LBIK10136U Heterologous Expression

Volume 2018/2019
Education

MSc Programme in Biology-Biotechnology
MSc Programme in Biochemistry

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 / 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 (only theoretically)
    • Aspergillus (only theoretically)
    • Bacillus


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

Learning Outcome

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.

Literature

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 Biology-Biotechnology.
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.
  • Category
  • Hours
  • Colloquia
  • 28
  • Exam
  • 1
  • Lectures
  • 24
  • Practical exercises
  • 140
  • Preparation
  • 219
  • Total
  • 412
Written
Oral
Continuous feedback during the course of the semester
Credit
15 ECTS
Type of assessment
Oral examination, 25 min. (no preparation)
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
External censorship
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 assesment

See desription of learning outcomes