Wu Yifeng, Zhou Yangbin, Song Jiaping, Hu Xiaojian, Ding Yu, Zhang Zhihong
Department of Physiology and Biophysics, School of Life Sciences, Fudan University, Shanghai 200433, China.
Biochem Mol Biol Educ. 2008 Jan;36(1):43-54. doi: 10.1002/bmb.117.
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.
我们设计了一门实验室课程，利用绿色和红色荧光蛋白（GFP和RFP）来展示蛋白质科学中的克隆、表达、色谱纯化、结晶和蛋白酶切割实验。通过PCR扩增增强型绿色荧光蛋白（EGFP）和单体红色荧光蛋白（mDsRed）的编码序列，并将其克隆到pMAL（MBP-EGFP）或pT7His（His(10)-mDsRed）原核表达载体中。然后通过IPTG诱导或自诱导在Rosetta (DE3) pLysS中表达荧光蛋白。我们通过亲和色谱（直链淀粉和金属离子螯合柱）、阴离子交换色谱（High Q柱）、尺寸排阻色谱（Sephacryl S-200柱）和疏水相互作用色谱（甲基疏水相互作用色谱柱）来纯化荧光蛋白，以展示蛋白质纯化技术。纯化后，用TEV蛋白酶切割融合蛋白MBP-EGFP。通过悬滴气相扩散技术使重组mDsRed蛋白结晶，向学生展示结晶的基本操作。使用荧光蛋白时，整个过程可以用肉眼实时监测。表达、纯化、结晶和蛋白酶切割的展示变得更加生动有趣，极大地加深了学生对现代蛋白质科学技术的理解。
