title: Purification of recombinant aprotinin from transgenic corn germ fraction using ion exchange and hydrophobic interaction chromatography.
abstract: 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.
authors.affiliation: Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA. qzhong@utk.edu
authors.forename: Qixin
authors.initials: Q
authors.lastname: Zhong
authors.forename: Li
authors.initials: L
authors.lastname: Xu
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhang
authors.forename: Charles E
authors.initials: CE
authors.lastname: Glatz
mesh_terms: D007611:Aprotinin / Q000235:genetics* / Q000302:isolation & purification*; D001709:Biotechnology; D002852:Chromatography, Ion Exchange; D057927:Hydrophobic and Hydrophilic Interactions; D030821:Plants, Genetically Modified; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D003313:Zea mays / Q000737:chemistry* / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D007611:Aprotinin