7.6倍的纯化，产率为76.4%，并且没有十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳可检测到的天然玉米胰蛋白酶抑制剂。经过HIC步骤（2.7倍的步骤纯化，产率为44.0%）后，最终产物的比活是亲和纯化抑肽酶的75.3%。
