Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1998-12-24
2001-02-20
Ketter, James (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
Reexamination Certificate
active
06190908
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The field of this invention is phage display of polypeptides. More particularly, this invention relates to a modified filamentous phage that allows for displaying polypeptides on the surface in a range of densities.
BACKGROUND OF THE INVENTION
Phage display of antibodies was initially based on systems developed for the display of peptides (Smith,
Science
228, 1315-7, 1985). Antibody single chain variable domains were fused to the coat protein gene (gpIII), (McCafferty, et al.,
Nature
348, 552-554, 1990) resulting in all the gpIII molecules displaying fusion antibodies. However, the fusion of a polypeptide to the gpIII reduced the ability of the phage to infect bacteria and secondly the multivalent display at the tip of the phage resulted in avidity selection rather than affinity discrimination. Utilizing a phagemid vector (to present the gpIII-fusion) and helper phage rescue (to introduce the wild type gpIII), the valency of fusion display was reduced and infectivity restored (Bass, et al,
Proteins: Structure, Function, and Genetics
8, 309-314, 1990). Likewise, the display of heterodimeric polypeptides such as antibody F(ab) fragments as either major (gpVIII) (Kang, et al.,
Proc. Natl Acad. Sci. USA
88, 4363-66, 1991), or minor (gpIII) (Barbas, et al.,
Proc. Natl. Acad. Sci. USA
88, 7978-82, 1991; Garrard, et al.,
Bio/Technol
. 9, 1373-77, 1991) coat protein fusions has successfully utilized phagemid with helper phage rescue.
Phage display of antibody fragments and other polypeptides has gained acceptance as a useful tool in contemporary molecular immunology. The density of polypeptide display per filamentous phage particle is influenced by the choice of which phage coat protein is used as fusion partner and the type of vector system used. Molecules expressed from nucleotide sequences fused with the sole copy of gpIII on the phage genome such as fd or M13 resulted in a multivalent cluster display (tri-penta valent) and reduced infectivity of bacteria (McCafferty et al.,
Nature
348, 552-554, 1990; Smith,
Science
228, 1315-7, 1985). Multivalent binding of phage with ligand would favor avidity selection and limit the ability to discriminate between modest gains in affinity (Cwirla, et al.,
Proc. Natl Acad. Sci. USA
87, 6378-82., 1990). This may be desirable when attempting to isolate ligand binding molecules of lower affinity. Phagemid vectors encoding phage coat protein fusion polypeptides used in conjunction with helper phage rescue, generated phage with restored infectivity and reduced valency permitting enrichment for high affinity interactions (Bass, S., et al.
Proteins: Structure, Function, and Genetics
8, 309-314, 1990).
Both the high and low density display systems have uses in accessing ligands against target receptors or tissues. It would be desirable to create a phage display system in which the density of the displayed fusion moieties on the phage particle could be modulated from a few displayed copies to less than 1 per phage. To achieve this with existing vectors requires shuttling of inserts between gpIII phage and gpVIII/gpIII phagemid vectors. However this may also be attained by utilizing a single M13 phage based vector with a synthetic second copy of the gene encoding gpIII or gpVIII (i.e. pseudo wild type) as a fusion partner (Huse, et al.,
J. Immunol
. 149, 3914-20, 1992), and manipulating the phage growth conditions to favor low or moderate rates of fusion incorporation into the phage filament. Incorporating the display expression cassette onto the phage genome may also have the added benefits of fusion expression being synchronous with phage morphogenesis. The present invention describe a phage vector in which a polypeptide is displayed on the phage surface. This display system was used to investigate the modulation of display fusion on phage resulting in optimal phage display as determined by relative panning enrichment efficacy.
BRIEF SUMMARY OF THE INVENTION
The present invention discloses a phage vector for the display of polypeptides on the surface of a modified filamentous phage which permits facile manipulation of the valency of display. The gene encoding the polypeptide is fused to a synthetic copy of a major coat protein gene which permits incorporation into the phage during assembly of the filament.
Thus, in one aspect, the present invention provides a modified filamentous phage expression vector. That vector includes a gene encoding a wild type major coat protein of the phage; a leaky, inducible promoter; a gene encoding a synthetic major coat protein of the phage; and a directional cloning site for receiving a nucleotide insert. The insert is a nucleotide that includes a sequence that encodes a translation initiation site, contains a leader sequence that directs polypeptide expression to a bacterial periplasmic membrane and a polypeptide encoding sequence. The directional cloning site is situated between the promoter and the gene encoding the synthetic major coat protein of the phage, such that the polypeptide is expressed as a fusion protein with the synthetic major coat protein.
In preferred embodiments, the translation initiation site is a ribosome binding site, the promotor is the lac promoter, the leader sequence is ompA, the wild type major coat protein of the phage is gpVIII, the synthetic major coat protein of the phage is a synthetic gpVIII, and the polypeptide is a ligand-binding heterodimeric antibody. An especially preferred filamentous phage is M13. A preferred modified M13 vector of this invention is designated herein as JC-M13-88.
Preferably, the nucleotide insert of the modified filamentous phage is obtained from a pre-selection open reading frame expression and secretion plasmid (pORFES), preferably pORFES II or pORFES IV.
In a related aspect, the present invention provides a process for expressing a polypeptide. The process includes the steps of (a) inserting a nucleotide sequence containing a translation initiation site encoding region, a leader sequence that targets expression of a polypeptide to a bacterial periplasmic membrane and a polypeptide coding sequence that into a directional cloning site of a filamentous phage that contains a gene encoding a wild type phage major coat protein, an inducible promoter and a gene that encodes a synthetic phage major coat protein wherein the directional cloning site is located between the inducible promoter and the gene encoding the synthetic phage major coat protein; and (b) propagating the filamentous phage from step (a) in a bacterium. Preferred translation initiation sites, promoters, leader sequences, polypeptides and major coat proteins are the same as set forth above. A preferred directional cloning site comprises a pair of restriction enzyme sites. Exemplary such enzyme sites are XbaI and HindIII.
The density of the polypeptides displayed on phage may be modulated by phage altering growth conditions. Propagation is preferably carried out at a temperature of from about 25° C. to about 37° C. in the absence or presence of inducers that induce expression by way of the inducible promoter. Lowering the temperature of phage propagation reduced the overall phage yield, yet increased the quality of the antibody display. Likewise the addition of inducers during phage propagation reduced the phage yield but led to enhanced the recovery of phage during panning.
REFERENCES:
patent: 5427908 (1995-06-01), Dower et al.
patent: 5658727 (1997-08-01), Barbas et al.
Ketter James
Northrup Thomas E.
The Scripps Research Institute
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