Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Patent
1996-09-23
2000-03-21
Brusca, John S.
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
435 71, 435 691, 435 9141, 4352523, 43525233, 536 231, 536 234, 536 237, C12Q 168, C12P 2100, C12N 121, C12N 511
Patent
active
060401419
DESCRIPTION:
BRIEF SUMMARY
The present invention relates to bacteria for preparing stable fusion proteins from a carrier protein and a passenger protein, the bacteria possessing the genetic marker fpt. This genetic marker permits the improved preparation of protein fusions having a destabilizing effect on bacteria. The present invention in particular relates to bacteria for preparing fusion proteins, the bacteria stably presenting the fusion proteins on their surface and possessing the markers ompT.sup.-- and dsbA.sup.-- in addition to the genetic marker fpt. Moreover, the present invention relates to the identification of bacteria, which surface-present heterologous proteins having an affinity to a binding partner and methods for constructing vectors encoding these proteins. Finally, the present invention also relates to bacteria which stably present at least one fusion protein on their surface and possess the genetic markers fpt, ompT.sup.-- and dsbA.sup.--, and their use for instance for diagnostic purposes. For example, the method of the invention in particular permits the formation of protein fusions which are composed of portions of heavy and light antibody domains and portions of the Iga.beta. transport protein, and their export through the bacterial cell envelope. In a specific embodiment, recombinant antibody fusions with binding activity can be presented on the bacterial surface of Escherichia coli cells.
Specific interactions between receptor molecules and ligands constitute a basic phenomenon of biological systems. Even extremely complex processes in highly developed organisms can be reduced to simple molecular interactions and can be analyzed and effectively influenced on this level. The specificity of such interactions is expressed in the affinity between the biomolecules involved, i.e. the binding forces between the receptors and the ligands.
In order for biological processes to be specifically investigated and manipulated, it is necessary to prepare analogues possessing defined binding properties and being suitable for the most different receptors and ligands. For this purpose, artificial systems are developed permitting the simple preparation of a multitude of receptor or ligand structures on the one hand and the selection and propagation of molecules possessing defined binding properties on the other hand. Such systems, in which biomolecules having defined properties can be obtained by evolution-like mechanisms can be realized in different ways. According to one strategy which closely adheres to the principles of living systems, for instance the principle of clonal selection in the immune system of mammals, a biomolecule presented on the cell surface is coupled with the propagation apparatus of living cells in the form of the encoding genetic information. The cells do not only assume the function of producing structurally variable biomolecules but also enable the selection of suitable molecules by presenting the biomolecules on their surface, and in the process of their cell division also enable the propagation of the underlying genetic information.
The physical basis of such a system is formed by both the producer cells and the genetic information, determining the blueprint of a biomolecule. The producer cells have to be capable of propagation, and at the same time must be capable of expressing heterologous genetic information and presenting the corresponding biomolecules accessibly on their surface. Ideally, they are easy to genetically manipulate, possess physico-chemical and genetic stability and are undemanding in respect of their growth conditions. A suitable microorganism meeting these requirements is Escherichia coli. Suitable biomolecules are preferably proteins. The underlying genetic information can extend from a few codons up to several genes, depending on whether a short peptide, a protein or a receptor consisting of several protein molecules is encoded. With the use of conventional methods of molecular biology it is possible to produce a multitude of protein variants on the genetic level and to expres
Klauser Thomas
Kramer Joachim
Meyer Thomas F.
Pohlner Johannes
Brusca John S.
Max-Planck-Gesellschaft zur Forderung der Wissenschaften e.v.
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