Invented by Patrick S. Daugherty, Paul H. Bessette, Jeffrey Rice, University of California

Polypeptide display libraries are a powerful tool for identifying and optimizing protein-protein interactions, drug targets, and therapeutic antibodies. These libraries consist of large collections of polypeptides, displayed on the surface of a bacteriophage, yeast, or other cell type, that can be screened for binding to a target of interest. The market for polypeptide display libraries and methods for making and using them has grown rapidly in recent years, driven by advances in biotechnology and the increasing demand for new drugs and therapies. One of the key advantages of polypeptide display libraries is their ability to generate high-affinity binders to a wide range of targets, including proteins, peptides, small molecules, and even cells. This is achieved by screening large numbers of polypeptides in parallel, allowing for the identification of rare but potent binders that might be missed by traditional screening methods. In addition, polypeptide display libraries can be used to optimize the properties of existing binders, such as their affinity, specificity, and stability, through iterative rounds of selection and mutagenesis. There are several types of polypeptide display libraries available on the market, each with its own strengths and limitations. Phage display libraries, for example, are widely used for the discovery of therapeutic antibodies, due to their ability to generate high-affinity binders with low immunogenicity. Yeast display libraries, on the other hand, are better suited for the discovery of protein-protein interactions, as they can display larger and more complex proteins than phage display. Other types of display libraries include bacterial display, ribosome display, and mRNA display, each with their own unique features and applications. The methods for making and using polypeptide display libraries have also evolved rapidly in recent years, with new technologies and protocols being developed to improve their efficiency and versatility. For example, advances in DNA synthesis and assembly have made it possible to generate large libraries of polypeptides with high diversity and complexity, while new screening methods, such as high-throughput flow cytometry and next-generation sequencing, have enabled faster and more accurate identification of binders. The market for polypeptide display libraries and methods for making and using them is expected to continue growing in the coming years, driven by the increasing demand for new drugs and therapies, as well as the expanding applications of polypeptide display in fields such as diagnostics, biologics, and synthetic biology. Key players in this market include biotech and pharmaceutical companies, academic research institutions, and contract research organizations, who are all investing in the development and commercialization of new polypeptide display technologies and applications. In conclusion, polypeptide display libraries and methods for making and using them are a rapidly growing and highly promising area of biotechnology, with the potential to revolutionize drug discovery and development. As new technologies and applications continue to emerge, the market for polypeptide display is expected to expand even further, creating new opportunities for innovation and growth in the biotech industry.

The University of California invention works as follows

Disclosed are expression vectors that display a passenger protein on the outer surface a biological entity. The displayed passenger polypeptide can interact or bind with a given drug ligand, as disclosed in this document. Methods for making and using expression vectors are also disclosed. Methods of making and using N/C terminal-fusion expression vectors are also disclosed.

Background for Polypeptide display library and methods for making and using them

1. “1.

The invention generally refers to bacterial polypeptide displays libraries and methods for making and using them.


Polypeptide-display technologies have significantly impacted basic research applications, both applied and applied,” says Clackson, T. and J. T. Clackson and J. A. Wells (1994). Trends in Biotech. 12(5):173-184; Shusta, E. V., and others. (1999) Curr. Opin. Biotechnol. 10(2):117-122. Kodadek (2001) Chem. Biol. 8(2):105-158; Lee, S. W., et al. (2002) Science 296 (5569):892-859; and Nixon (A. E.) (2002) Curr. Pharm. Biotechnol. 3(1):1-12. These methods are strong because they can generate libraries that contain billions of molecules by using the biosynthetic machinery in the cell and then to identify rare, desired polypeptides through selection or high-throughput screening. To engineer and isolate peptides for molecular recognition, display libraries have been extensively used. The display of peptides on the surfaces of filamentous bacteriaophage (phage display) has been proven to be a flexible and efficient method for isolating peptide ligands that can be bound to diverse targets. Scott, J. K. and G. P. Smith (1990). Science 249(4967).:386-904. Norris, J. D., et al. (1999) Science 285(5428):744-765; Arap, W., et al. (1998) Science 279(5349).:377-806; and Whaley S. R. et al. (2000) Nature 405 (6787):665-668

Polypeptide display systems are mRNA and/or ribosome display and eukaryotic viruses display. They also include bacterial and yeast cell surfaces display. See Wilson, D. S., et al. 2001 PNAS USA 98(7):3750-3511; Muller, O. J., et al. (2003) Nat. Biotechnol. 3:312; Bupp K., and M. J. Roth (2002) Mol. Ther. 5(3):329-3513; Georgiou, G., et al., (1997) Nat. Biotechnol. 15(1):29-3414; Boder, E. T. Wittrup (1997) Nature Biotech. 15(6):553-557. Surface display methods are attractive since they enable application of fluorescence-activated cell sorting (FACS) for library analysis and screening. See Daugherty, P. S., et al. (2000) J. Immunol. Methods 243(1):211-2716; Georgiou G. (2000) Adv. Protein Chem. 55:293-315; Daugherty, P. S., et al. (2000) PNAS USA 97(5):2029-3418; Olsen, M. J., et al. Methods Mol. Biol. Biol.

Phage display is the localization and fusion of peptides to coat proteins. Scott, J. K., and G. P. Smith (1990). Science 249(4967).:386-390. Lowman, H. B., (1991) Biochem. 30(45):10832-10838. In general, polypeptides that have a specific function of binding can be isolated by incubating with a target and washing away non-binding bacteria. Then, the bound phage is eluted. Finally, the phage population can be re-amplified by infecting a new culture of bacteria. Unfortunately, phage display has some undesirable properties. See Zahn, G. (1999) Protein Eng. 12(12):1031-1034. For example, phage display is limited to about a few thousand copies of the displayed polypeptide per phage or less, thereby precluding the use of sensitive fluorescence-activated cell sorting (FACS) methodologies for isolating the desired sequences. It is also difficult for phage to remove or recover from immobilized target molecules, leading to clonal loss. Infection is also required for phage display. This means that viruses that don’t bind to a cell and enter it are lost in the early stages of the process. This can lead to lower quality results, such as affinity of isolated binding molecules. Additionally, phage display selections can be time-consuming and take approximately two to three weeks to isolate phage display polypeptides that will bind to a target.

Most importantly, phage display requires the investigator to be familiar with routine phage manipulation techniques including infections, phage amplifications and tittering. Due to their relative infectivity and assembly efficiency, as well as their toxicity to host cells, phage display can result in Darwinian outgrowth. The third factor is that enrichment ratios are relatively low, which slows down the rate at which binding clones can become enriched.

Other formats and methods include mRNA, ribosome, polysome, cukaryotic viral display and bacterial, yeast and mammalian surface display. See Matthcakis, L. C., et al. (1994) PNAS USA 91(19): 9022-9026; Wilson, D. S., et al. (2001) PNAS USA 98(7):3750-3755; Shusta, E. V., el al. (1999) Curr. Opin. Biotech. 10(2):117-122. Boder, E. T. Wittrup and K. D. Wittrup (1997) Nature Biotech. 15(6):553-557. There are many alternative display technologies that have been reported to be able to display on the surface microorganisms. They can also be used as a general strategy to isolate protein binding peptides. However, there has not been any reported success. See Maurer, J., el al. (1997) J. Bacteriol. 179(3):794-804; Samuelson, P., el al. (1995) J. Bacteriol. 177(6):1470-1476; Robert, A., et al. (1996) FEBS Letters 390(3): 327-333; Stathopoulos, C., et al. (1996) Appl. Microbiol. & Biotech. 45(1-2): 112-119; Georgiou, G., et al., (1996) Protein Engineering 9(2):239-247; Haddad, D., et al., (1995) FEMS Immunol. & Medical Microbiol. 12(3-4):175-186; Pallesen, L., et al., (1995) Microbiol. 141(Pt. 11): 2839?2848, Xu Z. and S. Y. Lee (1999). Appl. Environ. Microbiol. 65(11),:5142-5147; Wernerus H. and S. Stahl 2002 FEMS Microbiol. Lett. Lett. (2000) Int. J. Med. Microbiol. 290(3):223-230. Some of these systems were used in the prior art for library screening, but they did not yield high affinity protein binding proteins. See Brown, S. (1992) PNAS USA 89(18):8651-8655; Lang H., et al. (2000) Eur. J. Biochem. 267(1);163-170; Klemm and M. A. Schembri ((2000) Int. J. Med. Microbiol. 290(3):215-221. Klemm, P., and M. A. Schembri (2000 Microbiol. 146(Pt 12):3025-3032; Kjaergaard, K., et al. (2000) Appl. Environ. Microbiol. 66(1), 10-14; Schembri M. A. (1999) FEMS Microbiol. Lett. 170(2):363-371; Benhar, I., et al. (2000) J. Mol. Biol. Biol. (2000) Adv. Exp. Med. Biol. 485:133-136.

Prior art expression vectors for polypeptide displays libraries using host cells have a number of problems. Prior art methods have several problems. (1) Small peptides cannot be expressed; (2) large libraries cannot not be selected; (3) polypeptides displayed on the outer surface of the membrane do not interact or bind with certain molecules or targets properly; (4) analysis of expression on fimbrial and flagella causes some polypeptides to be lost due to mechanical shearing.

Protein display on bacterial cells has the potential to speed up and simplify the process of isolating ligands. This is because experimental procedures with bacteria are highly efficient and can be performed using FACS. See Daugherty, P. S., et al. (2000) J. Immunol. Methods 243(1):211-2720 Brown, S. (1992). PNAS USA 89 (18):8651-8521 Francisco, J. A., et al. (1993) PNAS USA 90(22):10444-10448; Taschncr, S., et al. (2002) Biochem. J. 367(Pt 2):393-402; Etz, H., et al. (2001) J. Bacteriol. 183(23). 6924-6935. Camaj, P., and et al. (2001) Biol. Chem. 382(12):1669-1677. Although there are many bacterial display systems, their utility has been limited by technical limitations, such as inaccessibility on cells, inability display high-quality sequences and adverse effects on cell viability and growth. Francisco, J. A., et al. (1993) PNAS USA 90(22):10444-10822; Lu, Z., et al. (1995) Biotechnology, NY 13(4):366-7223 Klemm, P. and M. A. Schembri. (2000) Microbiology, 146(Pt 12),:3025-3224 Christmann, A., and et al. (1999) Protein Eng. 12(9):797-80625; Lee, S. Y., et al. Trends Biotechnol. 21(1):45-52; Lu, Z., et al. (1995) Biotechnology (NY) 13(4):366-7225; Lee, S. Y., et al. Trends Biotechnol. 21(1):45-5226; Camaj, P., et al. (2001) Biol. Chem. 382(12), 1669-1677; Schembri, M. A. et al. (2000) Infect. Immun. 68(5):2638-2646.

These techniques cannot be used to isolate high affinity peptide-ligand ligands. These techniques also do not allow peptide exposure to the cell surface that is suitable for binding to analytes such as antibodies, proteins and viruses. These display formats do not work with some isolation methods because the peptides they produce don’t bind to large molecules or other surfaces such as magnetic particles. Prior art also decreases cell viability and alters the membrane permeability, which can reduce process efficiency. Routine isolation of high affinity, peptide-ligand ligands for arbitrary targets of protein has yet to be demonstrated. See Camaj, P., et al., (2001) Biol. Chem. 382(12),:1669-7727. Tripp, B. C. et al. (2001) Protein Eng. 14(5):367-377; Lang, H., et al. (2000) Eur. J. Biochem. 267(1):163-170; Lang, H., et al. (2000) Adv. Exp. Med. Biol. 485:133-136 Klemm, P., and M. A. Schembri ((2000) Int. J. Med. Microbiol. 290(3): 215-221. Klemm, P., and M. A. Schembri ((2000) Microbiol. 146(Pt 12):3025-302; Kjaergaard, K., et al. (2000) Appl. Environ. Microbiol. 66(1):10-14; Schembri, M. A., et al. (1999) FEMS Microbiol. Lett. 170(2):363-371; Benhar, I., et al. (2000) Mol. Biol. 301(4):893-904; Kjaergaard, K., et al. (2001) Appl. Environ. Microbiol. Microbiol. (2000) Exp. Med. Biol. 485:133-136.

Also in the prior art, polypeptides are most commonly displayed on cell surface as insertional or?sandwich? fusions. Into the outer membrane or extracellular apparatus, e.g. fimbria and flagella proteins or, less often, as fusions with truncated hybrid proteins thought to be localized at the cell surface. Se Pallesen, L., et al. (1995) Microbiol. 141(Pt. 11):2839-48. Etz, H., and et al. (2001) J. Bacteriol. 183(23):6924-6935. The LppOmpA and icc nucleation proteins (InP) are two examples of this latter. See Georgiou, G., et al. (1997) Nat. Biotechnol. 15(1):29-34. The display of polypeptides has been made possible by the OmpA, OmpC and OmpF outer membrane proteins. See Xu. Z., and S. Y. Lee (1999). Appl. Environ. Microbiol. 65(11):5142-5147; Taschner, S., et al. (2002) Biochem. J. 367(Pt 2):393-402.

However the C- and N-termini (of these?carriers?) are not located on the cell surface. The cell surface does not contain proteins, so it is impossible to display polypeptides in terminal fusions. Proteins that are incapable of folding in an insertional context are not able to be fused to their N and C termini. Protein sequences that are not capable of folding in the insertional fusion context, when their C and N termini fuse to the?carrier, can’t be effectively displayed as insertions. The restriction on insertional fusions also hinders the display of large numbers of proteins encoded in cDNA libraries on cells’ surface.

Prior art methods attempted to solve the problem of insertional-fusion displays by truncating outer protein sequences so that the resulting new termini might appear on the cell surface. See Lee, et al. Trends in Biotech. 23(1):45-52; Georgiou, et al. (1997) Nat. Biotech. 15(1):29-34. These methods were used to develop the LppOmpA system, which allows the targeting of peptides or polypeptides at the outer membrane of bacteria. See Francisco, et al. (1992) PNAS USA 89(7):2913. LppOmpA expression vectors include expression vectors that use LppOmpA, araBAD promoter and chloramphenicol resistance. Daugherty and al. (1999) Protein Engineer. 12(7):613-621. LppOmpA expression vector encodes an OmpA protein that results at amino acid residue159. The LppOmpA expression vector’s ability to express polypeptides from large collections is severely limited by the following: i) the decreased structural stability of modified OmpA proteins, iii] intolerance to expression of high temperatures, and iv). Most importantly, it is unable to display polypeptides on cells in a way compatible with binding large proteins without compromising viability or growth rate. See Christman, A. and colleagues, 1999. Pot. Eng. 12 (9):797.

Expression vectors used in the prior art are also problematic. (1) The polypeptides created by the expression vectors cannot be bound externally to proteins, cells or surfaces, (2) the expression Vectors doesn’t allow surface presentation large polypeptides, (3) any expressed protein can only interact small molecules that pass through outer membranes and into the periplasmic area. These issues have prevented the general application of this technology to isolating high affinity binding peptides. See e.g., Stathopoulos, C. (1996) Applied Microbiol. Biotech. 45 (1-2) 112. Earhart CF. (2000) Methods Enzymol. (326):506-16; Francisco, J. (1994) Annal. NY Acad. Sci. 745:372 and Bessette P. H. et al. (2004) Prot. Eng. (In Press).

There is a need for a robust display method that requires minimal technical knowledge, is less labor-intensive, and speeds up the process of ligand separation from weeks to days, as compared with the prior art methods.

The invention concerns expression vectors that display polypeptides on the outer surface of biological entities within a carrier protein loop.

In certain embodiments, the invention provides an expression vector capable to express and display a particular passenger polypeptide on the outer surface of a biologic entity within a carrier proteins loop that is capable interacting with a specific ligand.

Some embodiments expose the carrier protein loop, leaving either an N-terminus or a C-terminus on the outer surfaces. The peptide linker can be used to fuse the native C-terminus with the native N-terminus in some embodiments. The ligand may make the C-terminus and N-terminus visible to the outer surface. In some embodiments the C terminus is fused with the N terminus. In some embodiments the N terminus is fused with the C terminus. OmpX is a preferred carrier protein.

In some embodiments, a carrier protein is a bacterial outside membrane protein. Some preferred embodiments use OmpA and OmpX as the bacterial outer membrane proteins. Some preferred embodiments of the polypeptide are expressed in the OmpA’s first extracellular loop. Some preferred embodiments of the polypeptide are expressed in OmpX’s second extracellular loop. Some preferred embodiments of the polypeptide are expressed in the third extracellular Loop of OmpX.

In some embodiments, the polypeptide may be streptavidin or T7 binding proteine.

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