Heterologous Expression
PhD School at the Faculty of SCIENCE at University of Copenhagen
This is a specialised course where 50% of the seats are reserved to PhD students enrolled at the Faculty of SCIENCE at UCPH and 50% of the seats are reserved to other applicants. Seats will be allocated on a first-come, first-served basis and according to the applicable rules.
Anyone can apply for the course, but if you are not a PhD student at a Danish university (except CBS), you will be placed on the waiting list until enrollment deadline. After the enrollment deadline, available seats will be allocated to applicants on the waiting list.
Aim and Content
Large-scale production of technical enzymes, peptide- or protein-based pharmaceuticals is performed using specially engineered host organisms. The aim of this course is to educate students in processes to be considered in association with heterologous expression. Upon course completion, students will be able to design a strategy for expression of a desired gene for different purposes, choosing amongst a series of relevant heterologous expression hosts, including gram+ and gram- bacteria, yeast, filamentous fungi, plant, algae, mammalian cells and xenopus oocytes. This includes reflections about yield, quality, and downstream applications (research/large-scale production) of desired protein products.
Selected theoretical topics of major relevance to different heterologous gene expression will be addressed, including
A) The intelligent choice of a host organism;
B) Cloning strategies envisioned by in silico simulations;
C) Promoter strength and induction strategies;
D) Gene copy number and silencing problems in heterologous hosts;
E) Use of autonomous plasmids vs. chromosomal integration;
F) mRNA stability and introns;
G) Choice and placement of purification tags;
H) Post-translational modifications and stability of the protein product;
I) Secretion of proteins and the unfolded protein response;
J) Engineering alternative/new post-translational modifications in different host organisms;
K) Inclusion bodies and protein folding in bacteria;
L) Expression of membrane proteins compared to soluble proteins;
M) Design of drug screening assays;
N) Heterologous expression for antibody production;
O) Expression of toxic proteins;
P) Transient vs. stable expression strategies in higher eukaryotes;
Q) Metabolic engineering for metabolite production;
R) Fermentation and large-scale production.
The course will also address the peculiarities of different production hosts, including
1) Escherichia coli;
2) Saccharomyces cerevisiae;
3) Pichia pastoris;
4) Xenopus oocytes;
5) Aspergillus;
6) Bacillus;
7) Mammalian cell lines;
8) Algae;
9) Xenopus oocytes;
10) Plants.
Learning outcomes
Intended learning outcome for the students who complete the course:
Knowledge:
• Knowledge on the feasibility of common prokaryotic and eukaryotic organisms as heterologous expression hosts,
• Knowledge on the use of specific hosts for protein or metabolite production, and/or as tools for specific research and development applications.
• Knowledge of host physiology and expression methods relevant for heterologous gene expression for different applications
• Feasibility of hosts for research and/or large-scale production
• Physiological changes induced in a host during heterologous gene expression and palliative measures
• Knowledge of the effects of post-translational modifications on protein production, stability, function, and downstream processing/use.
Skills:
• Ability to choose a heterologous expression host for a specific gene based on the characteristics of both the host and the desired result of the expression strategy
• Ability to design metabolic engineering strategies to modify hosts for specific needs
• Experience with evaluation and quality assessment of the result of a heterologous expression strategy
• Ability to identify drawbacks of a strategy and design new steps leading to improvement
• Critical use of relevant literature for development of heterologous expression strategies
Competences:
• Outline a suitable expression strategy for a given gene including host, expression system, expression conditions and evaluation of outcomes
• Design strategies for host/gene modification/improvement for a specific goal in their own PhD projects
• Solving challenges with own expression strategies by critical consideration of related published results
• Design large-scale screening strategies for different purposes, including optimization of protein expression, drug screening, metabolic engineering for metabolite production, protein-protein interactions, and biosensor development.
Target Group
Students in Health Science, Pharma, Veterinary, Biology, Biotechnology, or Biosolutions research projects that need to obtain expression products from specified genes, modify a host for expression or set up a large screening system.
This course offers an insight into a rational approach to solving existing challenges and basic knowledge to discuss product production strategies in an interdisciplinary context.
Recommended Academic Qualifications
Master of Science students with basic cell biology knowledge at the start of their PhD-study.
Research Area
Health Science, Pharma, Veterinary, Biology, Biotechnology, and Biosolutions.
Teaching and Learning Methods
The course is divided into 8 thematic weeks, each presenting a different heterologous expression host and different research/production strategies. Each week is organized under a common structure including lectures and journal club discussions of published results. Lectures will cover the main characteristics and use of common host expression organisms. When possible, guest lectures will be invited to introduce the students to industrially relevant organisms. Journal club discussions will allow the students to get a deeper insight into the state of the art for heterologous expression in the organism under focus each week. In some weeks, the students will be asked to design and present model expression strategies in the form of short pitches.
Students will be provided with eight case examples (one per week), including unpublished experimental results, that will be the basis for preparation of short reports. In these, the students will be asked to reflect upon the host, the product, and the expression strategy and propose solutions to common bottlenecks based on literature searches and the knowledge provided in lectures and journal clubs.
The case example will focus on a specific common topic related to heterologous expression, selected amongst A) Expression and assembly of protein complexes in different hosts; B) Modification of host for introduction of specific post-translational modifications, including detection/evaluation of results; C) Expression of secreted proteins and bottlenecks for secretion in different hosts; D) Expression of membrane proteins in yeast and downstream processing by detergent use; E) Design of biosensors; F) Protein-protein interaction screenings; H) Electrophysiological characterization and substrate assignment for metabolite-transporters; I) Design of drug screening assays in mammalian cell and yeast cells; J) Metabolic engineering for metabolite production in algae; K) Large-scale protein production in microorganisms and downstream processing; and L) Strategies for large-scale and toxic protein production in plant systems.
Students will receive feedback on each submitted report to clarify concepts and facilitate further reflection.
Type of Assessment
Students must hand in and get approval from teachers for at least seven out of eight reports. Evaluation of each report will be in a passed/not passed basis according to the learning outcomes of the course. In addition, students must present and discuss one original article selected by the teachers during one of the journal club events. Students are also expected to be present in at least 7 out of 8 journal club sessions and participate actively in the discussion.
Literature
Each week, the students will be provided with a specific literature list presenting the state of the art for heterologous expression in each host. The material will be uploaded to the university platform Absalon at least 1 week prior to the corresponding teaching week
Course coordinator
Professor Anja Thoe Fuglsang, Department of Plant and Environmental Sciences, atf@plen.ku.dk.
Guest Lecturers
The course will include a series of experts on different aspects of heterologous expression from the Departments of Plant and Environmental Sciences and Food Science at the Faculty of Science and the Department of Veterinary and Animal Science at the Faculty of Health. If possible, industrial experts will be invited as guest lectures in the week devoted to large-scale fermentation.
Dates
2/2-12/4-26
Expected frequency
The course will be available once every year.
Course location
The course will be held at the Frederiksberg Campus of the University of Copenhagen
Course fee
• PhD student enrolled at SCIENCE: 0 DKK
• PhD student from Danish PhD school Open market: 0 DKK
• PhD student from Danish PhD school not Open market: 6000 DKK
• PhD student from foreign university: 6000 DKK
• Master's student from Danish university: 0 DKK
• Master's student from foreign university: 6000 DKK
• Non-PhD student employed at a university (e.g., postdocs): 6000 DKK
• Non-PhD student not employed at a university (e.g., from a private company): 16.800 DKK
Cancellation policy
• Cancellations made up to two weeks before the course starts are free of charge.
• Cancellations made less than two weeks before the course starts will be charged a fee of DKK 3.000
• Participants with less than 80% attendance cannot pass the course and will be charged a fee of DKK 5.000
• No-show will result in a fee of DKK 5.000
• Participants who fail to hand in any mandatory exams or assignments cannot pass the course and will be charged a fee of DKK 5.000
Course fee and participant fee
PhD courses offered at the Faculty of SCIENCE have course fees corresponding to different participant types.
In addition to the course fee, there might also be a participant fee.
If the course has a participant fee, this will apply to all participants regardless of participant
type - and in addition to the course fee.