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529-0046-00L 6 Credits BSC D-CHAB , D-BIOL
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Protein-derived Activity Probes for Ubiquitin Pathways

Lecturers & Examiners: Prof. Dr. Jeffrey W. Bode
Enrolment limited to max 6 students. The enrolment is done by the D-BIOL study administration.
VVZ CR n/a

Last Updated: 2026-02-05 16:23:34

Abstract

In this block course we will prepare proteins carrying electrophilic groups to interrogate intracellular pathways. These probes will be prepared by recombinant protein expression & purification followed by site-specific protein modification with electrophilic probes. Their reactivity will be tested in mammalian whole cell lysates and analyzed by proteolytic digestion and tandem mass spectrometry.

Objective

Students should learn how to design protein-based activity probes, express and isolate the proteins from E. coli, conduct site-specific protein modification, evaluate these probes in mammalian cell lysates using a proteomics workflow, and analyze and identify known or unknown proteins trapped by the activity probes. After the course participants should be able to apply gained skills in future chemical and molecular biology lab courses and projects. Individual reports describing the experiments and obtained results must be prepared by the end of the course.

Content

This course is based on a genuine research area and emerging technique. By working in pairs participants will recombinantly express and purify different ubiquitin mutants (UbV) in parallel. Based on the previous phage display screen, the chosen variants possess tunable affinity towards ubiquitin pathway proteases. Expression of the variants as intein fusions will enable site-specific C-terminal chemical modification either by direct aminolysis of the thioesters or by acylation of C-terminal hydrazides with activated carboxylic acids. The resulted constructs bearing electrophilic warheads will be utilized in mammalian whole cell lysates to covalently crosslink with cysteine UBL domain bearing proteases. The trapped proteases will be enriched by immunoprecipitation and analyzed by proteolytic digestion and tandem MS/MS with support from Functional Genomics Center Zurich (FGCZ). Results obtained from an individual UbV pull-down will be discussed and compared in the seminars. We expect to develop novel protein activity-based probes for various ubiquitin proteases (deubiquitinating enzymes).

Resources

Lecture Notes

All the required theoretical and experimental details will be discussed in class. Relevant research papers will be recommended for reading during the seminars.

General Information

Language
English
Levels
BSC
Frequency
Yearly recurring

Examination

Type
graded semester performance
Student performance will be evaluated by a written report and an oral presentation.

Course Components

Type Title Time & Place Hours
practical/laboratory course Protein-derived Activity Probes for Ubiquitin Pathways
Permission from lecturers required for all students. Block course in the second quarter of spring semester.
No time listed 100 h semesterly

Offered In