Investigating Activity of Engineered Proteins on Inorganic Substrates for Downstream Biomedical Applications
Abstract
As the proteomics field continues to expand for biomedical research purposes, scientists are looking to integrate cross-disciplinary tools for studying protein structures, functions, and interactions. One protein of interest, calmodulin (CaM), is a calcium-binding messenger protein with over a hundred downstream binding partners, and is involved in a host of physiological processes. To facilitate biophysical studies of calmodulin, researchers have designed site-specific labeling processes for use in bioconjugation applications while maintaining high levels of protein activity. Here, I present a platform for the highly efficient and selective immobilization of calmodulin onto different surfaces using azide-alkyne cycloaddition “click” chemistries. In particular, chemoenzymatically modified calmodulin is conjugated to sepharose resin beads for use in a novel affinity purification technique. I then present a method for in situ conjugation of calmodulin onto gold nanoparticles with consistent orientation and high levels activity. Protein Bioconjugation directly from clarified lysates represents a significant advance in what is normally a multi-step conjugation process. We anticipate these methods to be translatable to other proteins of interest and used in other downstream protein characterization assays.
Degree
M.S.B.M.E.
Advisors
Kinzer-Ursem, Purdue University.
Subject Area
Molecular biology|Biomedical engineering
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