Date of Award
Doctor of Philosophy
Cheng S. Lee
Rapid growth in the biotechnology industry has led to a dramatic increase in attention to the protein folding problem. Understanding of protein folding is essential to the production of biopharmaceuticals since commercial production of recombinant proteins often requires a protein refolding process to recover high yields;Capillary zone electrophoresis (CZE) equipped with laser induced fluorescence detection (LIFD) is developed as a tool for monitoring the refolding of a model protein, phage P22 tailspike endorhamnosidase. Intermediates on the folding pathway are separated in CZE and monitored by their intrinsic tryptophan fluorescence. Monitoring refolding of tailspike by traditional methods, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorescence spectrophotometry, are used to confirm the refolding kinetics and yield as measured by CZE. The agreement between these methods shows CZE to be an effective technique for simple, rapid detection of changes in protein conformation;Effects of temperature on the refolding kinetics and yield of tailspike are investigated using CZE-LIFD. Refolding kinetics are extremely slowed at 0°C and results in a loss of the protrimer in the refolding pathway. Results obtained from CZE-LIFD and polyacrylamide gel electrophoresis indicate the loss of folding intermediates through the adsorption of polypeptide chains onto the wall of the refolding vial;By increasing the reaction temperature at different stages of tailspike refolding, structured monomeric species are identified as the critical intermediates at the junction between productive and aggregation pathways of tailspike protein. Optimal refolding kinetics and yields of tailspike endorhamnosidase can be achieved by increasing the refolding temperature from 10 to 35°C only after passage of the stage for accumulation of the thermolabile intermediate;Capillary isoelectric focusing-electrospray ionization mass spectrometry (CIEF-ESIMS) is developed as another technique for monitoring protein folding intermediates. After the initiation of the refolding process, the reduced and denatured disulfide bonded protein, ribonuclease A (RNase A), is blocked at different refolding stages by alkylation of free cysteines with iodoacetate. This alkylation reaction results in the introduction of charge (-1) and mass (58) differences for each alkylation site, providing the means for predictable separation and direct identification of intermediates by CIEF-ESIMS.
Digital Repository @ Iowa State University, http://lib.dr.iastate.edu/
Pamela Kay Jensen
Jensen, Pamela Kay, "Monitoring protein refolding using diverse separation and spectroscopy techniques " (1997). Retrospective Theses and Dissertations. 11993.