Parking a bit more on Discerna -
The Company
Our company was founded in June 2001 to exploit discoveries in ribosome display made in the laboratories of the Babraham Institute (nr. Cambridge, UK).
We were formed as Babraham's first spin-out; a 50:50 joint venture between Babraham Bioscience Technologies Ltd and KS Biomedix Holdings Plc (LSE: KSB).
Intellectual Property
We possess a world wide exclusive license from The Babraham Institute to the ribosome display technology pioneered initially by Mingyue He and Mike Taussig. Since acquiring the license, Discerna has made further improvements to the technology which have broadened the scope of an already strong patent estate. The company now has a complementary patent portfolio containing four patent families. Further information on any of our patents can be obtained by contacting info@discerna.co.uk.
(http://www.discerna.co.uk)
Ribosome display technology
Ribosome display is an in vitro protein display system (1,2). The concept behind display methods is to link individual proteins to their genes (‘phenotype linked to genotype’) and to create libraries of such linkages. Selection of the protein from such a library simultaneously captures the gene, which can then be expressed or manipulated. A familiar example is phage display, which has been widely commercialised for making antibodies. The distinguishing features of ribosome display are that (a) it is a wholly cell free technology and (b) the link between the protein and the gene (mRNA) is made through a ribosome as a protein-ribosome-mRNA complex. Forming the complexes requires a modification of the mRNA, namely deletion of the stop codon. Under such conditions, nascent proteins are not released from the ribosome at the end of translation, remaining associated with their specific encoding mRNA molecule (Figure 1). Hence, libraries of protein-ribosome-mRNA complexes can be produced. The DNA is prepared by PCR and, since cloning is not involved, the restriction imposed by bacterial transformation is avoided, enabling rapid production of very large libraries (e.g. >1012 members). While we have used ribosome display primarily as a means of rapidly obtaining antibodies, it is adaptable to any type of protein, as well as to peptides. When displaying antibodies, we call the complexes ‘ARMs’ (antibody-ribosome-mRNA) and refer to the system as ARM ribosome display (1,2).
In the ribosome display cycle (Figure 2), a library of DNA molecules is converted into one of complexes by means of a eukaryotic cell-free transcription/translation system (from rabbit reticulocytes).The library can be prepared from different sources, such as cellular mRNA or cDNA, or as an artificial combinatorial library in which gene segments are assembled together, or as a library of mutants of existing genes in order to search for improved function. We have been expressing single chain antibody fragments called VH/K in which the VH domain is linked to the complete light chain (K). Individual complexes are selected through a binding reaction, such as to an antigen-coupled beads, and the genetic information is recovered using reverse transcription (RT) PCR. This recovery is performed directly on the selected ribosome complex without (as others have described) first disrupting the complex and recovering the mRNA. This give ARM display particularly sensitivity and speed.
We have used ARM display for cell-free selection of fully human monoclonal antibodies (3). Our approach has been to use the special resource of transgenic mice, created by Marianne Brüggemann (Babraham) and Michael Neuberger (MRC, Cambridge), which carry human heavy (H) and light (L) chain gene loci on a background in which the endogenous H and L genes have been knocked out. When immunised with an antigen which can include human proteins, the mice respond by making human antibodies. We use the spleen cells of such immune mice as a source of human VH and VL genes, from which single chain VH/K libraries are constructed for ARM ribosome display. After selection over a variable number of cycles, the products are expressed as soluble proteins in E. coli. In this way we have selected human antibodies against progesterone and gone on to improve their specificity and affinity by mutation and selection in the ARM display system.
We envisage a number of potential applications of ribosome display in selection of proteins from libraries and in protein engineering, among them isolation of novel ‘scaffolds’, in which existing protein domains are adapted to bind targets, protein evolution by continuous mutation and selection and, in the area of functional genomics, the use of ribosome display in conjunction with protein arrays to identify protein-protein interactions and in gene discovery.
1. He, M. and Taussig, M.J. (1997) Antibody-ribosome-mRNA (ARM) complexes as efficient selection particles for in vitro display and evolution of antibody combining sites. Nucleic Acids Research 24:5132-5134.
2. Taussig, M.J., Groves, M.T.A., Menges, M., Liu, H. and He, M. (2000) ARM complexes for in vitro display and evolution of antibody combining sites. In Monoclonal Antibodies: A Practical Approach (Eds. P. Shepherd, C. Dean) Oxford University Press, pp. 91-110
3. He, M., Menges, M., Groves, M.A.T., Corps, E., Liu, H, Brüggemann, M. and Taussig, M.J. (1999) Selection of a human anti-progesterone antibody fragment from a transgenic mouse library by ARM ribosome display. Journal of Immunological Methods 231:105-117. |
