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Recovery and purification of recombinant protein from transgenic corn by aqueous two-phase partitioning /

by Gu, Zhengrong.

Abstract (Summary)
Aqueous two-phase (ATP) partitioning was paired in a variety of ways to use of transgenic corn for protein expression. Systems were developed to selectively partition corn proteins from classes of potential recombinant proteins as well as the specific case of dog gastric lipase. More fundamentally, ATP partitioning was used to expand 2D gel electrophoretic characterization of protein size and pI to the third dimension of hydrophobicity with the usefulness of the method illustrated for corn extracts. ATP partitioning in polyethylene glycol (PEG)-salt systems separates proteins primarily according to their surface hydrophobicity differences. The average hydrophobicity of pH 7 extracts of endosperm proteins is higher than germ protein. The hydrophobic resolution of the phases depends on molecular weight (MW) of PEG and NaCl addition. Carrying out extraction and partitioning in one step benefits separation by reducing the extracted host proteins. Removing oil, by dry milling to remove the germ or hexane extraction, increased the extraction of the hydrophobic protein recombinant dog gastric lipase (r-DGL) expressed in the endosperm and reduces surfactant requirements. Germ removal and extraction at pH 3 decrease host protein burden. The recovery of r-DGL was increased by decreasing particle size and optimizing extraction time. ATP parameters for optimal purification of r-DGL included PEG MW, phase forming salt, NaCl addition, Triton X-100 concentration and phase ratio. Countercurrent ATP partitioning could theoretically achieve purification equivalent to cation exchange chromatography by using three to seven stages, but the concentration factor would remain lower. The surface hydrophobicity, isoelectric point (pI) and MW of the aqueous soluble corn germ proteins extracted at pH 4 and pH 7 was characterized by separately quantifying proteins partitioning in the top and bottom phase of polyethylene glycol (PEG) 3350 (15.7%)-Na?SO? (8.9%)-NaCl (3%) aqueous two-phase (ATP) systems with two dimensional (2D) electrophoresis. The pI and MW of host proteins were obtained through 2D electrophoresis directly. The partition coefficients of individual proteins, which were obtained by quantifying the proteins spots in the 2D gels of the top and the bottom phase samples, were used to estimate their hydrophobicities according to a surface hydrophobicity scale based on the partition coefficients of several proteins with known surface hydrophobicities in the same ATP system. This 3D mapping method can be used to optimize separation of target proteins from specified hosts or to choose suitable hosts and host tissue for targeted expression.
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School:Iowa State University

School Location:USA - Iowa

Source Type:Master's Thesis

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