NSCPHD1034 Heterologous Gene Expression
MSc Programme in Biology-Biotechnology
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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.
- Escherichia coli
- Saccharomyces cerevisiae
- Pichia pastoris
- Xenopus oocytes
- Mammalian cell lines
- 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/Design of drug screening assays.
After completing the course the student should have acquired the
-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)
- 15 ECTS
- Type of assessment
- Continuous assessmentApproval of the reports and presentations.
- Exam registration requirements
Active participation in the course.
- Only certain aids allowed
Reports from practicals, cases and notes.
- Marking scale
- passed/not passed
- Censorship form
- No external censorship
There can not be dispensated for the requirement of laboratory exercises, and students who do not fulfil the requirement has to follow the course the following study year.
- Practical exercises
- Theory exercises
- Project work