KBC/PRP Preparation of Recombinant Proteins

Guarantor: David Zalabák
Lecturer: Ondřej Plíhal
Exercise: 3 hours/week
Credits: 2
Summer semester
Form of course completion: course credit

Main goal of this course is to teach students the techniques of heterologous protein expression. In the framework of this course, students will learn how to prepare and purify recombinant proteins in E. coli, and also how to biochemically characterize them.

• Introductory lecture. Preparing the bacterial inoculum (E. coli) carrying the expression vector. Preparing cultivation media and sterile glass flasks.
• Setting up the heterologous protein expression, IPTG induction, preparing buffers, solutions etc.
• Harvesting bacterial cells, cell lysis and protein purification using affinity chromatography.
• Dialysis and concentration of protein sample using ultrafiltration.
• Analysis of protein sample using SDS-PAGE method to test protein purity and molecular weight. Protein quantification using Bradford assay.