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  Methods to screen peptide display libraries using minicell displayUSPTO Application #: 20070072221Title: Methods to screen peptide display libraries using minicell display Abstract: A minicell display method has been developed which has significant advantages for screening peptide libraries for candidates that can bind and effectively modulate a particular biological process. The method, based on the small, anucleate minicell, has increased versatility in generating unique sequences to screen as well as increasing the size of the peptides to be screened. In vivo mutagenesis, at the level of protein synthesis, as well as DNA replication, increases diversification of the library to be screened and therefore substantially increases the number of potential peptides that can modulate a particular biological response or mechanism. (end of abstract) Agent: Cooley Godward Kronish LLP Attn: Patent Group - Washington, DC, US Inventor: Samy Ashkar USPTO Applicaton #: 20070072221 - Class: 435006000 (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 Nucleic Acid The Patent Description & Claims data below is from USPTO Patent Application 20070072221. Brief Patent Description - Full Patent Description - Patent Application Claims
CROSS REFERENCE TO RELATED APPLICATIONS [0001] Priority is claimed to U.S. Provisional Application Ser. No. 60/274,039 filed on Mar. 7, 2001, and U.S. Provisional Application Ser. No. 60/306,946 filed on Jul. 20, 2001. FIELD OF THE INVENTION [0002] The present invention is generally in the field of high throughput peptide screening, and in particular relates to a minicell display technology for generation and screening of random peptides. BACKGROUND OF THE INVENTION [0003] The interaction between cognate proteins in receptor-ligand complexes, enzyme substrate reactions and antibody-antigen binding reactions has furthered the understanding of the molecular interactions required to effect a response in a wide range of processes. The search for new peptide molecules which can bind to selected targets and effectively modulate a particular biological process is at the forefront of agricultural, biological, and medicinal research. [0004] There are several examples of methods that use peptides or nucleotides to develop libraries of potential receptor, enzyme, or antibody interacting peptides. Over the course of the last two decades these libraries have been incorporated into systems that allow the expression of random peptides on the surface of different phage or bacteria. Many publications have reported the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected target. See, e.g, Cwirla et al., Proc. Natl. Acad. Sci. USA 87, 6378-6382 (1990); Devlin et al., Science 249, 404-406 (1990), Scott & Smith, Science 249, 386-388 (1990); U.S. Pat. No. 5,571,698 to Ladner et al. A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the target polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome which encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target bind to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means. [0005] In addition to providing a method for selecting peptides that interact with target molecules, phage display has been used to direct filamentous phage to target cells using peptides, genetically fused to phage coat proteins, that bind integrin proteins on the surface of mammalian cells. This method of phage display has had a profound influence on gene therapy applications and their attempts to target cells in a specific manner. [0006] Another approach to obtaining surface expressed foreign proteins has been the use of bacterial native membrane proteins as carriers for foreign protein. In general, many attempts to develop methods of anchoring proteins on a bacterial surface have focused on fusion of the desired recombinant polypeptide to a native protein that is normally exposed on the cell's exterior with the hope that the resulting hybrid will also be localized on the surface. However, in most cases, the foreign protein interferes with localization, and thus, the fusion protein is unable to reach the cell surface. These fusions either end up at incorrect cellular locations or become anchored in the membrane with a secreted protein domain facing the periplasm. See Murphy, et al., J. Bacteriol., 172:2736 (1990). [0007] Recent advances in bacterial display methods have circumvented this problem by using fusion proteins comprising pilin protein (TraA) or a portion thereof and a heterologous polypeptide displaying the library peptide on the outer surface of the bacterial host cell capable of forming pilus. See U.S. Pat. No. 5,516,637 to Huang et al. The pilus is anchored to the cell surface of the bacteria and is naturally solvent exposed. [0008] Alternatively, the FLITRX.TM. (Invitrogen Corp.) random peptide library uses the bacterial flagellar protein, FliC, and thioredoxin, TrxA, to display a random peptide library of dodecamers on the surface of E.coli in a conformationally constrained manner. See Lu et al., BioTechnology, 13:366 (1995). These systems have been applied to antibody epitope mapping, the development and construction of live bacterial vaccine delivery systems, and the generation of whole-cell bio-adsorbants for environmental clean-up purposes and diagnostics. Peptide sequences that bind to tumor specific targets on tumor derived epithelial cells have also been identified using the FLITRX.TM. system. See Brown et al., Annals of Surgical Oncology, 7(10):743 (2000). [0009] Although the phage and bacterial display systems have provided unique routes to elucidating new peptides which can bind target molecules with new or enhanced binding properties, there are several important limitations that need to be considered. Minimal changes in the structural conformation of the phage coat protein to which the peptide is genetically fused are tolerable. Problems arise when larger peptide inserts (more than 100 amino acids) disrupt the function of the coat protein and therefore phage assembly. Heterologous peptides have been displayed on bacteria using both fimbriae as well as flagellar filaments. Insert size constraints affect the applicability of these systems as well. To date, the largest peptides to be displayed in fimbriae range from 50 to 60 amino acids, while-the functional expression of adhesive peptides fused to the FliC flagellin of Escherichia coli appears to be restricted to 302 amino acids. See Westerlund-Wikstrom 2000. [0010] Amino acid analogs have been used to replace chemically reactive residues and improve the stability of the synthetic peptide as well as to modulate the affinity of drug peptide compounds for their targets. A limitation of the phage and bacterial display systems resides in the inability of these systems to incorporate amino acid analogs into peptide libraries in vivo. In vivo, amino acid analogs disrupt the cellular machinery used to incorporate natural amino acids into essential proteins as well as the growing peptide chain of interest. Phage and bacterial display both rely on the protein synthesis machinery of the bacterial cell to synthesize proteins essential for viability, synthesize the peptide library, and amplify or propagate the phage or bacterial pool harboring the peptide of interest. Technically cumbersome protocols can be time consuming when attempting the in vitro translation methods frequently used to incorporate amino acid analogs into a peptide sequence. [0011] The method of propagating the phage or bacterial pool requires expression of the peptide of interest. Peptides that are toxic to the bacterial cell and therefore lethal cannot be screened for in phage or bacterial display systems. This eliminates a potentially large segment of peptides that otherwise would be of interest. [0012] Phage and bacterial display also rely upon cumbersome and time consuming techniques in order to keep conditions optimal for cell growth and cell viability. Bacterial cells are relatively large and care must be taken while screening for target interacting peptides. Affinity chromatography is a common method used to separate non-binding peptides from binding peptides and care must be taken to prevent plugging and the non-specific retention of bacteria in the column. Candidate peptide displaying phage are generally amplified or propagated and therefore require the use of the cellular transcriptional, translational, and replication machinery of bacteria to synthesize the packaging proteins of the phage as well as the peptide of interest. Infecting bacterial cells, harvesting the phage, and re-infecting several rounds is very time consuming. The bacterial cell display system also requires optimal growth conditions to ensure safe passage of the plasmid encoded peptide from generation to generation and for subsequent re-screening. [0013] Oligonucleotide-mediated mutagenesis has been utilized to further characterize selected peptides. Generally, oligonucleotide-mediated mutagenesis is used to introduce very specific mutations into the gene of interest. Although the selection of specific mutations to be introduced into the gene is usually based on published reports describing the effects of the mutations on the activity or function of other homologous proteins, it is still difficult to predict the affect of the mutation or substitution. [0014] It is often advantageous to increase the spontaneous mutation frequency of the peptide library in vivo. Increasing the diversity of a population of peptides displayed on a bacterial surface has proven to be a very useful tool for identifying those with a particular effect. Spontaneous mutations maintain evolutionary pressure on the peptide library and maximize the screening of unique sequences. [0015] A display system that is amenable to the uncomplicated nature of cloning and amplification of DNA sequences using the genetics of bacteria, for example E. coli, to increase the variability and size of the peptides within the library is desirable. There is a need to generate novel peptide libraries in a system that will allow the in vivo incorporation of amino acids analogs into the oligonucleotide sequence such that its genetic and biochemical characteristics are altered. There is a need for generating peptides that may otherwise be eliminated by virtue of their toxicity in phage or bacterial display systems. There is also a need to manipulate the oligonucleotide in vivo and yet alleviate the requirement to ensure optimal growth conditions for cell viability. [0016] It is therefore an object of this invention to provide an effective and rapid method for the systematic preparation of novel peptide substrates having altered functional and binding activity and to address the shortcomings inherent in the phage and bacterial display methods currently practiced in the art. BRIEF SUMMARY OF THE INVENTION [0017] Methods for selecting oligonucleotides and peptides of interest, and generating and screening large mini-cell display libraries for peptides with desired functional and binding characteristics have been developed. These methods include selecting new and unique target interacting peptides from minicell display libraries of random oligonucleotides that are expressed as gene fusions to a protein such as the 17K antigen of Rickettsia rickettsii. [0018] The plasmid or expression vector encoded oligonucleotide fusion or gene fusion product is preferably localized to the minicell outer membrane forming what is referred to as a "display minicell". Briefly, the method consists of first constructing a library wherein the library consists of a replicable expression vector includes an inducible transcriptional regulatory element operably linked to a gene fusion, where the gene fusion includes: [0019] (i) a first gene encoding at least a portion of a bacterial outer membrane protein; and [0020] (ii) a second gene or oligonucleotide encoding a potential "substrate" peptide interacting with a target molecule. The 3' end of the first gene is linked to the 5' end of the second gene or oligonucleotide, thereby forming a chimeric gene. The chimeric gene encodes a chimeric protein. The linkage between the first and second gene may be direct, or indirect via a linker molecule or oligonucleotide. The second gene or oligonucleotide is obtained from a library of random oligonucleotides constructed by degenerate polymerase chain reaction (PCR), a method well known within the art, or other amplification method. Continue reading... Full patent description for Methods to screen peptide display libraries using minicell display Brief Patent Description - Full Patent Description - Patent Application Claims Click on the above for other options relating to this Methods to screen peptide display libraries using minicell display patent application. Patent Applications in related categories: 20080108057 - Allelic imbalance in the diagnosis and prognosis of cancer - Methods for assessing the extent of allelic imbalance in a genomic nucleic acid sample. 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