Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving virus or bacteriophage
Reexamination Certificate
2001-03-15
2004-06-22
Wang, Andrew (Department: 1639)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving virus or bacteriophage
C435S007100, C435S071200
Reexamination Certificate
active
06753136
ABSTRACT:
BACKGROUND OF THE INVENTION
The present invention relates to methods for displaying (poly)peptides/proteins on the surface of bacteriophage particles by attaching the (poly)peptide/proteins via disulfide bonds. A number of documents are cited throughout this specification. The disclosure content of these documents is herewith incorporated by reference in their entirety.
Smith first demonstrated in 1985 that filamentous phage tolerate foreign protein fragments inserted in their gene III protein (pIII), and could show that the protein fragments are presented on the phage surface (Smith, 1985). Ladner extended that concept to the screening of repertoires of (poly)peptides and/or proteins displayed on the surface of phage (WO 88/06630; WO 90/02809) and, since then, phage display has experienced a dramatic progress and resulted in substantial achievements.
Various formats have been developed to construct and screen (poly)peptide/protein phage-display libraries, and a large number of review articles and monographs cover and summarise these developments (e.g., Kay et al., 1996; Dunn, 1996; McGregor, 1996).
Most often, filamentous phage-based systems have been used.
Initially proposed as display of single-chain Fv (scFv) fragments (WO 88/06630; see additionally WO 92/01047), the method has rapidly been expanded to the display of bovine pancreatic trypsin inhibitor (BPTI) (WO 90/02809), peptide libraries (WO 91/19818), human growth hormone (WO 92/09690), and of various other proteins including the display of multimeric proteins such as Fab fragments (WO 91/17271; WO 92/01047).
To anchor the peptide or protein to the filamentous bacteriophage surface, mostly genetic fusions to phage coat proteins are employed. Preferred are fusions to gene III protein (Parmley & Smith, 1988) or fragments thereof (Bass et al., 1990), and gene VIII protein (Greenwood et al., 1991). In one case, gene VI has been used (Jespers et al., 1995), and recently, a combination of gene VII and gene IX has been used for the display of Fv fragments (Gao et al., 1999).
Furthermore, phage display has also been achieved on phage lambda. In that case, gene V protein (Maruyama et al., 1994), gene J protein, and gene D protein (Sternberg & Hoess, 1995; Mikawa et al., 1996) have been used.
Besides using genetic fusions, foreign peptides or proteins have been attached to phage surfaces via association domains. In WO 91/17271, it was suggested to use a tag displayed on phage and a tag binding ligand fused to the peptide/protein to be displayed to achieve a non-covalent display.
A similar concept was pursued for the display of cDNA libraries (Crameri & Suter, 1993). There the jun/fos interaction was used to mediate the display of cDNA fragments. In their construct, additional cysteine residues flanking both ends of jun as well as fos further stabilised the interaction by forming two disulfide bonds
When screening phage display libraries in biopanning the problem remains how best to recover phage which have bound to the desired target. Normally, this is achieved by elution with appropriate buffers, either by using a pH- or salt gradient, or by specific elution using soluble target. However, the most interesting binders which bind with high affinity to the target might be lost by that approach. Several alternative methods have been devised which try to overcome that problem, either by providing a cleavage signal between the (poly)peptide/protein being displayed and its fusion partner, or between the target of interest and its carrier which anchors the target to a solid surface.
Furthermore, all the approaches referred to hereinabove require to use fusion proteins comprising at least part of a phage coat protein and a foreign (poly)peptide/protein. Especially in the case of using gene III as partner for peptides/proteins to be displayed, this leads to several problems. First, the expression product of gene III is toxic to the host cell, which requires tight regulation of gene III fusion proteins. Second, expression of gene III products can make host cells resistant to infection with helper phage required for the production of progeny phage particles. And finally, recombination events between gene III fusion constructs and wild type copies of gene III lead to undesired artefacts. Furthermore, since at least the C-terminal domain of the gene III protein comprising about 190 amino acids has to be used in order to achieve incorporation of the fusion protein into the phage coat, the size of the vectors comprising the nucleic acid sequences is rather larger, leading to a decrease in transformation efficiency. Transformation efficiency, however, is a crucial factor for the production of very large libraries. Additionally, for the characterisation of (poly)peptide/proteins obtained after selection from a phage display library, the (poly)peptide/protein are usually recloned into expression vectors in order to remove the phage coat protein fusion partner, or in order to create new fusion proteins such as by fusion to enzymes for detection or to multimerisation domains. It would be advantageously to have a system which would allow direct expression without recloning, and direct coupling of the (poly)peptide/protein to other moieties.
Furthermore, most of these approaches (except for the work of Jespers et al. (1995), WO 91/17271, and Crameri & Suter (1993) mentioned hereinabove) are limited to the presentation of (poly)peptides/proteins having a free N-terminus, since the (poly)peptides/proteins have to be fused at the C-terminus with a phage coat protein. Especially in the case of cDNA libraries, or in the case of proteins requiring a free C-terminus to be functional, it would be highly desirable to have a simple method which doesn't require the generation of C-terminal fusions.
SUMMARY OF THE INVENTION
Thus, the technical problem underlying the present invention is to develop a simple, reliable system which enables the presentation of (poly)peptides/proteins on phage particles without the need to use fusion proteins with phage coat proteins. Additionally, there is a need for a method which allows to recover tightly binding (poly)peptides/proteins in a more reliable way.
The solution to this technical problem is achieved by providing the embodiments characterised in the claims. Accordingly, the present invention allows to easily create and screen large libraries of (poly)peptides/proteins displayed on the surface of bacteriophage particles. The technical approach of the present invention, i.e. linking (poly)peptides/proteins by disulfide bonds to the surface of phage particles, is neither provided nor suggested by the prior art.
Thus, the present invention relates to a method for displaying a (poly)peptide/protein on the surface of a bacteriophage particle comprising:
causing or allowing the attachment of said (poly)peptide/protein after expression to a member of the protein coat of said bacteriophage particle, wherein said attachment is caused by the formation of a disulfide bond between a first cysteine residue comprised in said (poly)peptide/protein and a second cysteine residue comprised in said member of the protein coat.
DETAILED DESCRIPTION OF THE INVENTION
In the context of the present invention, the term “bacteriophage” relates to bacterial viruses forming packages consisting of a protein coat containing nucleic acid required for the replication of the phages. The nucleic acid may be DNA or RNA, either double or single stranded, linear or circular. Bacteriophage such as phage lambda or filamentous phage (such as M13, fd, or fl) are well known to the artisan of ordinary skill in the art. In the context of the present invention, the term “bacteriophage particles” refers to the particles according to the present invention, i.e. to particles displaying a (poly)peptide/protein via a disulfide bonds. During the assembly of bacteriophages, the coat proteins may package different nucleic acid sequences, provided that they comprise a packaging signal. In the context of the present invention, the term “nucleic acid sequences” contained
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Heller Ehrman White and McAuliffe
Morphosys AG
Wang Andrew
LandOfFree
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