Evolution by selection begins at the molecular level within the cell: a series of mutations occur that are able to become fixed in the population, and finally lead to a protein sequence that performs a novel function for the organism. ß-lactamase, a bacterial protein that counteracts ß-lactam antibiotics such as ampicillin, is a significant and well-studied protein, making it an excellent model for studying the biochemical pathway of protein evolution. It has been hypothesized that the relative fitness of mutant proteins is primarily determined by three factors: catalytic activity at the active site, stability of overall folding structure, and localization in the cell. We expressed and purified wild-type and mutant alleles of the TEM-1 ß-lactamase gene to study stability and localization of this protein. All of these mutations were outside the protein's active site, removing catalytic activity at the active site as a variable. We isolated proteins from periplasmic, cytosol-soluble and insoluble fractions using established biochemical methods. Subsequently these protein samples be analyzed using transverse urea gradient gel electrophoresis and Western blot to determine stability of these alleles, and using ELISA to quantitatively determine localization.
Spangle, Dylan,
"Stability and location of mutant proteins in bacterial cells"
(2015).
Summer Research Symposium.
Brown Digital Repository. Brown University Library.
https://doi.org/10.26300/a40q-2727
Each year, Brown University showcases the research of its undergraduates at the Summer Research Symposium. More than half of the student-researchers are UTRA recipients, while others receive funding from a variety of Brown-administered and national programs and fellowships and go …
