Chemistry: molecular biology and microbiology – Micro-organism – per se ; compositions thereof; proces of... – Bacteria or actinomycetales; media therefor
Reexamination Certificate
1998-09-30
2001-12-25
Brusca, John S. (Department: 1631)
Chemistry: molecular biology and microbiology
Micro-organism, per se ; compositions thereof; proces of...
Bacteria or actinomycetales; media therefor
C435S320100, C536S023400, C536S023700, C536S024100
Reexamination Certificate
active
06333187
ABSTRACT:
This invention relates to phagemid vectors suitable for phage display. Phage display is a tool to identify peptide variants with a particular binding specificity. Phage display comprises a collection of peptides. Each of these peptides is expressed on a filamentous bacteriophage particle as fusion to one of the coat proteins of single strand DNA phages like f1, fd and M13, and each of them is encoded by the corresponding DNA molecule inside that particle. By exposing the collection to a target, and selectively amplifying those members of the collection that bind to the target genes can be isolated encoding peptides that have binding affinity for the target. The principle of phage display is described extensively, for instance in U.S. Pat. No. 5,223,409. The variant binding peptides can for instance be derived from antibodies (Hoogenboom et al., 1991), proteinese inhibitors (Roberts et al., 1992), cDNAs (Jespers, 1995) or random peptides (Cwirla, 1991). The invention is also relevant for other selection procedures that involve phages like Selectively Infective Phage (SIP) and Selection and Amplification of Phage (SAP) as described by Duenas et al., 1994, Gramatikoff et al., 1995 and Krebber et al., 1995. The main power of phage display is rapid screening of vast numbers of variants.
Various cases of large libraries dominated by deletion mutants have been reported (Smiley & Benkovic, 1994; Tan et al., 1994; Bradbury, 1998). Such DNAs may easily appear in the course of alternating selection and phage amplification stages, since they reproduce much faster than intact phagemids. This may particularly be the case when the protein of interest is not indifferent to
E.coli,
but retards its growth (Krebber et al., 1996). Proteins may have toxic effects in their native fold, like in the case of lysozyme (Maenaka et al., 1996), or inhibit growth when aberrantly folded, as has been reported for some antibodies (Knappik & Pluckthun, 1996). A more general problem may be that already a low level of expression of the bacteriophage coat protein (g3p), to which the protein of interest is fused, retards growth of
E. coli.
In addition, g3p alters the properties of the
E. coli
outer membrane so that it can no longer be infected by helperphage (Boeke et al., 1982; Rakenjae et al., 1997).
The existing phagemid vector systems for phage display, most of them based on pHEN1 (Hoogenboom et al., 1991), generally use the promoter of the
E. coli
lac-operon. This system is preferred over the natural g3p promoter, since the lac-promoter can at least partially be repressed during library generation by addition of glucose, and induced during phage synthesis by removal of glucose. In spite of this control, several reports indicate a high incidence of deletion phages. The present inventors carried out some experiments to be described in the experimental part, which yielded only mutants that carry deletions or amber stopcodons.
To overcome the above problem Krebber et al. (1996) use a phagemid vector which carries the lacl repressor gene and a transcription termination signal just upstream of the lac-promoter. This provides improved control over the lac-promoter, as judged from Western blots and bacterial morphology.
EP 0.699.760 discloses a phagemid vector system for phage display which uses the lambda P
L
-promoter and a bacterial strain carrying a lysogenic lambda phage. This patent application does not comprise a comparison to other systems neither are any advantages mentioned.
According to the present invention the above problem has been solved by a collection of phagemids comprising
i) a promoter operatively linked to a gene coding for a translational fusion between a peptide and a filamentous single strand DNA bacteriophage coat protein or a part thereof, which promoter is induced by expression of gene IV of a filamentous bacteriophage,
ii) a replication origin derived from a filamentous single strand DNA bacteriophage, and
iii) a plasmid replication origin.
Hereinafter the gene coding for a translational fusion between a peptide and a filamentous single strand DNA bacteriophage coat protein or a part thereof will be shortly referred to as the fusion gene. The fusion protein is encoded by this fusion gene.
According to the invention the commonly used lac promoter is replaced by a promoter which is repressed under normal growth conditions, and induced by expression of gene IV of a filamentous bacteriophage.
According to a preferred embodiment the promoter is the promoter of the
E. coli
phage-shock-protein (psp) operon or a homologous promoter from a different organism.
The psp-promoter comprises the region spanning 200 bp upstream of the
E. coli
pspA gene. The promoter is induced when gene IV of filamentous bacteriophages is expressed. The promoter is also triggered by expression of gene IV-protein homologues, like the pulD gene product of
Klebsiella oxytoca.
The actual mechanism of induction is not fully understood, but a proposed pathway is discussed in Model et al., 1997. The organization of the psp promoter is alike a sigma 54 promoter (reviewed in Morett et al., 1993). Sigma 54 promoters have been found in
E.coli,
Salmonella, Klebsiella, Rhizobium, Pseudomonas and Azotobacter species. It is therefore possible that psp promoters are present in these organisms, particularly in
Salmonella typhimurium
and Shigella spp. since these are closely related to
E. coli.
The system using the above defined promoter has two technical advantages. Firstly, it provides satisfactory control over the encoded gene-fusions, as judged from super-infection levels, and growthcurves as well as in a complete phage display selection experiment. Secondly, handling procedures for switching on the promoter after helperphage infection, which normally involves delicate washing steps in order to remove glucose, are no longer required. This may for instance be important for large scale production of phage libraries in fermentor devices or in automated phage display procedures.
According to a further preferred embodiment of the invention the plasmid replication origin is a low-copynumber origin of replication. Preferably the low-copynumber origin of replication is derived from pBR322. Using this plasmid replication origin the phagemid copynumber is lowered to about 30 per cell, instead of about 500 as in the case of pUC-derived vectors like pB3 (Sambrook et al., 1989).
According to yet another preferred embodiment the phagemid comprises the rop gene from pBR322, which reduces the copynumber an additional twofold. The order of the various elements to be present on the phagemids of the invention is not critical, but preferably the phagemids comprise in a 5′ to 3′ direction: the plasmid replication origin and the promoter operatively linked to the fusion gene.
If the rop gene is present, it is suitably present between the plasmid replication origin and the promoter operatively linked to the fusion gene.
In a further preferred embodiment the promoter is operatively linked to the leader sequence of the polB gene of
Erwinia carotovora
and the fusion gene.
The phagemids of the invention generally comprise at least one transcription terminator sequence.
Protease inhibitors (PI's) are part of the defensive response of plants towards feeding insects (Ryan, 1990). They are produced in large quantities in wounded leaves, and reversibly bind to the insect digestive proteases (Jongsma & Boller, 1997). It appears that some insects have overcome this defense of their host plants. These insects have available a set of proteases which is insensitive to the inhibitors of the host plant. This set is induced when plant PIs are ingested, while other, presumably sensitive proteases are down-regulated (Jongsma et al., 1995a; Bown et al., 1997). To control such insect pests, it is the inventors' aim to complement the plant's arsenal with PIs that do inhibit these insensitive proteases. The strategy to generate these complementary inhibitors is by modifying existing plant inhibitors, using phage display to select v
Beekwilder Jules
Bosch Dirk
Jongsma Maarten
Jovanovic Goran
Rakonjac Jasna
Brusca John S.
Centrum Voor Plantenveredelings-en
Smith , Gambrell & Russell, LLP
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