title: Using green and red fluorescent proteins to teach protein expression, purification, and crystallization.
abstract: We have designed a laboratory curriculum using the green and red fluorescent proteins (GFP and RFP) to visualize the cloning, expression, chromatography purification, crystallization, and protease-cleavage experiments of protein science. The EGFP and DsRed monomer (mDsRed)-coding sequences were amplified by PCR and cloned into pMAL (MBP-EGFP) or pT7His (His(10) -mDsRed) prokaryotic expression vectors. Then the fluorescent proteins were expressed in Rosetta (DE3) pLysS by IPTG induction or autoinduction. We purified the fluorescent proteins by affinity chromatography (Amylose and metal ion-chelating column), anion-exchange chromatography (High Q column), size exclusive chromatography (Sephacryl S-200 column), and hydrophobic interaction chromatography (Methyl HIC column) to exhibit the protein-purification techniques. After purification, the fusion protein MBP-EGFP was cleaved by TEV protease. The recombinant mDsRed protein was crystallized by hanging drop vapor diffusion technique to show students the basic operation of crystallization. The whole procedure can be monitored real time by naked eyes when using fluorescent proteins. The demonstration of expression, purification, crystallization, and protease cleavage became much more vivid and interesting, which greatly deepened the students' understanding of modern protein-science techniques.
authors: Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China.
authors: Yifeng
authors: Y
authors: Wu
authors: Yangbin
authors: Y
authors: Zhou
authors: Jiaping
authors: J
authors: Song
authors: Xiaojian
authors: X
authors: Hu
authors: Yu
authors: Y
authors: Ding
authors: Zhihong
authors: Z
authors: Zhang