This work was supported by the Brazilian research funding institution, Fundação de Amparo à Pesquisa do Estado de São Paulo (Centro de Biotecnologia Molecular Estrutural-Centro de Pesquisa Inovação e Difusão-Proc. 98/14138-2). César Adolfo Sommer is a fellowship recipient of Coordenação de Aperfeiçoamento de Pessoal de Nível Superior. The cost of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Teaching molecular biology to undergraduate biology students: An illustration of protein expression and purification*
Article first published online: 3 NOV 2006
Copyright © 2004 International Union of Biochemistry and Molecular Biology, Inc.
Biochemistry and Molecular Biology Education
Volume 32, Issue 1, pages 7–10, January 2004
How to Cite
Sommer, C. A., Silva, F. H. and Novo, M. T. M. (2004), Teaching molecular biology to undergraduate biology students: An illustration of protein expression and purification. Biochem. Mol. Biol. Educ., 32: 7–10. doi: 10.1002/bmb.2004.494032010290
- Issue published online: 3 NOV 2006
- Article first published online: 3 NOV 2006
- Manuscript Revised: 25 AUG 2003
- Manuscript Received: 11 JUL 2003
- Green fluorescent protein;
- protein expression;
- protein purification;
- laboratory classes
Practical classes on protein expression and purification were given to undergraduate biology students enrolled in the elective course “Introduction to Genetic Engineering.” The heterologous expression of the green fluorescent protein (GFP)* of Aequorea victoria is an interesting system for didactic purposes because it can be viewed easily during experiments. The students were provided with basic information about the molecular features and applications of the GFP in molecular biology, the available heterologous expression systems, and the theoretical and experimental details of GFP expression in Escherichia coli and its purification. E. coli BL21-competent cells were transformed with the pET28a expression vector containing the GFP gene fused to a histidine (His) tag. During the induction of a transformed clone by isopropylthiogalactoside, a time course for GFP expression was analyzed by SDS-PAGE, and the expression was also visualized by the increasing green fluorescence of the bacterial culture. After cellular disruption, protein purification was illustrated by affinity chromatography of the His-tagged protein in a nickel column. Eluted fractions containing imidazole in increasing concentrations were analyzed visually and also by SDS-PAGE, demonstrating the role of imidazole in protein recovery by competition with nonspecific proteins and the His-tagged protein. The results obtained and the experimental factors involved in protein expression, solubilization, and folding were discussed following the laboratory experiments. These practical classes allowed several current approaches to molecular biology to be demonstrated rapidly and helped underscore some of the topics taught during the course.