Expression, purification, and analysis of unknown translation factors from Escherichia coli: A synthesis approach

Abstract

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.

Protein analysis using real‐time PCR instrumentation: Incorporation in an integrated, inquiry‐based project

“…The E. coli cloning strain XL1‐Blue was transformed with the translation factor expression plasmids , kindly provided by P. C. Spiegel, and maintained as frozen stocks. Plasmid preparation was done using the Zyppy Plasmid Miniprep kit (Zymo Research).…”

“…The project in BIOC 312 combines elements of two published student laboratory projects . Each group begins the project with a culture of a standard cloning strain of E. coli carrying a plasmid for expression of a different translation factor.…”

“…Except for the fact that they are told the identity of their factor, they proceed essentially as described by Walter et al . through the purification of their “wild‐type” translation factor (not a true wild‐type protein as it has a 6× histidine tag at the N‐terminus). In this part of the project, students learn methods for plasmid purification, characterization by restriction digestion and agarose gel electrophoresis, bacterial transformation, recombinant protein expression, cell harvesting and disruption, and affinity chromatography.…”

“…The original translation factor project includes a series of protein unfolding exercises on the wild‐type translation factor using circular dichroism and intrinsic fluorescence to monitor protein conformation. In BIOC 312, all students complete a similar analysis using the reporter dye SYPRO orange and a real‐time PCR instrument to monitor conformation during thermal denaturation.…”

Preparative Protein Production from Inclusion Bodies and Crystallization: A Seven-Week Biochemistry Sequence

“…6 Student-directed inquiry has been introduced into the protein purification process either at the point of protein selection and purification design 7 or using themodynamic analysis to characterize unknown translation factors. 8 …”