Degree Name

MS (Master of Science)

Program

Biomedical Sciences

Date of Award

12-2020

Committee Chair or Co-Chairs

Valentin Yakubenko

Committee Members

Patrick Bradshaw, Jonathan Peterson

Abstract

Macrophage integrin aDb2 promotes macrophage retention and accumulation within inflamed tissue, a key event in development of chronic inflammation. Recently, the P5 peptide was identified as a specific inhibitor for integrin aDb2 interaction with 2-(ω-carboxyethyl) pyrole (CEP), a ligand at inflammatory sites. This thesis aims to identify integrin aD I-domain amino acids involved in binding P5 peptide and likewise to CEP. We propose that non-conserved, basic amino acids of the integrin aDb2 I-domain are responsible for binding to P5 peptide and likewise to CEP. Eight amino acids were analyzed by generating six mutant aD I-domains: K180[A], R189[Q], K205[L], HHK223-225[NIT], K233[A], and K246[A]. Mutagenic constructs were created using PCR site-directed mutagenesis, then transformed into E.coli BL21 cells for IPTG-induced protein expression. Of the 6 mutant I-domains analyzed, amino acid K246 was critical in binding to P5 peptide and CEP through ForteBio Protein-Protein Assay, as well as to CEP by cell adhesion assay.

Document Type

Thesis - embargo

Copyright

Copyright by the authors.

Available for download on Monday, November 04, 2024

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