| Phage display of intact domains at high copy number -> Monitor Keywords |
|
|
  Phage display of intact domains at high copy numberUSPTO Application #: 20060068379Title: Phage display of intact domains at high copy number Abstract: Disclosed is a phage display system in which the molecules to be displayed (i.e., molecules of interest) are bound to dispensable capsid polypeptides such as SOC (small outer capsid) and HOC (highly antigenic outer capsid) polypeptides that are, in turn, bound to a surface lattice protein, such as those on the surface of a virion or polyhead. Also disclosed are methods of displaying a molecule of interest, methods of immunizing a patient by administering a displayed antigen, and methods of treating a patient who has a disorder associated with aberrent expression or activity of a biological molecule. In the latter instance, the method includes administering a displayed polypeptide, such as an immunoglobulin molecule or an enzyme, that is capable of specifically interacting with the aberrent biological molecule. (end of abstract) Agent: National Institute Of Health C/o Needle & Rosenberg, P.C. - Atlanta, GA, US Inventors: Alasdair C. Steven, Paul T. Wingfield, Lindsay W. Black, Zhaojun Ren USPTO Applicaton #: 20060068379 - Class: 435005000 (USPTO) Related Patent Categories: Chemistry: Molecular Biology And Microbiology, Measuring Or Testing Process Involving Enzymes Or Micro-organisms; Composition Or Test Strip Therefore; Processes Of Forming Such Composition Or Test Strip, Involving Virus Or Bacteriophage The Patent Description & Claims data below is from USPTO Patent Application 20060068379. Brief Patent Description - Full Patent Description - Patent Application Claims
[0001] The work described herein was carried out, in part, at the National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health. The government therefore may have certain rights in the invention. BACKGROUND OF THE INVENTION [0002] The field of the invention is phage display. [0003] Filamentous phage-based display systems (as described, for example, in Smith, Science 228:1315-1317, 1985) have found widespread use in molecular biology, including many immunologic applications such as antigen presentation and the immuno-isolation of desired recombinants by "biopanning" (Marks et al., J. Biol. Chem. 267:16007-16010, 1992; Smith et al., Gene 128:37-42, 1993; Williamson et al., Proc. Natl. Acad. Sci. USA, 90:4141-4145, 1993). However, with filamentous phages, peptides that may be displayed from the major coat protein are limited in size to 6-10 amino acid residues (Kishchenko et al., J. Mol. Biol. 241:208-213, 1994; Iannolo et al., J. Mol. Biol. 248:835-844, 1995), although somewhat longer peptides can be displayed by co-assembly with the wild-type coat protein (Perhan et al., FEMS Microbiol. Rev. 17:25-31, 1995). Full-length polypeptides can be displayed on minor phage proteins, but only at very low copy number (Parmley and Smith, Gene 73:305-318, 1988). Moreover, the requirement that the fusion protein should pass through the secretion system of Escherichia coli may pose problems of toxicity for the host, or for correct folding of the displayed protein (Skerra and Pluckthun, Protein Eng. 4:971-979, 1991). SUMMARY OF THE INVENTION [0004] Described herein is a phage display system in which the molecules to be displayed (i.e., molecules of interest) are bound to dispensable capsid polypeptides such as SOC (small outer capsid) and HOC (highly antigenic outer capsid) polypeptides that are, in turn, bound to a surface lattice protein, such as those on the surface of a virion or polyhead. Polyheads are tubular capsid variants, and their formation is described below. Also described below are various ways in which a molecule of interest can be displayed. For example, a chimeric polypeptide that includes a dispensable polypeptide and a polypeptide of interest can be expressed in Escherichia coli, purified, and then bound in vitro to separately isolated surface lattice proteins. The surface lattice proteins can be those on the surface of a capsid or polyhead from which the wild type dispensable polypeptides have been deleted. Similarly, a chimera that contains a dispensable polypeptide and a synthetic molecule of interest can be prepared in vitro and bound to surface lattice proteins. In another embodiment, a positive selection vector forces integration of a gene that encodes a dispensable polypeptide and a polypeptide of interest into the genome of a phage from which the wild type dispensable polypeptide is deleted. For example, a modified soc gene can be integrated into a soc-deleted T4 genome, leading to in vivo binding of the display molecule on progeny virions. More than one type of dispensable polypeptide can be used as part of the chimera for displaying one or more molecules of interest. For example the surface lattice proteins of a phage may be bound to a chimera that contains SOC and a chimera that contains HOC. [0005] The display system has been successfully demonstrated for three molecules of interest that vary in their length and character: (1) a tetrapeptide; (2) the 43 amino acid residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). [0006] The display system of the invention is capable of presenting approximately 1,000 copies or more of the displayed molecule of interest per capsid and 10,000 copies or more per polyhead of V3-sized domains. Appropriate binding between the fusion protein and the surface lattice was apparent in averaged electron micrographs of polyheads. Furthermore, phage displaying the V3 loop domain of gp120 were highly antigenic in mice and produced antibodies reactive with native gp120. In addition, phage displaying SOC-VP1 were isolated from a 1:106 mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated. Therefore, the system described herein can be used in numerous immunologic applications, including treatments that rely on antigen or antibody persentation, and in the isolation of various polypeptides and other pharmacophores. The fact that the molecules of interest are displayed at high copy number greatly facilitates these applications, and the fact that the molecules of interest are displayed on surface lattice proteins that form regular arrays allows analysis of their structure as well. BRIEF DESCRIPTION OF THE DRAWING [0007] FIGS. 1A-1D are a series of schematic diagrams illustrating the principle of the novel display system described herein. A segment of the surface lattice (dashed boxes) of a virion (illustrated on the left of FIG. 1A) and a polyhead (illustrated on the right of FIG. 1A) is shown in FIG. 1B. The display platform (in this case, a SOC-less capsid or polyhead) and SOC (small outer capsid) and chimeras that contain SOC are shown in FIG. 1C. Chimeric proteins are shown bound to the display platform in FIG. 1D. The viability of the phage display system is demonstrated for three polypeptides, which are illustrated in FIG. 1C and FIG. 1D as soc-cys, where cys=Cys-Leu-Asn-Ser, as soc-v3, where SOC is fused to a 47-residue polypeptide, and soc-vp1, where SOC is fused to a 312-residue polypeptide. [0008] FIGS. 2A-2B are schematic diagrams of vectors constructed as described herein and a region of the T4-Z1 phage that has been altered by homologous recombination in a phage-plasmid cross. The expression vectors shown in FIG. 2A are: pE-SOC, into which a 248 base pair SOC-encoding sequence has been inserted (panel i); pE-S11, into which a 259 base pair sequence encoding SOC and the tetrapeptide Cys-Leu-Asn-Ser has been inserted (panel ii); pE-V3, which, in addition to the SOC-tetrapeptide sequence, encodes 43 amino acids of V3 (gp120) (panel iii); and pE-VP1, which, in addition to the SOC-tetrapeptide sequence, encodes 312 amino acids of the poliovirus capsid protein, VP1 (panel iv). FIG. 2B illustrates pRH, a phage integration plasmid that contains a portion of the phage T4 lysozyme gene e' at the 5' end of the modified soc gene, and a portion of the 3' denV' gene of T4 (panel i). Homologous recombination with the T4-Z1 phage, which is deleted for genes soc, IPIII, IPII, and part of e'', is shown in panel ii. Following recombination, which forms an intact gene e and transfers the modified soc gene into the phage genome, an egg white lysozyme-independent phage plaque selects for the soc gene (panel iii). [0009] FIG. 3 is a photograph of an SDS-polyacrylamide gel, stained to reveal protein. Lane 1 contains molecular weight standards; lanes 2 and 5 contain wild-type T4 phage; lane 3 contains soc-deleted T4 phage; lane 4 contains the integration phage Z1, which lacks viral proteins SOC, IPIII, IPII, and ALT; lane 6 contains SOC expressed from HMS174 bacteria containing the plasmid pE-SOC; and lane 7 contains lysates of HMS174 bacteria that lack the plasmid. [0010] FIGS. 4A and 4B are a pair of photographs of an SDS-polyacrylamide gel, stained to reveal protein (FIG. 4A) and a Western blot (FIG. 4B) of the same samples. The samples analyzed by SDS-PAGE in FIG. 4A include: E. coli overexpressing SOC-V3 (.about.14 kDa, pEV3, lane 2); recombination phage lacking SOC and IpIII (Z1, lane 3); phage purified by high-speed centrifugation followed by CsCl density gradient centrifugation (Z1 SOC-V3, CsCl, lane 4) or by the first step only (Z1 SOC-V3; lane 5); and wild-type T4 phage (T4, lane 6). Molecular weight standards were run in lane 1. The same samples were examined following Western blotting using antiserum against gp120 of HIV-1 (FIG. 4B). [0011] FIGS. 5A and 5B are a pair of photographs of an SDS-polyacrylamide gel, stained to reveal protein (FIG. 5A), and a Western blot (FIG. 5B) of the same samples. SOC-V3 protein was separated by CsCl gradient centrifugation of T4-SOC.sup.+ phage. Protein extracts of bacteria induced for SOC-V3 (PE-V3, lane 4) or SOC (PE-SOC, lane 5) were prepared as described herein; a mixture of SOC-V3 extract and T4-SOC.sup.+ phage (T4+SOC-V3, lane 3) was centrifuged at 35,000 rpm for 20 hours. Visible and widely separated bands containing phage (.rho..about.1.5) (T4 CsCl, lane 1) and protein (.rho..about.1.35) (SOC-V3 CsCl, lane 2) were collected, dialyzed, and analyzed by SDS-PAGE. Lane 6 contains molecular weight standards of 3.0, 6.5, 14.3, 21.5, 30, and 46 kDa. The same samples were examined following Western blotting using antiserum against gp120 of HIV-1 (FIG. 5B). [0012] FIG. 6 is a line graph illustrating induction of anti-gp120 antibodies by inoculation of mice with T4 SOC-V3-displaying phage. Individual responses of five animals are shown (.smallcircle., .box-solid., .quadrature., .tangle-solidup., .DELTA.), as is the baseline of the pre-bleed titer of one mouse (.cndot.), which was typical of the whole group. Pooled sera data (open diamond) represent results obtained after pooling the sera collected from all five mice on day 120, following a second boost on day 104. [0013] FIG. 7 is a photograph of an SDS-polyacrylamide gel, through which the following samples had been electrophoresed: T4 polyheads complemented with purified SOC (lane b), control (lane c), and SOC-V3 (lane d). Purified SOC-V3 is shown in lane (e), and molecular weight standards (97, 66, 43, 31, 22, and 14 kDa) appear in lane (a). [0014] FIGS. 8A-8E are electron micrographs of T4 polyheads (cleaved/expanded type) after complementation with SOC-V3 (FIG. 8A; Bar=100 nm), an individual polyhead at higher magnification (FIG. 8B, inset within FIG. 8A; Bar=50 nm), computer-filtered image of the surface lattice at .about.3 nm resolution (FIG. 8C; Bar=15 nm), the undecorated gp23* surface lattice (FIG. 8D), and the lattice decorated with wild-type SOC (FIG. 8E; reproduced from Ross et al., J. Mol. Biol. 183:353-364, 1985; Bar=15 nm). Triplets of SOC binding sites surround the points of local three-fold symmetry in the hexagonal surface lattice (FIG. 8E). In the SOC-V3 binding experiment (FIG. 8C), these sites are occupied by stain-excluding units that are somewhat larger than those observed with wild-type SOC, consistent with the greater size of the SOC-V3 fusion protein. Thus, occupancy of the SOC sites by SOC-V3 molecules is complete or close to it, because the peak density above background of the SOC-V3-related units in FIG. 8C is, on average, 10-15% higher than for the gp23* related units. [0015] FIGS. 9A and 9B are a pair of photographs of an SDS-polyacrylamide gel, stained to reveal protein (FIG. 9A), and a Western blot (FIG. 9B) of the same samples. Molecular weight standards are shown in lane 1 of FIG. 9A. The samples examined in FIG. 9A are as follows; CsCl-purified virion of the recombination phage Z1 (Z1; lane 2), the soc-vp1 Z1 integrant, purified by high-speed centrifugation (Z1 SOC-VP1; lane 3), the soc-vp1 Z1 integrant, purified by high-speed centrifugation and additionally by CsCl gradient centrifugation (Z1 SOC-VP1, CsCl; lane 4), products of the expression vector pE-VP1 (pE-VP1; lane 6) and the recombination ventor pRH-VP1 (pRH-VP1; lane 5) have the expected apparent molecular weight of 43 kDa. The Western blot shown in FIG. 9B is stained with anti-VP1 antiserum. The samples are as described in FIG. 9A and, in addition, include purified poliovirus (lane 3) and the same amount of T4 (lane 4) as Z1 and Z1-soc-vp1 phages (lanes 1, 2, 5), as a negative control. [0016] FIGS. 10A and 10B are a pair of photographs of an agarose gel following a PCR assay of a biopanning procedure using an antibody against poliovirus VP1 and an initial mixture of 5.times.10.sup.9 delsoc and 5.times.10.sup.3 Z1-soc-vp1 (FIG. 10A), and a Western blot of the same phage samples stained with anti-VP1 antibody. In FIG. 10A, molecular weight standards are shown in lane 1. DNA derived from the soc gene (259 bp) can be detected in expression vector PE-VP1 (lane 3), T4 (lane 4), and Z1--soc-vp1 integrant (lane 6), but not in delsoc (lane 5), Z1 (before recombination; lane 10), or buffer used for single plaque test (lane 2). C1, C2 and C3 (shown in lanes 9, 8, and 7, respectively) are samples obtained following successive cycles of biopanning. The soc gene can be detected in a mixture of phages arising from the second cycle of precipitation followed by growth. In FIG. 10B, the same phage samples are assayed by Western blotting with the anti-VP1 antibody. DETAILED DESCRIPTION [0017] The invention features a system for displaying molecules of interest. The displayed molecules are bound to a dispensable polypeptide that can, in turn, bind a protein that is part of the lattice found on the surface of a virion or polyhead. [0018] The term chimera is used herein to describe the entity that is formed when a display molecule is bound to a dispensible polypeptide; when the display molecule is also a polypeptide, the chimera is referred to as a chimeric polypeptide. The terms "protein" and "polypeptide" are used herein to refer to any chain of two or more amino acid residues, regardless of the length of the chain or the presence or absence of post-translational modifications such as glycosylation or phosphorylation. [0019] The chimera can consist of numerous types of display molecules and dispensable polypeptides. For example, the dispensable polypeptides can be a small outer capsid (SOC) polypeptide or a highly antigenic outer capsid (HOC) polypeptide, such as those expressed on the surface of the bacteriophage T4. The dispensable polypeptide used according to the method of the invention need not be identical, either in length or sequence, to a wild-type dispensable polypeptide. It may, for example, contain mutations including substitutions, deletions, and/or additions of amino acid residues, provided these mutations do not destroy the ability of the dispensable polypeptide to bind a display molecule and a surface lattice protein. [0020] The T4 SOC polypeptide and the related polypeptide, HOC, have a number of features that allow them to function as vehicles for displaying molecules of interest. Any polypeptide that has one or more of these features may, therefore, be useful as a dispensable polypeptide in various embodiments of the present invention. These features include their arrangement on the surface of a virion or polyhead, the level at which they are expressed, their ability to withstand particular conditions of temperature and pH, and the degree to which the dispensable polypeptide is required for capsid morphogenesis. Continue reading... Full patent description for Phage display of intact domains at high copy number Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Phage display of intact domains at high copy number patent application. ###  How KEYWORD MONITOR works... a FREE service from FreshPatents1. Sign up (takes 30 seconds). 2. Fill in the keywords to be monitored. 3. Each week you receive an email with patent applications related to your keywords. Start now! - Receive info on patent apps like Phage display of intact domains at high copy number or other areas of interest. ### Previous Patent Application: Nanoparticles having oligonucleotides attached thereto and uses therefor Next Patent Application: Alternative pol kappa nucleotide and amino acid sequence and methods for using Industry Class: Chemistry: molecular biology and microbiology ### FreshPatents.com Support Thank you for viewing the Phage display of intact domains at high copy number patent info. IP-related news and info Results in 3.3593 seconds Other interesting Feshpatents.com categories: Software: Finance , AI , Databases , Development , Document , Navigation , Error |
||
