Identification of Unknown Fluorescent Proteins: A Colorful Lab Module for Teaching Gene-to-Protein Information Flow and Protein Structure

 

Aaron Coleman

Tested Studies in Laboratory Teaching, 2017, Volume 38

Abstract

Fluorescent proteins impart specific colors to coelenterate marine organisms such as jellyfish and corals. Green fluorescent protein (GFP; from the jellyfish Aequorea victoria) and DsRed (from the coral Discosoma sp) have been adapted for use as fluorescent tags for the real-time imaging of proteins in living cells, leading to a revolution in cell biology research. These proteins also make fun and engaging tools for teaching biology and biochemistry. Here we describe a lab module in which students identify unknown fluorescent proteins by performing spectrophotometry and SDS-PAGE. Students are assigned an unknown gene from a panel of 12 cDNAs encoding various fluorescent proteins derived from GFP or DsRed. These fluorescent proteins differ in their fluorescence properties and the degree to which they aggregate to form higher order complexes. The students receive the genes in a bacterial expression vector and proceed to express the fluorescent proteins in Escherichia coli (E. coli), followed by rapid purification via a poly-histidine tag added by the vector. Three pieces of evidence allow for identification of the unknown. The color of the bacterial colonies allows students to determine if their protein is a derivative of GFP or DsRed. The absorption spectrum of the fluorescent protein is measured on a spectrophotometer, allowing unknowns from the same parent protein to be identified by their excitation max. Some of the unknowns, however, have excitation maximums that are identical or too close to determine. These can only be distinguished by determining their subunit composition through running heated (denatures complexes) and unheated (complexes remain intact) samples on SDS-PAGE and analyzing the banding pattern of the stained gel. Correctly interpreting the gel requires students to understand how different length polypeptides are encoded on the gene, how these come together to form a dimer or tetramer, and how these would run under native and denaturing conditions.

Keywords:  green fluorescent protein, fluorescent protein

University of Houston (2016)