Graduate Thesis Or Dissertation

 

Identifying Purification and Storage Techniques for the Human Papillomavirus Type 16 Major Capsid Protein L1 Public Deposited

https://scholar.colorado.edu/concern/graduate_thesis_or_dissertations/qj72p7427
Abstract
  • Expression of human papillomavirus (HPV) major capsid protein L1 in Escherichia coli produces L1 proteins that can self­assemble into pentamers, with 72 pentamers forming a capsomere. With effective purification and storage techniques, these L1 pentamers could provide a more economic alternative for manufacturing HPV vaccines. Various techniques were evaluated in order to develop the most efficient method for L1 protein purification. Primary purification of L1 protein fused to Glutathione S­ transferase (GST) was accomplished by GST affinity chromatography. DEAE anion exchange chromatography and heparin affinity chromatography were then tested as secondary purification techniques. Results showed heparin chromatography removed L1 degradation products, while DEAE chromatography removed significant levels of endotoxin from the sample. Several storage methods, including ammonium sulfate precipitation and freezing, were also analyzed based on their ability to maintain L1 protein conformation. A concentration of 40% ammonium sulfate was determined to be the most effective at precipitating protein from solution. In addition, a conformation­ specific antibody that recognizes the neutralizing epitope of L1 was used to analyze resuspended proteins and showed high levels of binding, indicating that L1 protein conformation was maintained. Further analysis may prove that this is an effective technique for protein storage during transport, which would improve vaccine cost­ effectiveness.
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  • 2012
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  • 2019-11-17
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