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Teaching biochemistry and molecular biology using dihydrofolate reductase as an expression system
Author(s) -
Lau Joann,
Gilbert Michele
Publication year - 2012
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.26.1_supplement.620.5
Subject(s) - dihydrofolate reductase , biochemistry , microbiology and biotechnology , biology , mutagenesis , enzyme , amino acid , western blot , affinity chromatography , mutant , gene
The enzyme dihydrofolate reductase (DHFR) converts dihydrofolic acid to tetrahydrofolic acid, thereby regulating the synthesis of purine and certain amino acids. Since DHFR plays such an important role in cell proliferation and growth, it was the first enzyme targeted for chemotherapy. The objective of this project is to teach molecular and biochemical techniques by having students induce gene expression, purify and enzymatically assay for DHFR activity. During this semester‐long project, students induce expression in recombinant bacteria and purify the DHFR protein using IMAC (immobilized metal affinity chromatography) resin. Students check purity via SDS‐PAGE electrophoresis and identify the fraction (insoluble vs. soluble) containing DHFR. Additionally, they design PCR primers for site‐directed mutagenesis, verify PCR success via restriction enzyme digestion and agarose gel electrophoresis, transform into expression competent cells, and confirm the amino acid mutation by DNA sequencing. Students then examine specific activity of the wildtype DHFR protein and compare it with the mutant DHFR protein. They also determine protein concentrations using the Bradford assay and perform Western blot analysis to detect glutathione S‐transferase (GST) and histidine (His) tags. All student groups successfully purified DHFR and detected both GST and His tags by Western blot analysis. Site‐directed mutagenesis showed a decrease in specific activity ranging from 32–122% compared to the wild‐type protein. This project provides students with the opportunity to gain hands‐on experience with a multitude of laboratory techniques as well as the opportunity to design and implement cutting edge research approaches.