Expression, Purification, and Analysis of Unknown Translation Factors from Escherichia coli: A Synthesis Approach
Protein folding, Biophysical chemistry, Translation, Circular dichroism, Intrinsic tryptophan fluorescence
New approaches are currently being developed to expose biochemistry and molecular biology undergraduates to a more interactive learning environment. Here, we propose a unique project-based laboratory module, which incorporates exposure to biophysical chemistry approaches to address problems in protein chemistry. Each of the experiments described herein contributes to the stepwise process of isolating, identifying, and analyzing a protein involved in a central biological process, prokaryotic translation. Students are provided with expression plasmids that harbor an unknown translation factor, and it is their charge to complete a series of experiments that will allow them to develop hypotheses for discovering the identity of their unknown (from a list of potential candidates). Subsequent to the identification of their unknown translation factor, a series of protein unfolding exercises are performed employing circular dichroism and fluorescence spectroscopies, allowing students to directly calculate thermodynamic parameters centered around determining the equilibrium constant for unfolding as a function of denaturant (temperature or chemical). The conclusion of this multi-part laboratory exercise consists of both oral and written presentations, emphasizing synthesis of the roles of each translation factor during the stepwise process of translation.
Biochemistry and Molecular Biology Education
Required Publisher's Statement
Copyright © 2010 International Union of Biochemistry and Molecular Biology, Inc.
Article first published online: 28 JAN 2010
Walter, Justin D.; Littlefield, Peter; Delbecq, Scott P.; Prody, Gerry; and Spiegel, P. Clint, "Expression, Purification, and Analysis of Unknown Translation Factors from Escherichia coli: A Synthesis Approach" (2010). Chemistry Faculty and Staff Publications. 7.
Subjects - Topical (LCSH)
Proteins--Synthesis; Escherichia coli--Genetics; Genetic translation; Fluorescence spectroscopy; Physical biochemistry--Study and teaching--Activity programs
Biochemistry Commons, Chemistry Commons, Molecular Biology Commons