Ola Nilsson, the PhD student at the Clinical Immunology and Allergy unit spent 3 month at the Swiss Institute of Allergy and Asthma Research, Davos, Switzerland, where he was working under the supervision of Doctor Claudio Rhyner and Professor Reto Crameri. The purpose of the travel was to learn the methodology of the phage display technique, as well as the methods of creating a phage display library.
Purpose of the travel:
The purpose of the travel was a research visit to the lab of Prof. Reto Crameri to develop methodology for the phage display technique, as well as creating a phage display library. Although phage display is a state of the art technique for screening of complex protein and peptide libraries, it is still limited by the basic mechanics of E.coli. In the first project, the aim was to insert the glycosylation machinery of C.jejuni into E.coli and evaluate biopanning with glycosylated bacteriophages. The second project focused on fusing a tetracysteine-tag to the helper phage, which would make labeling of bacteriophages easy for both screening and titration purposes. This was to be performed by direct modification of the helper phage genome. The third project aimed to create a phage display library from human fibroblast cDNA. The library would then to be used for biopanning against bound IgE from patients with chronic urticaria and patients with chronic rhinosinusitis with nasal polyps, in order to find a relevant autoantigen to this disease.
Description of the acquired technique/methodology:
Phage display is a versatile technique that can be used for various applications within our own field of research. While we recently have developed a cat vaccine in collaboration with the group of Reto Crameri, the present project has focused on method development, as well as aiming to create a library which will be used identify novel autoantigens responsible for chronic urticaria and chronic rhinosinusitis with nasal polyps. The method involves state of the art cloning techniques, which is crucial when creating a library. Modifying a phage viron involves several critical steps, which will ultimately result in a modified helper phage with the desired tag. We also developed methodology for enriching glycosylated phage prior to biopanning, as well as detecting glycosylated phage in the output material.
Results of the visit/collaboration:
During my stay in Davos, I have fully learnt to construct a phage display library from cDNA, a technique that can be used to identify novel allergens and autoantigens involved in autoimmune disease. The library consistent of cDNA from human fibroblasts was successfully introduced into the in-house modified vector pJF3H, and the library can now be used for biopanning against patient IgE.
We developed a novel method for enrichment of glycosylated phage, using magnetic beads coated with lectin, which potently binds glycosylated phage. For detection of these phages, we developed a phage-ELISA, specifically designed to visualize the level of glycosylation of bacteriophages. Currently, a method is being developed for staining glycosylated phage for detection in immunoblot.
I have also learnt the method of attaching a tag of interest to protein VII on phage particles, and in this case, we have attached a tetracysteine tag. The construct will be used for visualization of the phage, as well as for possible development of a novel screening method that can replace phage titration with bacterial colonies.
Description of how the technique/methodology will be used at KI:
The research approach of Reto Crameri's group and that of our own group are similar in several ways, as we both use molecular approaches to better understand and ultimately to treat allergic disease. Methodology used and developed in Davos are at our disposal and phage display libraries, E.coli transformed with the glycosylation machinery and the modified helper phage will be transported to our own laboratory. We have future plans on creating cDNA phage display libraries that will be used to detect new allergens, both within the glycosylated and the non-glycosylated system.