by Lance Liotta, M.D., Ph.D., NCI, and Mark Sobel, M.D. Ph.D., NCI

Identifying an unknown protein ligand has traditionally been a daunting task. Typically, when a researcher has wanted to identify the protein that binds to a particular receptor, antibody, enzyme, or acceptor molecule of interest, he or she has had to resort to exhausting trial-and-error testing of suspected ligands, screening of expression libraries, or serial testing of peptide analogs or protein cleavage fragments. These traditional methods are being improved by a variety of highly sophisticated techniques in which large libraries of ligands are synthesized in parallel and screened against the binding partner (1,2). The new techniques fall into two categories: chemical methods and biological libraries (1). Chemical methods employ instrumentation to synthesize and screen randomly generated peptides. Biological libraries, the subject of this Hot Methods Clinic, utilize tens of millions of bacteriophage clones, each of which has a cDNA sequence cloned into the phage genome in such a way that the phage expresses a unique polypeptide on its surface (3,4). Using a method called "biopanning," the phage with the DNA encoding the protein of interest is isolated. The selected phage is then propagated in E.scherichia coli, and the amino acid sequence of the ligand is determined by sequencing the corresponding coding region of the viral DNA.

The technology for these biological, or phage, display libraries is rapidly accelerating and offers great promise as a research tool for the rapid cloning of protein peptides and entire, fully folded large protein ligands (2,5). Phage constructs may even include libraries of "phage antibodies" that could display an array of binding specificities large enough to recognize any possible antigen (4,6). Many NIH intramural scientists are gearing up to try phage display, but at this moment, none have the technique fully up and running. The purpose of this article will be to provide an introduction to this powerful technique. A future article will provide trouble-shooting tips when more NIH intramural scientists have experience with phage display.

Phage Display Libraries: What are they?

Biological libraries displayed by phage include peptide libraries and full protein libraries. Peptide phage display libraries are batches of filamentous bacteriophage virions each displaying one or more copies of a different short amino acid segment on its surface (3,7). For example, Scott and Smith assembled a 200 million clone hexapeptide epitope library and used two monoclonal antibodies (Mabs) to screen a 23 million clone subset and pull out a sequence that corresponded to the mobile region of the protein antigen myohemerythrin (3). Dower's group constructed a hexapeptide library of 300 million clones and screened with Mab 3-E7, which, like the opioid receptor, binds tightly to the amino terminal four residues (YGGF) of beta endorphin (3).

Protein phage display libraries are libraries of large, multidomain proteins bearing variations in selected amino acid residues displayed on the surface of a filamentous bacteriophage. These large, folded proteins have included functional domains of antibodies (5,6), hormones (4), lectins, and enzymes (7). Maruyama et al (7) have developed the [[lambda]]foo phage vector and used it to express functionally active [[beta]]-galactosidase ([[beta]]-gal) and a plant lectin, BPA. [[beta]]-gal and BPA are tetramers of four identical subunits and have large molecular masses, 465 kDa and 120 kDa, respectively.

How is the protein displayed?

Phage coats typically consist of two viral proteins: minor coat protein -- protein pIII -- and major coat protein, pVIII. pVIII forms the body of the phage. Copies of the minor coat protein are added at the trailing end of the emerging virion. Both pVIII and pIII are synthesized with short signal sequences that allow them to be transported to the inner membrane of the bacteria. The cDNA of interest is inserted into the phage genome to allow it to be synthesized as a fusion protein with the coat protein of the virus.

How is the variable protein library created?

A variable protein library can be created by generating combinations of randomly encoded amino acids (3). Hexamers (6mers) of oligonucleotides can be constructed with the degenerate sequence NN(G-or-T),NN(G-or-C), where N is any of the four nucleotides. The hexamers are randomly encorporated into the viral DNA. Culturing large batches of phages that have encorporated this degenerate code will generate phage clones bearing all 20 possible amino acids (and one possible stop codon) at encorporation sites. All possible combinations of 6mers can generate a library of more than a billion phage clones. The number of peptides encompassed by this technology exceeds by a factor of 100 to 1000 the number that can be screened by conventional expression systems.

How do you detect the desired protein that binds to your given target?

"Biopanning" (Figure 2) is one term used to describe the method for purifying the phage clone that expresses the epitope of interest. A specific example is the isolation of a peptide antigen epitope. The researcher starts with a monoclonal antibody for which an antigen epitope is sought. The antibody is biotinylated and mixed with the phage library. Only the phage clones that express the correct antigen protein will bind to the biotinylated antibody. To separate the phage bound to the antibodies, the mixture is incubated with plates coated with streptavidin. The biotinylated antibodies stick to the streptavidin and carry the bound phage with them. In a variation of this selection technique, the plate is coated directly with the binding partner protein. When panning is conducted, only the phage expressing the desired binding ligand sticks to the plate. The unbound phage are washed away, and the bound phage are subsequently eluted with acid. The eluted phage are amplified by growth in bacterial cells, then used in successive rounds of biopanning, infection, and propagation.

A commercial variation on the theme: Recombinant phage antibody system

One of the most exciting versions of the phage display system is the recombinant phage antibody system, described by Hoogenboom et al. (4) and recently commercialized by Pharmacia Biotech in Piscataway, N.J. Pharmacia offers this system as a kit for the cloning and expression of monovalent antibody fragments that bind to a known antigen for which a genetically known antibody is sought. The company's literature claims, "The system's integrated modular format greatly simplifies the task of isolating and cloning antibody genes and expressing and detecting their products." The starting material, prepared by the researcher, is mRNA from antibody-producing mouse hybridoma or spleen cells from mice that have been injected with the antigen for which an antibody is desired. The antibody variable heavy (VH) and light (VL) chain genes are separately amplified and assembled into a single chain SCFV fragment with a short, linker DNA. The VH and VL module is then cloned into an expression vector and ultimately, when tranfected into the phage, both VH and VL genes are expressed on the same polypeptide chain, fused with phage pIII protein. Once selected by biopanning on a plate coated with the known antigen, the phage expressing the correct antibody is then used to infect E. coli and produce large amounts of the antibody chains. Soluble antibodies can be produced in large quantities using the proper conditions. According to the company's literature, yields ranging from 0.2 to 10 mg/l of culture are possible. The soluble antibodies can then be labeled and used for immunoassay or immunoblots to detect the antigen.

What are the applications of phage display?

Phage display methodology is at an early stage in what can be expected to grow into a widely adopted technology. It offers great promise for identifiying of novel ligands that bind to a protein of interest, or for mapping the functional domains of known proteins. Clinical utility of phage display includes the development of new drugs through peptide-mimetics and the refinement of vaccine reagents. The recombinant phage antibody system could potentially be applied to analysis of antibody functional domains, sequence analysis of antibody genes, and large-scale production of antibodies for immunoassays or immunotherapy.

What are the limitations and challenges of phage display?

As can be seen from the sample protocol below, the methods have multiple steps and may be time-consuming to set up. In addition, both the short peptides selected by phage display and monovalent antibodies from phage cloning may produce antibodies and peptides of relatively low affinity (6). Short peptides may not contain the secondary and tertiary structure required for ligand recognition, and until Maruyama et al. (7) devised their protocol, large proteins with complicated 3-D structures could not be expressed. Unfortunately, phage bearing large fusion proteins may have reduced infectivity (7) and thus be difficult to produce and cultivate in quantity. Results may also be somewhat ambiguous but still potentially valuable in drug design: it is possible for a specific short peptide to mimic the binding epitope but still be different from the natural ligand (1,7).

Outline of a general protocol for the recombinant phage antibody library

This protocol is derived from the Pharmacia Biotech Recombinant Phage Antibody System Kit. It assumes that you have an antigen of interest and want to produce a genetically defined recombinant antibody that will recognize the antigen. The kit provides everything but your antigen and the spleen lymphocytes or hybridoma cells.

I. Construction of the library

1. Starting material can be either mouse hybridoma cells or spleen cells from mice that have been injected with the antigen for which an antibody is sought.

2. Isolate mRNA from the mouse spleen (lymphocyte) or hybridoma cells.

3. Synthesize cDNA from the mRNA.

4. Set up two PCR reaction tubes, one for amplifying cDNAs encoding the antibody's immunoglobulin heavy chain and the other for amplifying cDNAs encoding the light chain protein. The PCR reactions are primed with variable region probes.

5. The PCR products from these two reactions are purified by gel electrophoresis.

6. Using a linker fragment that is designed to anneal to the 3' end of the heavy chain and to the 5' end of the light chain PCR products, the purified heavy and light chain DNAs are assembled into a single gene. Using the 5' primer from the heavy chain PCR reaction and the 3' primer from the light chain PCR reaction, single antibody genes are amplified in another PCR reaction.

7. The assembled antibody genes are reamplified using modified 5' and 3' primers that now include different restriction sites to permit directed cloning. The particular restriction sites selected rarely occur within mouse antibody genes, thus guaranteeing that mostly intact antibody sequences will be cloned. After purification, the antibody DNA fragments are restricted to generate cohesive ends for ligation into the cloning vector.

8. The cloning vector is a phagemid (genetically engineered bacteriophage) that has appropriate restriction sites positioned such that the recombinant antibody genes will be cloned as fusion genes with a phage coat protein that is expressed on the phage surface.

9. The restricted antibody DNA fragments are ligated into this cloning vector, which has been appropriately restricted.

10. E. coli cells are transformed with the recombinant phagemids, thus generating millions of copies of candidate antibodies.

11. Transformed bacteria are infected with helper phage that rescue the phagemids, facilitating their reproduction and protein expression.

II. Detection of recombinant phage

1. An ELISA is used to detect recombinant antibodies that are expressed on the phage surface.

2. The antigen for which an antibody is being sought is coated onto the wells of a microtiter plate.

3. The recombinant phage are added to the wells, allowing the antigen to interact with the antibodies that are expressed on the surface of each phage.

4. The plate is washed.

5. Horseradish peroxidase-conjugated antibody that recognizes the phage coat protein is added to detect the presence of any antigen-bound recombinant phage. A positive color reaction in a well identifies a recombinant antibody-producing phage.

6. These phage are eluted from the ELISA-positive wells and are used to reinfect bacteria to purify the phage through multiple rounds of detection until a pure clonal population is achieved.

III. Production of recombinant antibody

1. If desired, the original phagemid vector can be replaced with a modified phagemid that will produce soluble antibody molecules with an epitope tag at the carboxy terminus. A monoclonal antibody is available that recognizes this epitope tag.

2. Soluble antibody molecules are produced by the phagemids and accumulate in the periplasm of the bacteria, and then leak into the culture medium. The bacterial culture medium is collected.

3. The epitope tag antibody is used to purify the recombinant antibody molecules from the culture medium via affinity chromatography.

References

G. P. Smith and J. K. Scott. "Libraries of peptides and proteins displayed on filamentous phage." In: Methods in Enzymology, Volume 217, Recombinant DNA part H, R. Wu, ed. San Diego: Academic Press, pp. 228 - 57 (1993)

Recombinant phage antibody system protocol and ordering information: 1994 Pharmacia Biotech Data File available by fax from Verna Frasca, Ph.D., phone 800-526-3593; fax 908-457-8100 or 800-329-3593.

Selected References

1. M. R. Pavia, T. K. Sawyer, and W. H. Moos. "The generation of molecular diversity." In: Bioorg. Med. Chem. Let., 3, 387 - 96 (1993)

2. I. N. Maruyama, H. I. Maruyama, and S. Brenner. "Lambda foo: a lambda phage vector for the expression of foreign proteins." Proc. Natl Acad. Sci. USA 91, 8273 - 77 (1994).

3. J. M.Scott and G. P. Smith. "Searching for peptide ligands with an epitope library." Science 249, 386 - 90 (1990).

4. C. F. Barbas III, A. S. Kang, R. A. Lerner, and S. J. Benkovic. "Assembly of combinatorial antibody libraries on phage surfaces: the gene III site." Proc. Natl. Acad. Sci. USA. 88, 7978 - 82 (1991).

5. S. Bass, R. Greene, and J. A.Wells. "Hormone phage: an enrichment method for variant proteins with altered binding properties." Prot. Struc. Function Genet. 8, 309 - 14 (1990).

6. H. R. Hoogenboom, A. D. Griffiths, K. S. Johnson, D. J. Chiswell, P. Hudson, and G. Winter. "Multi-subunit proteins on the surface of filamentous phage: methodologies for displaying antibody (Fab) heavy and light chains." Nucl. Acids Res. 19, 4133 - 37 (1991).

7. S. E. Cwirla, E. A. Peters, R. W. Barrett, and W. J. Dower. "Peptides on phage: a vast library of peptides for identifying ligands." Proc. Natl. Acad. Sci. USA 87, 6378 - 82 (1990).