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Using green and red fluorescent proteins to teach protein expression, purification, and crystallization
Author(s) -
Wu Yifeng,
Zhou Yangbin,
Song Jiaping,
Hu Xiaojian,
Ding Yu,
Zhang Zhihong
Publication year - 2008
Publication title -
biochemistry and molecular biology education
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.34
H-Index - 39
eISSN - 1539-3429
pISSN - 1470-8175
DOI - 10.1002/bmb.117
Subject(s) - green fluorescent protein , affinity chromatography , fusion protein , chromatography , protein purification , chemistry , protein crystallization , fluorescence , recombinant dna , biochemistry , target protein , protease , crystallization , microbiology and biotechnology , biology , gene , enzyme , physics , organic chemistry , quantum mechanics
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.