PhD courses at University of Copenhagen

Phage Display; Screening of proteins with DNA encoded peptide libraries...
Provider: Faculty of Health and Medical Sciences

Activity no.: 3139-14-00-00
	Enrollment deadline: 01/06/2015

Date and time	15.06.2015, at: 09:00 - 26.06.2015, at: 16:00
Regular seats	20
Course fee	11,160.00 kr.
Lecturers	Brian Lohse
ECTS credits	7.20


Enrolment Handling/Course Organiser	PhD administration E-mail address: phdkursus@sund.ku.dk

Aim and content This course is a part of the "open market" agreement and free of charge for PhD students enrolled at a Danish University (except students from CBS). COURSE DESCRIPTION This course aims primarily towards that the ph.d.-student achieves practical experience and theoretical knowledge of the Phage Display technology, covering some of the most used methods in molecular biology, e.g. virus infection of bacteria, DNA-encoding for peptide libraries and ELISA binding assays. On the experimental and practical level it is the aim of the course the students acquire the knowledge of methods that will enable them to do screening of different protein targets using large peptide libraries using important methods, which are fundamental in pharmacology and medicinal chemistry today. These techniques will demonstrate to the students how powerful molecular biology are to facilitate identification of new peptide lead structures in 10 days, which would take years if using conventional techniques e.g. synthetic small molecule chemistry. The participants will learn how to handle and grow bacteria, infecting them with virus and isolating hits. They will learn how to precipitate the phages and isolate their DNA, and sequence it. The ph.d.-students will also gain insight into the mechanisms by which virus can infect bacteria and how this infection pathway can be taken advantage of and turned into a powerful tool, to aid the researcher in identifying new lead structures that can be used in the pursuit of new pharmaceuticals. The ph.d.-student will gain a good understanding of how binding motifs and interactions, identified in the phage display can be elucidated, using techniques like Biacore and H/D-Exchange. Furthermore, the students are allowed to bring their own research project targets, which increase the value of such a course in the sense that the students have the possibility to gain new lead structures for their own projects. Problem Based and Research Based Learning will be the fundament throughout the course. These methods and phage display is cutting edge technology and used on a daily basis in the medicinal industry and in molecular drug research as an essential step towards obtaining new lead structures that can be developed into new potent biopharmaceuticals. Background Information What is phage display? Phage display is a powerful tool for selecting peptides, proteins, or antibodies with specific binding properties from a large number of variants. The technology consists in expressing peptides, proteins or antibody fragments at the surface of phage particles (Smith, 1985; Winter et al., 1994; Kay and Hoess,1996). The purpose is to screen for proteins with specific properties selected from libraries of DNA variants expressed into populations of protein variants on phages surfaces. What is a phage or bacteriophage? Phages are viruses that infect bacterial cells. Many of the vectors used in recombinant DNA research are phages that infect the standard recombinant DNA host: the bacterium Escherichia coli. There are several types of phage that have been used as vehicles for phage display including Ff filamentous phage, Lambda and T7. Each of these has advantages and disadvantages with respect to each particular application. The Ff phage family (M13 and its close relatives fd and fl) are excellent cloning vehicles because their size is not constrained by the DNA contained within them. The insertion of foreign sequences within their genome is accommodated simply by the assembly of longer phage particles. History of phage display Phage display technology was first introduced in 1985 by George Smith. It was used as an expression vector for presenting a foreign amino acid sequence accessible to binding an antibody. Since then, a large number of phage displayed peptide and protein libraries have been constructed, leading to various techniques for screening such libraries. Phage display technology has had a positive effect on works done in the fields of immunology, cell biology, pharmacology, and drug discovery. Technical description of phage display The starting point is usually a library of peptides, proteins or antibodies, which can be obtained commercially or constructed in house against a specific type of target. The construction of a phage display library is accomplished by inserting DNA fragments into phage or phagemid genomes which proteins are expressed on the phage coat. This creates a direct physical link between the DNA sequences and their encoding proteins. All five capsid proteins from the phage (pIII, pVI, pVII, pVIII and pIX) have been used to display proteins, peptides or antibodies, to varying degrees. The choice of pVIII or pIII is related to the choice of the type of display, either polyvalent or monovalent display. Applications of phage display Phage display can be used for selection of proteins, peptides, or antibodies with affinity and specificity to a molecule or protein of interest (see screening of phage display libraries). It can be helpful for preservation of unstable hybridoma clones. The technique is useful to identify molecules that can be recognized and internalized by eukaryotic cells. In addition, it can identify epitopes, mimotopes, functional and accessible sites from antigens (Mol Inmunol 1986; 23:709-15), and postransductional modifications in different molecules (Science. 1993; 260:1113-7). Phage display is an excellent tool for designing vaccines. It can facilitate the identification of peptides with applications in the prevention of diseases, for example, peptides of interest from a phage display peptide library can be captured with monoclonal antibodies against antigens from pathogens. Also phage particles displaying antigens on their surface can be utilized as vaccine like the antigens from Plasmodium falciparum (J Biol Chem 1988;263:4318-22).One of the most important application of phage display is to generate monoclonal antibodies and improve antibody affinity by affinity maduration. Advantages of phage display Phage display is a system for large scale study and selection of proteins based on their binding affinity and specificity. One advantage of phage display is the enormous diversity of variant proteins that can be represented. For example, phage display antibody libraries with diversities as high as 10e10 are routinely constructed. Phage display is highly flexible and selection may be performed in vivo or in vitro. In vitro selection enables phage displayed proteins to be screened not only against a wide range of biological targets but also inorganic ones. Phage display screening formats can be readily modified to manipulate selection conditions and stringencies. Phage display provides a means of rapidly screening large numbers of proteins against potential binding partners. As a rough guide, billions of clones can be screened within a week using phage display. Phage display libraries There are several types of Phage Display libraries including peptide libraries, protein libraries and antibody libraries. For example, peptides can be selected from peptide libraries, which commonly expressed variants of peptides of 7 to 20 amino acids in fusion with the protein pIII or pVIII of the phages. Pre-made or commercial peptide libraries are available for screening service in our website. Proteins of interest can be selected from a protein library like pre-made libraries of phages expressing on their surfaces proteins from microorganisms. The purpose of these libraries is to select proteins with a specific function or affinity to another molecule of interest. There is a system called Shotgun Phage display which consists in displaying proteins or domains from microorganisms in a multivalent form using pVIII -fusion. In this system, the genomic DNA is digested and cloned randomly in fusion with the gene of pVIII of the phage capsid (Comb Chem High Throughput Screen. 2001 Apr; 4(2):135-43.). There are other systems of phage display which used the protein pIII to display proteins or domains. Phage display antibodies can be selected from antibodies libraries which have been the main use of the system Phage display. In the antibodies libraries, the phage display antibodies are not expressed as complete antibodies, but as Fab or scFv fragments. Learning objectives A student who has met the objectives of the course will be able to: a. Use DNA-encoded libraries b. Screen a specific protein target c. Identify a potential binder and/or inhibitor Assessment The course is accessed based on 3 important elements: 1) Active participation in the lab. ( min. 90 %) 2) Approved report. 3) Multiple choice test (min. 50 % correct) The written test is a multiple choice test, consisting of 33 questions with 4 possibilities in each. All questions are weighted equally and the test lasts 2 hours. The approved report from each student will be graded and make up for 50 % of the final grade. The grade from the multiple choice test will make up for the rest of the final grade. Hence, the final grade will be the average of the two grades mentioned, following the 7th scale grade. Content and Curriculum The course covers the following subjects: Molecular Biology: • Recombinant DNA technique • DNA sequencing • Cell growth • Recombinant expression by transfection in host cells • ELISA Medicinal Chemistry • Peptide chemistry • Combinatorial peptide libraries • Fluorescense-based functional assays • Binding/ inhibition studies The instruction and lab. manual is written by the teacher (primary) responsible for the course and as a supplement relevant articles from the literature will be added. Participants The ph.d.-students are expected to have acquired the following competences and skills, before they start the course corresponding to the following courses/ modules: Biochemistry/ Bioorganic chemistry and/ or microbiology and lab experience within one of these fields. Language If all attendants speak Danish, then in Danish, otherwise in English. Form (For instance: Lectures, group work, discussions, poster presentations, exercises or others) The course is open for all SUND ph.d.-students and researcher and lab. Technicians from industry and ph.d.-Students from other universities will also be accepted. The course will be held outside the block structure, in order not to conflict with obligatory lab. courses. The students will be accessed based on active participation, constituting 90 %. More than 10 % absence will exclude the student from taking the written exam. The final evaluation is based upon an accepted report and a multiple choice test lasting 2 hours, which are equally weighted. Practical laboratory exercises alternating with review and discussion of underlying theory. Students work in teams of two. Dates: 15-20 and 23-26 of June.