Protein Purification from Corn Germ - PowerPoint PPT Presentation

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Protein Purification from Corn Germ

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Protein Purification from Corn Germ

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  1. Protein Purification from Corn Germ Danielle McConnell Department of Chemical Engineering Iowa State University

  2. Why extract Protein X from Transgenic Corn? • Could be a good alternative for a low calorie sweetener • 9500 times sweeter than sucrose on a molecular basis • Good extraction attributes (heat-stable and low molecular weight) • Transgenic corn is economically feasible, easy to scale up, and is highly available

  3. Extraction (part 1) pH determination • Isoelectric point of Protein is 5.72 • Tested pHs 4,5,6, and 7 • Total Protein Assays • pH 4 showed the least amount of total protein extracted

  4. Extraction (part 2) Determination of Heating Time • At 80 ° C Protein is thermo-stable • Total Protein Assays of precipitate at different times • At ten minutes the total protein is the smallest

  5. Cation Exchange Chromatography • Proteins bind to the negatively charged clusters by electrostatic forces • A SP-Sepharose column was used to run the ion exchange • According to the chromatogram (see next panel), peak 6 or fraction #38 displays the protein rich sample Cation Exchange Setup shown above

  6. Cation Exchange Chromatogram Conductivity vs. Time (fraction # at top of graph)

  7. Ultrafiltration • Used a used large and small molecular weight cutoff membranes to filter the extract • Protein should be left in the retentate of the small MW cutoff membrane • Total Protein Assays gave a preliminary evaluation of what was left in the retentate

  8. Ultrafiltration Flowchart

  9. Detects purity of Protein from injected sample Concentration can be found by integrating the peak area Protein X typically elutes around 15 minutes The HPLC chromatogram displays three elutions. The red line indicates a pure protein sample, the blue line represents pure protein from the cation exchange, and the black line portrays a sample of extract with spiked protein. Reverse Phase HPLC

  10. HPLC Chromatogram mV vs. Minutes

  11. Final Discussion: The final results of this project have not been determined, however, many progressive steps have been made in obtaining the protein from corn germ. Various extraction conditions (pH and salt conditions), filtration steps, and analysis of a quantification method (ELISA tests) are currently being researched to obtain a purified protein. The final steps of the protein sequence will be based on a combination of factors such as purity and yield which will be tested by reverse phase HPLC and the ELISA test. Diagram of complete Antibody setup for ELISA testing

  12. Acknowledgments: An extended gratification and thank you is made to the multitude of people who not only made this project possible but successful: • Dr. Charles Glatz • PWSE • Yandi Dharmadi, Zhengrong Gu, Maureen Griffin, Todd Menkhaus, and Jim Kupferschmit