LBIK10136U Heterologous Expression
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 / 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.
- Escherichia coli
- Saccharomyces cerevisiae
- Pichia pastoris
- Xenopus oocytes
- Mammalian cell lines
- Algae
- Higher plants
- Aspergillus (only theoretically)
- 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/
Virus induced expression /Design of drug screening
assays
After completing the course the student should have acquired the
following:
Knowledge:
-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.
Skills:
-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.
Competences:
-Transfer theory and principles regarding the usefulness of
different organisms as expression hosts to different work
situations.
-Make ethic 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)
- Category
- Hours
- Colloquia
- 28
- Exam
- 1
- Lectures
- 24
- Practical exercises
- 140
- Preparation
- 219
- Total
- 412
As
an exchange, guest and credit student - click here!
Continuing Education - click here!
- Credit
- 15 ECTS
- Type of assessment
- Oral examination, 30 minIn the first part the practical exercises will be discussed following one of the cases. It is important to demonstrate an overview of the topics that have been dicussed during the course. We expect you to be able to compare expression in the different organisms as well as discussing advantages/disadvantages when using a certian organism for a specific purpose.
- Exam registration requirements
- 7 out of 8 reports must be submitted
- Aid
- Only certain aids allowed
Reports from practicals, cases and notes.
- Marking scale
- 7-point grading scale
- Censorship form
- External censorship
Criteria for exam assesment
See desription of learning outcomes
Course information
- Language
- English
- Course code
- LBIK10136U
- Credit
- 15 ECTS
- Level
- Full Degree Master
- Duration
- 1 block
- Placement
- Block 3
- Schedule
- Lectures: Monday 9-12
Cases / Student presentations: Thursday 9-12
Lab work:
Monday and Thursday 12.30-16.30,
Tuesday, Wednesday and Friday 8.30-12.30
The exercises will be within this time, but the exacts number of days will vary between the different exercises. - Course capacity
- 45
- Continuing and further education
- Study board
- Study Board of Biomolecular Sciences and Technology
Contracting departments
- Department of Plant and Environmental Sciences
- Department of Food Science
Course responsibles
- Anja Thoe Fuglsang (3-65786a4474706972326f7932686f)
Lecturers
Anja Thoe Fuglsang
Rosa Lopez
Bent Larsen-Petersen
Poul Erik Jensen
Seong Wook
Barbara Halkier
Hussam Nour El Din Auis
Nils Arneborg
Thomas G. Pomorski