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首页> 外文期刊>Applied Microbiology and Biotechnology >Purification of recombinant aprotinin from transgenic corn germ fraction using ion exchange and hydrophobic interaction chromatography
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Purification of recombinant aprotinin from transgenic corn germ fraction using ion exchange and hydrophobic interaction chromatography

机译：使用离子交换和疏水相互作用色谱法从转基因玉米胚芽级分中纯化重组抑酶肽

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摘要

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.
机译：由于具有大的生产能力和低的生产成本的希望，植物作为蛋白质表达的宿主已引起人们的兴趣。然而，回收成本仍然是挑战，如从重组转基因玉米中去除天然玉米胰蛋白酶抑制剂的回收重组胰蛋白酶抑制蛋白（胰蛋白酶抑制剂）所示（Azzoni等人，Biotechnol Bioeng 80：268-276，2002）。当表达针对玉米籽粒部分时，干磨可以分离胚芽和胚乳部分。因此，仅需要提取含产物的馏分，从而降低了提取成本和提取物中的杂质含量。选择性萃取条件可以将杂质水平降低至低成本吸附剂可以导致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的方法获得可比的纯度。我们用顺序的胰蛋白酶亲和力和IMAC步骤代替了全谷物提取和重组抑肽酶的纯化，而替代了胚芽部分的提取和离子交换和疏水相互作用色谱（HIC）的纯化。使用在酸性pH值下进行胚芽提取并辅以热沉淀以去除其他宿主蛋白的方法，可将更高的比活度进料到色谱步骤中。阳离子交换步骤可进行7.6倍纯化，收率为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的天然玉米胰蛋白酶抑制剂。在HIC步骤（2.7x一步纯化，收率44.0％）之后，最终产品的比活是亲和纯化的抑肽酶的75.3％。

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