Heterologous Expression
Course content
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 /
Optimization 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
- Bacillus
- Higher plants (only theoretically)
- Aspergillus (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 protein complexes / 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 homologues from
thermophilic bacteria /Yeast two-hybrid system/ Split-Ubiquitin
system / Electrophysiological measurement on ion-transporters
/Design of drug screening assays/Metabolic engineering
MSc Programme in Biochemistry
MSc Programme in Biotechnology
MSc Programme in Biotechnology with a minor
subject
After completing the course, the student should have acquired
the following:
Knowledge:
-Describe the main features of E.coli, Bacillus, S.cerevisiae,
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-translational
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.
Digitalization
Design an expression strategy by "In silico cloning" using the programme CLC workbench. Information regarding the target enzyme is found by searching in public databases such as (NCBI, UniProt and TAIR)
Modelling and analysis of experimental data.
Calculation of fermentation kinetics based on published papers.
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.
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)
Qualifications within microbiology, molecular biology, cell
biology and biochemistry corresponding to a BSc in Biotechnology.
Academic qualifications equivalent to a BSc degree is
recommended.
- ECTS
- 15 ECTS
- Type of assessment
-
Oral examination, 25 min. (no preparation)
- Type of assessment details
- The exam starts off with discussing one of the practical exercises, based on the respective assignment. Thereafter one of the theoretical cases will be discussed. It is important to demonstrate an overview of the topics that have been covered and to discuss this using examples from both written assignments and cases. We expect you to be able to compare expression in the different organisms as well as discussing advantages/disadvantages of using a certain organism for a specific purpose.
- Exam registration requirements
-
6 out of 7 written assignments must be approved in order to attend the exam
- Aid
- Only certain aids allowed
Reports from practicals, cases and notes.
- Marking scale
- 7-point grading scale
- Censorship form
- No external censorship
Several internal examiners
- Re-exam
-
As the ordinary exam.
There cannot be dispensated for the requirement of laboratory exercises, and students who do not fulfil the requirement have to follow the course the following study year.
Criteria for exam assessment
See desription of learning outcomes
Single subject courses (day)
- Category
- Hours
- Lectures
- 24
- Class Instruction
- 28
- Preparation
- 219
- Laboratory
- 140
- Exam
- 1
- English
- 412
Kursusinformation
- Language
- English
- Course number
- LBIK10136U
- ECTS
- 15 ECTS
- Programme level
- Full Degree Master
- Duration
-
1 block
- Placement
- Block 3
- Schedulegroup
-
Lectures: Monday 9-12
Cases / Student presentations: Thursday 9-12
Lab work:
Monday and Thursday 13-17,
Tuesday and Wednesday 8.30-12.00
(a few Fridays, 9-12)
The lab exercises will be within this time, but there will be variations between the weeks. - Capacity
- 45
The number of places might be reduced if you register in the late-registration period (BSc and MSc) or as a credit or single subject student. - Studyboard
- Study Board for the Biological Area
Contracting department
- Department of Plant and Environmental Sciences
- Department of Food Science
- Department of Veterinary and Animal Sciences
Contracting faculty
- Faculty of Science
Course Coordinator
- Anja Thoe Fuglsang (3-71847650807c757e3e7b853e747b)
Teacher
Anja Thoe Fuglsang
Rosa Lopez
Bent Larsen-Petersen
Stephan Wenkel
Hussam Nour El Din Auis
Kirstine Callø
Nils Arneborg
Thomas G. Pomorski
Roseanna Hennessy
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