Peptide ligands that bind to staphylococcal enterotoxin B (SEB)

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Title: Peptide ligands that bind to staphylococcal enterotoxin B (SEB)
Author: Wang, Guangquan
Advisors: Steven W. Peretti, Committee Member
Zhilin Li, Committee Member
Robert M. Kelly, Committee Member
Peter K. Kilpatrick, Committee Member
Ruben G. Carbonell, Committee Chair
Abstract: Staphylococcal enterotoxin B (SEB) is a primary toxin that causes food poisoning. It also acts as a superantigen that interacts with the major histocompatibility complex class II molecule (MHCII) and T cell receptor (TCR) to activate large amounts of T cells leading to autoimmune diseases. Highly purified SEB is needed in research and can be used as a standard in current detection methods for SEB. SEB is also a contaminant in protein A purification from Staphylococcal aureus fermentation broth. Inexpensive, robust ligands with high affinity would be suitable replacements for antibodies in biosensors for the detection of SEB. Affinity adsorption processes using short peptides as ligands show great promise in purifying and detecting proteins in comparison with other methods due to their high stability and low cost. By screening a solid-phase combinatorial peptide library, a short peptide ligand, YYWLHH, has been discovered that binds with high affinity and selectivity to SEB, but only weakly to other staphylococcal enterotoxins that share sequence and structural homology with SEB. Using column affinity chromatography with an immobilized YYWLHH stationary phase, it was possible to separate SEB quantitatively from Staphylococcus aureus fermentation broth, a complex mixture of proteins, carbohydrates and other biomolecules. The immobilized peptide was also used to purify native SEB from a mixture containing denatured and hydrolyzed SEB, and showed little cross reactivity with other SEs. To our knowledge this is the first report of a highly specific short peptide ligand for SEB. Such a ligand is a potential candidate to replace antibodies for detection, removal and purification strategies for SEB. Modeling the transport and kinetic processes in peptide affinity chromatography allows for a direct measurement of the rate of binding of SEB to peptide ligands. It can also provide design parameters for columns that can be used to either remove or detect SEB as well as other pathogens. The mass transfer parameters of SEB were either measured using pulse experiments or determined from correlations. The adsorption isotherms of SEB on YYWLHH resins with different peptide densities were fitted to bi-Langmuir isotherms. The general rate (GR) model was used to fit experimental breakthrough curves to obtain the intrinsic rate constants of the adsorption-desorption kinetics of the protein on the peptide ligands. An analysis of the number of transfer units in the column revealed that both intraparticle mass transfer and intrinsic adsorption rates were important rate-limiting steps for adsorption to the resin particles. The substitution level of peptide on the resin's surface has a significant impact on the resin's performance. The effects of peptide density on both equilibrium (batch format) and dynamic adsorption (column format) were investigated. The results revealed that the binding mechanism might change from univalent to multivalent adsorption with an increase in peptide density, thus explaining the variation of dissociation constants, maximum capacity, and rate constants with increases in peptide density. Adsorption processes can play an important role in helping to remove infectious pathogens such as SEB from solution without affecting other desirable components in a product stream. However, little work has been done on the design of adsorption columns specifically for the removal of several logs of infectious components. This requirement is much more stringent than the normal design of affinity separation columns where at most two logs of removal are sufficient. A column design strategy was developed, aimed at the removal of several logs of an infectious agent from a known volume of a process stream in a fixed amount of time. Two design options are analyzed with the general rate (GR) model, one fixing the column length and varying the fluid velocity, the other fixing the fluid velocity and varying the column length. The results indicate that the reduction in pathogen concentration is highly dependent on the residence time in the column, which is in turn dependent on the flow rate and column geometry. The theory, with no adjustable parameters, is shown to predict with great accuracy the effect of residence time on the log removal of SEB from an aqueous stream using an affinity resin with the peptide YYWLHH. Using a conventional Enzyme-Linked Immunosorbent Assay (ELISA) method, up to 5 log removal of SEB was detected, and the experimental log removal results agreed well with the theoretical prediction. The detection of minute amounts of SEB using High Performance Liquid Chromatography (HPLC), ELISA or Mass Spectrometry (MS) requires a concentration step prior to analysis. This can be accomplished by solid-phase affinity extraction on a peptide ligand column. It was shown that the YYWLHH resin has a great potential in sample preparation for SEB detection due to its high affinity and capacity. The peptide column was shown to capture all the SEB in highly diluted samples and it was possible to release SEB in a small volume of elution buffer. The enrichment of SEB from the YYWLHH column enabled an ELISA method to be able to work on diluted samples that cannot be analyzed without the peptide-affinity solid-phase extraction. The detection sensitivity of the ELISA method was improved from ng/ml to pg/ml using the YYWLHH column for SEB concentration.
Date: 2004-05-24
Degree: PhD
Discipline: Chemical Engineering

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