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ERIC Number: EJ758957
Record Type: Journal
Publication Date: 2004-Jan
Pages: 4
Abstractor: Author
ISBN: N/A
ISSN: ISSN-1470-8175
Teaching Molecular Biology to Undergraduate Biology Students: An Illustration of Protein Expression and Purification
Sommer, Cesar Adolfo; Silva, Flavio Henrique; Novo, Maria Teresa Marques
Biochemistry and Molecular Biology Education, v32 n1 p7-10 Jan 2004
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.
John Wiley & Sons, Inc. Subscription Department, 111 River Street, Hoboken, NJ 07030-5774. Tel: 800-825-7550; Tel: 201-748-6645; Fax: 201-748-6021; e-mail: subinfo@wiley.com; Web site: https://secure.interscience.wiley.com/cgi-bin/jhome/112782101
Publication Type: Guides - Classroom - Teacher; Journal Articles; Reports - Descriptive
Education Level: Postsecondary Education
Audience: Teachers
Language: English
Sponsor: N/A
Authoring Institution: N/A