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 ionchelating 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.Keywords: Green fluorescent protein, red fluorescent protein DsRed monomer, chromatography purification, protein crystallization, TEV protease cleavage.We have introduced a course named ''modern techniques in protein structural and functional analysis'' to the senior undergraduate students or the first-year graduate students who studied biology but had not yet taken similar courses. The course focused on the whole process of cloning, expression, purification, functional characterization and structural analysis of a specific protein. Green and red fluorescent proteins (GFP and RFP) were used in the laboratory sections, which greatly increased the students' interests and minimized the specialized equipment usage at the same time.The GFP, a small protein comprised 238 amino acids from the jellyfish Aequorea victoria, emits green fluorescent light when exposed to blue light [1]. It is stable under most physiological environment as a weak dimer [2]. Nowadays, GFP has already become an excellent marker for gene expression and protein localization in cellular and molecular biology. It is also used to demonstrate the dynamic processes of many biological activities within the cell, including protein folding, and transport. The GFP gene can also be maintained through breeding, and it was used to develop versatile reporter systems for genetic analysis.Different mutants of GFP have been engineered over the last few years. The enhanced green fluorescent protein (EGFP) 1 was one of the most famous mutants with increased stability and fluorescence efficiency. EGFP shifts its major excitation peak from 395 to 488 nm while the peak emission remains at 507 nm [3]. Using commercial vectors such...

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