Heterologous Gene Expression
PhD School at the Faculty of SCIENCE at University of Copenhagen
The production of technical enzymes as well as of peptide- and protein-based pharmaceuticals are in large scale being performed in specially designed host organisms. The aim of the course is to educate the students in processes associated with heterologous expression. The students will upon completion of this course be able to design and perform a strategy for the expression of a given gene. This includes considerations about amount, quality and downstream applications of the product.
Topics that will be covered in the theoretical part of the course:
The intelligent choice of a host organism / Cloning strategies envisioned by an “in silico” multistep cloning / Promoter strength and induction / Copy number and silencing problems in heterologous hosts / Expression vectors / mRNA stability and introns / Choice of, and placement of purification tags / Stability of the product / Secretion of proteins and signal trapping / Post-translational modifications in different host organisms / Inclusion bodies and folding of proteins / Expression of membrane proteins compared to soluble proteins / Design of drug screening assays/ Heterologous expression for production of antibodies / Expression of toxic proteins / Transient expression / Optimisation of expression level / Fermentation and large scale production.
In the course we will work with a range of different expression organisms.
o Escherichia coli
o Saccharomyces cerevisiae
o Pichia pastoris
o Xenopus oocytes
o Mammalian cell lines
o Higher plants
o Aspergillus (only theoretically)
o Bacillus (only theoretically)
We will express different types of proteins, determine the amount and activity of produced protein and discuss ways to optimize the expression level.
In the practical part we will also cover a broad aspect of typical problems related to the production of recombinant protein.
Topics from the practical part of the course:
Expression and assembly of a multi subunit protein complex / The effect of alcohol and temperature on expression level / Expression of a secreted protein / Sub-cellular fractionation / Detection of post-translated modifications / The use of protein homologs from thermophilic bacteria /Yeast two-hybrid system/ Split-Ubiquitin system / Electrophysiological measurement on ion-transporters/Design of drug screening assays.
If you would like to participate please:
1) Register online by clicking on "Apply" and
2) Send an e-mail to Anja Thoe Fuglsang firstname.lastname@example.org stating your motivations (10 lines)
After completing the course the student should have acquired the following:
-Describe the main features of E.coli, Bacillus, S.cereviiae, P.pastoris, mammalian cell lines, Xenopus oocytes, Aspergillus, Algea and plants as expression hosts
-Describe the following parameters for the above mentioned expression systems: Expression levels, Type of post-translationel modifications, Mechanisms for secretion of the product, Stability of the product, Stability of the transformed expression host, Methods commonly used for transformation, Strategies for optimization of the expression level and quality of the product.
-Use the knowledge to design an appropriate strategy for the expression of the correct amount and quality of a given protein/peptide.
-Design a strategy for creating an optimal genetically modified expression host in relation to reduction of proteases, improvement of secondary modifications and efficient compartmentation of the desired product.
-Transfer theory and principles regarding the usefulness of different organisms as expression hosts to different work situations.
-Make considerations about the use of GM organisms for production of peptides and about the disease risks connected to a certain expression host.
The course is based on:
- Selected reviews
- Scientific papers for presentation and discussion
- Laboratory manual (will be available for the students one week before beginning of the course)
Teaching and learning methods
The course contains both a theoretical part and a practical part. In the theoretical part there will be lectures as well as student presentations based on cases and journals. A practical laboratory part is running several days during most weeks. There is a close connection between the topics covered in the theoretical cases and the practical work. The course will be divided into smaller parts build upon the different expression organisms.
Anja Thoe Fuglsang
Hussam Nour El Din Auis
Thomas G. Pomorski
Price: DKK 3.000,- covering lab. costs and course related materials
Course capacity 48 (including students from the master course LBIK10136U)
•Department of Plant and Environmental Sciences
•Department of Food Science
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