Lipchock James M, Ginther Patrick S, Douglas Bonnie B, Bird Kelly E, Patrick Loria J
Department of Chemistry, Washington College, Chestertown, Maryland, 21620.
Department of Chemistry, Yale University, New Haven, Connecticut, 06511.
Biochem Mol Biol Educ. 2017 Sep;45(5):403-410. doi: 10.1002/bmb.21056. Epub 2017 Mar 13.
Here, we present a 10-week project-oriented laboratory module designed to provide a course-based undergraduate research experience in biochemistry that emphasizes the importance of biomolecular structure and dynamics in enzyme function. This module explores the impact of mutagenesis on an important active site loop for a biomedically-relevant human enzyme, protein tyrosine phosphatase 1B (PTP1B). Over the course of the semester students guide their own mutant of PTP1B from conception to characterization in a cost-effective manner and gain exposure to fundamental techniques in biochemistry, including site-directed DNA mutagenesis, bacterial recombinant protein expression, affinity column purification, protein quantitation, SDS-PAGE, and enzyme kinetics. This project-based approach allows an instructor to simulate a research setting and prepare students for productive research beyond the classroom. Potential modifications to expand or contract this module are also provided. © 2017 by The International Union of Biochemistry and Molecular Biology, 45(5):403-410, 2017.
在此,我们展示了一个为期10周的以项目为导向的实验室模块,旨在提供基于课程的本科生物化学研究经验,强调生物分子结构和动力学在酶功能中的重要性。该模块探讨了诱变对一种与生物医学相关的人类酶——蛋白酪氨酸磷酸酶1B(PTP1B)的重要活性位点环的影响。在一个学期的时间里,学生们以经济高效的方式指导自己的PTP1B突变体从构思到表征,并接触到生物化学的基本技术,包括定点DNA诱变、细菌重组蛋白表达、亲和柱纯化、蛋白质定量、SDS-PAGE和酶动力学。这种基于项目的方法使教师能够模拟研究环境,并让学生为课堂之外的有效研究做好准备。还提供了对该模块进行扩展或缩减的潜在修改方法。© 2017年,国际生物化学与分子生物学联盟,45(5):403 - 410,2017。