Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-06-30
2003-08-19
Ponnaluri, Padmashri (Department: 1627)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S007100, C435S235100, C435S069100, C435S091500, C435S091500, C435S091500, C536S023400
Reexamination Certificate
active
06607881
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to methods for selecting specific ligand and receptor encoding sequences and to kits for carrying out the methods.
BACKGROUND OF THE INVENTION
There is a continuing need for highly efficient selection systems in the screening of protein libraries, such as antibody libraries. Current systems are based on the display of antibodies on the surface of microorganisms containing the gene of the antibody. Specific clones can then be selected with immobilized antigens, for instance by panning on microtiter plates (Parmley and Smith, 1988, Gene, 73, 305-318 and Barbas III et al, 1991, PNAS 88, 7978-7982), selection on magnetic beads (Hawkins et al, 1992, J. Mol. Biol. 226, 889-896), immunotubes (Marks et al, 1991, J. Mol. Biol, 222, 581-597), affinity chromatography (McCafferty et al, 1990, Nature, 348, 552-554), fluorescence assisted cell sorter (FACS), antigen specific precipitation (Kang et al, 1991, PNAS, 88, 4363-4366) and SAP (Dueñas and Borrebaeck, 1994, Bio/Technology 12, 999-1002).
Several antibody libraries have been constructed on the surface of phages, e.g. a bacteriophage such as fd (McCafferty et al, 1990, Nature, 348, 552-554) or M13 (Barbas III et al, 1991, PNAS, 88, 7978-7982). The possibility of expressing antibodies (scFv) on the surface of bacteria has also been demonstrated by fusions to bacterial membrane proteins like Lpp-Omp A (Francisco et al, 1993, PNAS, 90, 10444-10448) and PAL (Fuchs et al, 1991, Bio/Technology, 9, 1369-1372). For a recent review of antibody display systems and the screening of antibody libraries, see Little, 1994, Biotech. Adv., 12, 539-555.
Antigen libraries have been constructed following essentially the same principles as antibody libraries, e.g. peptide libraries on the surface of bacteriophages (Smith, 1985, Science, 228, 1315-1317). Expression of antigens on the surface of bacteria has been demonstrated by fusions to LamB (Charbit et al, 1988, Gene, 70, 181-189 and Bradbury et al, 1993, Bio/Technology, 1565-1568), Omp A (Pistor and Hoborn, 1989, Klin. Wochenschr., 66, 110-116), fimbriae (Hedegaard and Klemm, 1989, Gene, 85, 115-124 and Hofnung, 1991, Methods Cell Biol., 34, 77-105), IgA protease &bgr; domain (Klauser et al, 1990, EMBO J., 9, 1991-1999) and flagellae (Newton et al, 1989, Science, 244, 70-72).
However, many of the prior art selection steps, such as panning and affinity chromatography, are not very efficient, and even if the yield of antibody or antigen is reasonable, these techniques do not provide any information about the nucleic acid sequence encoding it.
SUMMARY OF THE INVENTION
In the past, it has not been possible to combine a ligand library with a receptor library in order not only to clone and select one of the specific binding pair members and their corresponding nucleic acid sequences encoding them, but actually both.
The present invention provides an efficient screening technique to obtain corresponding ligand/receptor molecules and establishes a physical and logical connection between the two and their encoding sequences. This has the advantage that it is simple and rapid and opens up possibilities of applications, such as detecting new ligands and/or receptors, where both are unknown, as well as improvements regarding epitope mapping, localization of gene products and drug design.
Accordingly, in one aspect, the present invention provides a method for selecting nucleic acid sequences encoding ligand and receptor molecules capable of specific binding to each other, the method comprising:
(a) expressing in a host microorganism nucleic acid encoding a surface molecule which is operably linked to the expression of nucleic acid encoding a ligand or receptor molecule, or functional fragments thereof, so that the microorganism expresses the surface molecule and the ligand or receptor molecule as a fusion and displays it on its surface, wherein the host microorganism is modified so that it does not display the surface molecule other than as a fusion with the ligand or receptor molecule;
(b) contacting the modified host microorganism of step (a) with one or more replicable genetic units capable of expressing nucleic acid encoding ligand or receptor molecules, or functional fragments thereof, the ligand or receptor molecules being candidates for specific binding to the molecules displayed by the host microorganisms of step (a) and being expressed as fusions with a surface protein of the replicable genetic unit, wherein binding of the surface displayed ligand and receptor molecules mediates the transfer of the nucleic acid encoding the ligand or receptor from the replicable genetic unit to the microorganism; and,
(c) selecting the host microorganisms containing the nucleic acid sequences encoding the ligand and receptor molecules, or functional fragments thereof.
Thus, in this aspect, the invention provides a method for selecting nucleic acid sequences encoding ligand and receptor molecules, the selection arising from modification to the host microorganism so that infection or transfer of the nucleic acid from the replicable genetic unit takes place when binding of the receptor and ligand molecules occurs. Thus, infection via the normal route, e.g. for
E. coli
via wild type pili, is prevented by a modification to the host microorganism to prevent the display of a given type of surface molecule so that the only surface molecules of that type that are displayed are those fused to the receptor or ligand.
In a preferred aspect, the host microorganism expresses and displays modified ligand molecules, with the replicable genetic units expressing the candidate receptor molecules. Thus, this provides a method for selecting specific ligand and receptor encoding sequences wherein:
(a) the expression of a surface molecule encoding sequence is combined with a ligand encoding sequence, or a sequence encoding a functional fragment thereof, so that the host microorganism expresses modified ligand molecules on its surface;
(b) infecting, or in other ways transferring DNA to, the modified host organism of step (a) with a genetically modified replicable genetic unit capable of expressing a receptor, or a functional fragment thereof, fused to a surface protein of the replicable genetic unit; and,
(c) selecting infected host organisms containing specific ligand and receptor encoding sequences or sequences encoding functional fragments thereof.
In the above aspects, the method of the invention optionally includes the additional step of:
(d) isolating the nucleic acid sequences encoding the ligand or receptor molecules.
Conveniently, the isolation can be achieved by associating different selection markers (e.g. different antibiotic resistance markers) with the nucleic acid sequences encoding the ligand and receptor molecules. Thus, the host microorganisms containing nucleic acid sequences encoding a specific binding pair can be selected by growing them in the presence of both antibiotics. After this, the vectors containing nucleic acid encoding the ligand and the receptor can be separated from each other by omitting one of the antibiotics from the growing medium.
In different embodiments, this method can be used to screen:
(a) microorganisms displaying one type of ligand or receptor molecule against libraries of replicable genetic units displaying different receptor or ligand molecules;
(b) libraries of microorganisms displaying different ligand or receptor molecules against replicable genetic units displaying one type of receptor or ligand molecule; or,
(c) libraries of microorganisms displaying different ligand or receptor molecules against libraries of replicable genetic units displaying different receptor or ligand molecules.
In the above aspects, the host microorganisms do not express the normal (wild type) surface molecules that are used to display ligand or receptor molecules as fusions on its surface. This means that the replicable genetic units used to display candidate binding partners of the ligand or receptor molecule will only infect the microorganisms when binding between
Borrebaeck Carl
Malmborg Anki
Söderlind Eskil
Bioinvent International AB
Dann Dorfman Herrell and Skillman
Ponnaluri Padmashri
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