WO2001042454A2

WO2001042454A2 - Methods for detecting compound interactions using phage display

Info

Publication number: WO2001042454A2
Application number: PCT/CA2000/001496
Authority: WO
Inventors: Peter D. Duck
Original assignee: Select Therapeutics Inc.
Priority date: 1999-12-09
Filing date: 2000-12-11
Publication date: 2001-06-14
Also published as: WO2001042454A3; AU1979301A

Abstract

Phage display technology is used to detect interactions between compounds. In one aspect, the invention features a method for determining compounds which interact with each other by providing a genetic package (GP), which comprises a compound associated with the surface of the GP and encoded within the genetic material of the GP. The GP is incapable of independently replicating in a host cell, but capable of replicating in a host cell after coinfection with a second GP. The second GP comprises another compound associated with its surface and encoded within its genetic material. The second GP is also incapable of independently replicating in a host cell, but capable of replicating after coinfection with the first GP. The first GP and the second GP are contacted with host cells under conditions such that coinfection occurs substantially only when the surface compound of the first GP interacts with the surface compound of the second GP, the GPs are allowed to replicate, and the genetic material of at least one of the GPs is then recovered.

Description

METHODS FOR DETECTING COMPOUND INTERACTIONS USING PHAGE

DISPLAY

Related Applications This application claims priority to U.S. Provisional Patent Application Serial

No. 60/170,083, entitled "Methods for Detecting Compound Interactions Using Phage Display," filed on December 9, 1999. The entire contents of this provisional application are incorporated herein by reference.

Background of the Invention

Phage display is a technique for the expression or 'display' of a peptide or protein on the surface of a phage. This is accomplished by insertion of a gene or a gene fragment in a phage surface protein gene. Provided that the reading frame is correct and that the insert does not interfere with the essential functions of the surface protein, the insert will result in a fusion protein on the phage surface. If the peptide is well exposed on the phage surface, it will be available to interact with other compounds present in the system. The insertion of random oligonucleotide sequences provides a means of constructing extensive peptide libraries that may be screened to select peptides with specific affinities or activities. Phage display of combinatorial libraries allows one to test a myriad of structures for binding affinities without detailed information about the structures being tested.

Summary of the Invention

The invention generally relates to methods for detecting compound interactions through a novel use of phage display technology.

In one aspect, the invention features a method for determining compounds which interact with each other by providing a genetic package (GP), which comprises a compound associated with the surface of the GP and encoded within the genetic material of the GP. The GP is incapable of independently replicating in a host cell, but capable of replicating in a host cell after coinfection with a second GP. The second GP comprises another compound associated with its surface and encoded within its genetic material. The second GP is also incapable of independently replicating in a host cell, but capable of replicating after coinfection with the first GP. The first GP and the second GP are contacted with host cells under conditions such that coinfection occurs substantially only when the surface compound of the first GP interacts with the surface compound of the second GP, the GPs are allowed to replicate, and the genetic material of at least one of the GPs is then recovered. In one aspect, the replicated GPs will be subjected to additional rounds of host cell infection and recovery. For example, replicated first and second GPs will be contacted with host cells, under conditions such that coinfection occurs substantially only when the surface compound of the first replicated GP interacts with the surface compound of the second replicated GP, and allowing for replication of the replicated GPs. The conditions for the additional coinfection may be the same or different from previous coinfections. For example, the salt concentration may be adjusted or the concentration of GPs may also be adjusted. Furthermore, the host cells also might be the same or different. In one aspect, the rate of coinfection of host cells by GPs without surface compound interactions approaches zero.

In a preferred aspect, the invention features a method for detecting peptides which bind to each other by providing a first population of filamentous bacteriophage, which display on their surfaces a diverse library of peptides which are encoded within the bacteriophage's gene III (or gene 3), and which are only capable of replicating in a host cell after coinfection with an individual filamentous bacteriophage from a second population. A second population of filamentous bacteriophage is also provided. The second population of filamentous bacteriophage also display on their surfaces a diverse library of peptides which is also encoded within gene III of the second population of filamentous bacteriophage. The second population is also only capable of replicating in a host cell through coinfection with a filamentous bacteriophage from the first population. The first and second filamentous bacteriophage populations are then combined with E. coli host cells under conditions such that coinfection substantially occurs only when a peptide displayed on the surface of first population of filamentous bacteriophage binds to a peptide displayed on the surface of the second population, replication of the filamentous bacteriophage is allowed, and the genetic material of at least one of replicated filamentous bacteriophage is then recovered. In an advantageous embodiment, the replicated filamentous bacteriophage are reexposed to the host cells at least once. In another aspect, the invention features a pair of GPs which individually have insufficient genetic material for replication, but complement each other such that replication may occur after coinfection of the same cell. The invention also features the genetic material of the replicated GPs as well as the surface compounds which interact and cause the formation of the combined GP pair. Brief Description of the Drawings

Figure 1 is a schematic diagram of the co-infection process of the invention. It shows certain steps of double infection-selection of two complementing phage strains. Figure 2 is a plasmid map of pCANTAB 5E.

Detailed Description of the Invention

The presently disclosed invention provides novel methods of identifying compounds which interact with each other through the use of genetic packages (GPs) and phage display technology. Although many other techniques have been used to determine the binding affinities of various compounds, no techniques have been developed that can screen for the binding of two independent libraries of peptides and then directly amplify the genes of interest. Phage display technology has been used to select for proteins that bind to a specific target ligand or receptor (e.g., U.S. Patent No. 5,514,548). The term "genetic package" or "GP" refers to an entity, such as a virus, or a bacteriophage which can be replicated following the infection ofa suitable host cell. For example, the GP may be one of a class of viruses known as bacteriophage, which infect bacterial cells. In another embodiment, the GP can be a lambda type phage. In an embodiment, the GP is a filamentous bacteriophage. Filamentous bacteriophages have small native genomes into which large libraries, consisting of a number of different genes or gene fragments, are easily inserted. Filamentous bacteriophages are also known as viral particles or virions. They are stable to potential elution conditions, such as low pH and they accumulate to high titers (e.g., such about 1012 mL), such that every clone in a gene library can be well represented. Preferred filamentous phages include M13, fd, fl, Ifl, Ike, Zj/2, Ff, Xf, Pfl, and

Pf3. More preferably, the filamentous bacteriophages can be fl, fd and Ml 3. These filamentous bacteriophage are F-specific and have evolved a method of propagation which does not kill the host cell. They are commonly used as vehicles for recombinant DNA. Bacteriophage fd is particularly advantageous, because biologically active foreign sequences can be easily inserted into its gene III.

The present invention employs at least two populations of GPs. Each population of GPs has incomplete genetic material such that a single GP is unable to replicate itself after independent infection of a cell or after coinfection with another GP of the same population. In order for replication to take place, coinfection of a host cell must take place by at least two GPs from separate populations. The genetic material of the two populations complements each other such that replication is enabled. Coinfection is facilitated by interaction between surface compounds of the GPs. In an embodiment, the host cells are E. coli.

The term "interact" is intended to include'interactions such as ionic, covalent, hydrophobic, etc. and combinations thereof. The term "binding" is intended to include interactions between peptides which result in coinfection of host cells.

Figure 1 is a scheme of the coinfection method. In Figure 1, two populations of GPs are mixed. One of the populations of GPs is tetracycline resistant and the other is ampicillin resistant. The two populations of GPs are mixed and the surface proteins are allowed to interact. The GPs are allowed to infect host cells. After exposure to both antibiotics, only the cells which were coinfected for both ampicillin and tetracycline resistant populations of GPs will be selected. The coinfected host cells will have both plasmids when characterized by agarose gel electrophoresis.

The term "surface compound" is intended to include compounds associated with the surface of the GP. These compounds may be peptides, peptide derivatives, nucleic acids, nucleic acid derivatives, biological cofactors, or other inorganic or organic compounds. In one embodiment, at least one of the surface compounds may be immunoglobins, antigens, cell surface receptors, cell surface receptor binding compounds, or fragments thereof. In another embodiment, these surface compounds may be a specific compound for all of the GPs of at least one population, e.g. an antigen, a cell surface receptor, etc., for a given population of GPs. In an embodiment, both the GPs and the surface compounds may be generated through phage display.

The term "phage display" is intended to refer to a technique for the expression or 'display' of a peptide or protein on the surface ofa filamentous bacteriophage or other GP. This is accomplished by insertion of a gene or a gene fragment in a phage surface protein gene. Provided that the reading frame is correct and that the insert does not interfere with the essential functions of the surface protein, the insert will result in a fusion protein on the phage surface. If the peptide is well exposed on the phage surface, it will be available to act as a ligand, enzyme, immunogen or otherwise actively participate in a biochemical process. The insertion of random oligonucleotide sequences provides a means of constructing extensive peptide libraries that may be screened to select peptides with specific affinities or activities. The separation of phage particles expressing different peptide inserts in the phage surface protein is accomplished by affinity selection in a method called panning. This strategy allows one to test a myriad of structures for optimal function without detailed information about the relationship between the function and the structure.

-4-

SUBSTITUTΕ SHEET (RULE 26) For example, in filamentous bacteriophages, two genes have been used extensively for phage display, gene III (gill or gene3) and gene NIII (gNIII). Gene III encodes for a protein at the proximal end of the phage, protein III (pill). Protein III is required for infection of E. coli and binds to the end of the pili of the bacteria. Gene VIII encodes a major coat protein, protein VIII, (pVIII), and is therefore present in approximately 2700 copies in comparison with gene III that is present in 3-5 copies depending on the phage used (Ladner et al. PCT publication WO 90/02909; Garrard et al, PCT publication WO 92/09690; Marks et al. (1992) J. Biol. Chem. 267:16007- 16010; Griffiths et al. (1993) EMBO J 12:725-734; Clackson et al. (1991) Nature 352:624-628; and Barbas et al. (1992) PNAS 89:4457-4461). In a further embodiment, the surface compounds are encoded within gene III or gene IV of at least one of the GPs. In another preferred embodiment, cleavage sequences may be incorporated in to the surface compound. The incorporation of cleavage sequences into the surface compounds would allow for rapid recovery of the surface compounds after being treated with an endonuclease.

In a preferred embodiment, a technique known as "polyvalent phage display" may be used. In "polyvalent phage display," small foreign DNA fragments are inserted near the amino-terminal end between the amino- and carboxy-terminal domains into the phage surface protein gene. In this method the peptide is expressed in multiple copies on the phage produced, i.e. the peptide is expressed in all copies of the gene product. In polyvalent display, the fusion using pill cannot have a too large insert, since larger peptides than 6-10 amino acids will probably interfere with the packaging process.

In a preferred embodiment, the surface compounds for at least one population of GPs comprise a genetically diverse combinatorial library of compounds, which may be generated by methods well known in the art, such as the split synthesis method (See e.g., Cormack and Struhl (1993) Science 262:244-248, incorporated herein by reference).

The use of combinatorial libraries to identify compounds which bind to a specific ligand is well established (see, e.g., M.A. Gallop et al., (1994) J. Med. Chem. 37:1233; and E.M. Gordon et al., (1994) J. Med. Chem. 37:1385; and references cited therein). Combinatorial libraries displayed on phages commonly contain about 10⁶-10¹² member peptides. Such peptide libraries can comprise all possible peptides of a given length (e.g., every one of the twenty natural amino acid residues at each position of a hexamer), or a subset of all possible peptides. The structure of selected peptides can be determined with relative ease by standard sequencing methodologies (e.g., sequencing of the peptides themselves or of a nucleic acid molecule encoding the peptide). Following selection of one or more peptide library members that bind to each other, the amino acid sequence of at least one of the peptides can be determined according to standard methods. For example, the amino acid sequence of at least one of the peptides may be determined by determining the nucleotide sequence of a nucleic acid molecule encoding the peptide and translating the encoded peptide using the genetic code. Nucleotide sequencing can be performed by standard methods (e.g., dideoxynucleotide sequencing or Maxam-Gilbert sequencing, either manually or using automated nucleic acid sequencers). Alternatively, the amino acid sequence of at least one of the selected peptides can be determined by direct amino acid sequencing of the peptide (e.g., by Edman microsequencing, either manually or using automated peptide sequencers).

In one embodiment, the recovered GPs may be reexposed, at least once, to the host cells before being analyzed. The additional rounds of replication may enhance the selectivity of the coinfection process, by reducing the probability of random coinfection by two GPs from separate populations with out surface compound interaction. Also, the GPs may be subjected to mutagenic conditions which may contribute to greater genetic diversity amongst the populations.

In another aspect, the invention also features a method for detecting peptides which bind to each other by providing a first population of filamentous bacteriophage, which display on their surfaces a diverse library of peptides which are encoded within the bacteriophage's gene III, and which are only capable of replicating in a host cell after coinfection with an individual filamentous bacteriophage from a second population. A second population of filamentous bacteriophage is also provided. The second population of filamentous bacteriophage also display on their surfaces a diverse library of peptides which is also encoded within gene III of the second population of filamentous bacteriophage. The second population is also only capable of replicating in a host cell through coinfection with a filamentous bacteriophage from the first population. The first and second filamentous bacteriophage populations are then combined with host cells under conditions such that coinfection substantially occurs only when a peptide displayed on the surface of first population of filamentous bacteriophage binds to a peptide displayed on the surface of the second population, replication of the filamentous bacteriophage is allowed, and the genetic material of at least one of replicated filamentous bacteriophage is then recovered. Furthermore, the method may also include combining replicated first and second filamentous bacteriophage populations with host cells under conditions such that coinfection substantially occurs only when a peptide displayed on the surface of the replicated first population of filamentous bacteriophage binds to a peptide displayed on the surface of the replicated second population of filamentous bacteriophage; and allowing for replication of the replicated filamentous bacteriophages.

EXEMPLIFICATION OF THE INVENTION

Materials

In the Examples, one of the populations of genetic packages used included RNase as its surface compound. The description and source of the materials used in these examples are described in Table 1. RNase was cloned as a gene III fusion into a phage display pCANTAB 5E vector containing ampicillin resistance gene as one population of GPs as described below.

Table 1

Item Description Source fUSE 5 The gene-III reading frame is disrupted m fUSE5, abolishing all pill functions, G. Smith vector including infectivity There are two Sfil cloning sites in fUSE5, which have non- ldentical, non-complementary 3-base 3 ^' overhanging ends; this allows directional cloning after removal of the stuffer that lies between these sites in the vector. fUSE5/6- Primary library: Scott & Smith, Science 249, 386-390, 1990 G. Smith mer library Vector: fUSE5 (foreign 6-mer displayed on all 5 copies of pill)

Number of primary clones: 2 * 10⁸

Number of transductant clones amplified 3.7 * 10"

Size of phage DNA. 9225 bases

Concentration of physical particles: 1 62 * 10¹⁴ virions/ml

Approximate infectivity: 5%

Buffer: TBS, Volume: 15 μl

K91BluKan lacZΔM15; probably is lacfi Sex: (Hfr-C) G. Smith

Phenotype: stable Hfr, even without selection, excellent for propagatmg filamentous phage; gives large plaques with wt phage, small but visible plaques with infective members of fd-tet family; requires (only) thiamine m minimal medium, LacZ^"; kanamycm resistant , LacY"; probably LacIV (super-repressor) lacZ Ω-donor pCANTAB Phagemid vector for expression of pill fusions, contains ampicillin resistance Pharmacia 5E marker (kit Cat.# 27-9401-01, see p 509 of Amersham Pharmacia 2000 Catalogue)

M13KO7 Deπvative of Ml 3 with a mutated version of gene II, a plasmid origin of Pharmacia replication and the Kan resistance gene at the Aval site of Ml 3. Provided with kit (Cat.# 27-9401-01)

Stain TGI | Used with helper phage M13K07 for production of phage displaying the RNase- Pharmacia genelll fusion. Provided with kit (Cat.# 27-9401-01).

EXAMPLE 1 : Cloning of Bovine Pancreatic RNase Fragment as a Gene3 Fusion

In this Example, a bovine pancreatic RNase fragment was used as the surface compound for a population of GPs. The RNase fragment was cloned into Gene3 of the filamentous bacteriophage. Preparation of RNase fragment specific primers with the Sfi I and Not I restriction sites

DNA encoding the 104-aa fragment of bovine pancreatic RNase was amplified by PCR using the 2 primers SRC-107 and SRC-108. The sense primer SRC107 with Sfi I site and M13 tail for sequencing of PCR product is shown in Table 2. The antisense primer SRC108 with Not I site at 5' end is shown in Table 2. The restriction site sequences are underlined.

Table 2

PCR Amplification of RNase Gene Fragment from Calf Thymus DNA. A standard condition of PCR containing 100 ng of Calf Thymus DNA (Sigma, Saint

Louis, MO, USA) per reaction and 50 μl reaction volume were used. Other PCR conditions included 35 cycles, an annealing temperature of 56°C, and 2 mM MgCl₂. Analysis of 15 μl of the PCR reaction, using a 1.5% agarose gel, showed the expected product of approximately 360 basepairs.

Restriction digestion of the PCR product with Clal showed the expected 277 basepair fragment (SEQ ID NO. 3). The 84 basepair fragment was not visible in this experiment but was detected in other experiments. The PCR reaction was scaled up to 3x200 μl reactions. The amplicons were purified using a Qiagen kit. The DNA concentration was estimated by gel electrophoresis. The PCR fragment was cut with Sfil and Notl and purified by agarose gel electrophoresis and a Qiagen kit.

Cloning of RNase encoding fragment into pCANTAB 5E

Cloning of the RNase encoding fragment into pCANTAB 5E (Figure 2) was performed essentially as recommended by the kit supplier (Amersham-Pharmacia). Transformation into competent DHA5 cells (Life Technologies) was performed. Screening of positive clones was performed using standard plasmid preparation (Sambrook).

DNA prepared from single colonies cut with Hindlll and EcoRI showed the expected size. The RNase encoding fragment was cloned into pCANTAB 5E for sequencing. Sequencing of the RNase cloned fragment shows the expected sequence of the RNase at the 5 'end and also showed the presence of Clal restriction site (SEQ ID NO. 4). Competent TGI cells were transformed with a pCANTAB 5E vector containing the cloned RNase-encoding fragment and referred to as pSRC-1 GPs.

Amplification of 6 mer Phage Display Library in a Population of GPs The 6 mer phage display library was amplified using the following procedure. 1- liter culture flasks containing 50 mL terrific broth (TB) were innoculated with 1 mL of an overnight culture of K91BluKan in LB containing 100 μg/mL kanamycin. The flasks were agitated vigorously at 37° until the OD₆₀₀of the 1/10 dilution reached ~0.2 corresponding to the late log phase. The agitation was decreased for five minutes to allow the sheared pili to regenerate, before 5 μL of the library to be amplified was added. The final concentration of the GPs (10¹⁰ virions/ml) was roughly comparable to the concentration of the viable host cells. Gentle agitation was continued for fifteen minutes after the addition of the GPs. The culture was then poured into a pre-warmed 3-liter fernbach flask containing 0.5 liter of LB supplemented with 0.22 μg/ml tetracycline. The fernbach flask was agitated vigorously for 35 minutes at 37°. Tetracycline was then added to bringing the concentration of the antibiotic up to 18 μg/ml. A 7-μl sample from each flask was then removed to be diluted. The flasks were agitated vigorously over night. 200 μL of 10^" and 10^"5 serial dilutions of the two 7-μLsamples from the previous step on LB plates containing 40 μg/mL tetracycline and 100 μg/mL kanamycin. The colonies were counted the next day. A colony count of -100 on the 10^"5 plates indicates a typical infectivity of about -5 x 10¹⁰ infected cells per culture.

The GPs were then isolated from the culture by PEG precipitation three times using the following protocol (Expression module/Recombinant Phage Antibody system product insert, Pharmacia Biotech document XY-040-00-08). 2 ml of PEG/NaCl was added to 10 ml of the GPs, mixed well and placed on ice for 30-60 minutes. The GPs were recovered by centrifuging the PEG/NaCl mixtures at 10,000 g (Allegra) for 20 min at 4 °C. The supernatant was then discarded and the pellet was resuspended in 1 mL TE, filtered through a 0.45 μm filter and stored at 2-8°C.

The titer of the GPs was determined using standard methods (Kay et al 1996. Phage display of peptides and proteins. A laboratory manual. Academic Press).

EXAMPLE 2: Phage Amplification and Determination of Titer Amplification of 6 mer library GPs was performed as described in Example 1.

Amplification of pSRC-1 GPs was performed using methods previously described (Expression module/Recombinant Phage Antibody system product insert, Pharmacia Biotech document XY-040-00-08).

A single colony of pSRC-1 GPs of TGI containing RNase A clone 1-1 was grown in 4 ml LB overnight. 100 μl of this culture was added to 10 ml of 2YT-G (+glucose, -Ampicillin). The culture was incubated for 1 hour at 37°C with shaking at 250 rpm to an optical density of about 1. 20 μl of 50 mg/ml Amp and 4xl0¹⁰ pfu of M13KO7 was then added to the culture. The culture was then incubated for 1 hour at 37°C with shaking at 250 rpm, before being centrifuged at 1000 g in an Allegra centrifuge for 10 min to sediment the cells. The supernatant was removed. The entire remaining sample was then resuspended in 10 ml of 2 YT-AK (2YT+100μg/ml Ampicillin and 50 μg/ml Kanamycin, no glucose). The culture was incubated overnight at 37°C with shaking at 250 rpm. The culture was then centrifuged at 1000 g for 20 min using an Allegra centrifuge. The supernatant, containing the GPs, was then transferred to 50 ml tubes, filter sterilized and the GPs were PEG precipitated.

Determination of phage titer was performed as described in the literature (Kay et al 1996. Phage display of peptides and proteins. A laboratory manual. Academic Press). The titer was estimated to 1.5xlO^u virions/ml for pSRC-1 GPs and 10¹⁰ for the 6 mer library GPs and 10'° for the fUSE5 control.

EXAMPLE 3: Co-Infection of TGI using 6 mer library GPs and pSRC-1 GPs The purpose of this example was to co-infect TGI cells using 10 μl of both 6 mer library GPs and pSRC-1 GPs.

The TGI cells were coinfected by streaking a master culture of the bacterial strain onto minimal media M9 agar plate. The plate was then incubated for 24-36 hours at 37 °C. A single colony from a minimal medium plate was then grown in 4 ml LB overnight at 37°C at 250 rpm. 1.5 ml of LB medium is incubated with 50 μl of the inoculated LB culture in a 14 ml tube. The

GPs were then added to the host cell culture (e.g., E. coli) after being incubated for ten minutes.

Negative controls of each population of GPs were also done concurrently. For example, the

6mer GP was cultured by itself, and the pSRS-1 was cultured by itself as well.

The cultures were then incubated for 4 hours at 37°C with constant agitation and diluted appropriately. The diluted cultures were then plated on 3 LB plates with antibiotics (a-

LB+Amp, b-LB+Tet, c-LB+Amp+Tet ) and incubated overnight at 37°. The colonies were then counted on each of the plates.

Colonies from the LB+Amp+Tet were chosen and streaked on the same media. The

GPs were then added for a second round of infection selection. For this example, dilutions of 10°, 10^"4 and 10^"6 were made. For the 10° dilution, all cells were plated. The results are shown in Table 3. Table 3

Positive and negative controls were as expected. 48 colonies were sub-cultured from the co-infected culture plates onto a new Tet+ Amp plate. The absence of growth on the Tet+ Amp plate suggested that the cells were not coinfected with both the populations of GPs.

EXAMPLE 4: Co-infection of TGI using 6mer and pSRC-1 phage and plating at a lower dilution.

TGI cells were co-infected using 10 μl of both 6 mer library and the pSRC-1 GPs. The GP incubation time was 10 min, and dilutions of 10°, 10² and 10^"4 were made. 10 μl of 6 mer and pSRC-1 GPs were plated separately, and served as controls. The results are shown in Table 4.

Table 4

Both the positive and negative controls were as expected. However, no positive coinfection colonies were observed at the 10^"2 dilution.

Example 5: Co-infection of TGI using 6mer and pSRC-1 phage, effect of incubation time

TGI cells were infected using 10 μl of both the 6 mer and pSRC-1 GPs. The GP incubation times were 10 min and 60 min in separate tubes, and dilutions of 10°, 10^"1 and 10^"2 were made. 10 μL of 6 mer and pSRC-1 GPs were plated separately, and served as controls. For the 10°, all cells were plated. The results are shown in Table 5.

Table 5

Negative controls were as expected at the 3 dilutions used. Colonies were observed in the coinfected sample as were observed in the previous experiments. The GP incubation time did not appear to effect the number of colonies infected.

Example 6: Co-infection of TGI Host Cells using 6mer and pSRC-1 GPs

TGI host cells were coinfected using lOOμl of 6 mer and 10 μl of pSRC-1 GPs. The phage incubation time was 60min, and dilutions of only 10° were made. A pooled control was added which included 6 mer and pSRC-1 GPs incubated separately, and then pooled just before plating. 10 μl of pSRC-1 GPs were also plated separately, and served as controls. Cells in this example were recovered in 500 μl LB media and plated in 5 independent plates.

Table 6 shows the results of the "pooled example. The 6 Mer/ pSCR-1 "pooled" control results are not shown because the number of colonies on the plate were too numerous to count.

Table 6

32 isolated colonies from the co-infected sample plates were sub-cultured onto a new Tet+ Amp plate. 11 colonies grew on the plate. It was hypothesized that these colonies were co-infected by the two populations of GPs. These 11 colonies were then grown overnight in LB + Tet+ Amp. All of the samples grew in the overnight culture suggesting that the Amp and Tet traits are stable.

Plasmids were isolated from these colonies. Initial characterization of the plasmids was performed using agarose gel electrophoresis. The results showed that at least one colony had a similar profile to the Tet+Amp mixed control, suggesting that the colony was co-infected by both GPs.

Example 7: Comparison of Co-infection using TGI and K91BK Host Cells

TGI and K91BK host cells were coinfected with lμl of pSRC-1 and 10 μl of 6 mer GPs. The cells were incubated for 60 min and 10° dilutions were made. A pooled control was added which included 6 mer and pSRC-1 GPs incubated separately, using both TGI and K91BK host cells then pooling samples just before plating. None of the K91BK host cell co- infections or control cultures contained growth. The results of the co-infection and controls using TGI host cells are shown in Table 7.

Table 7

# of colonies observed with TGI cells at 10° dilution

From this example, 100 colonies from both sample and control plates were sub-cultured onto a new Tet+Amp plate. Only 27 out of 100 colonies were successfully sub-cultured suggesting that the Amp and Tet traits in these 27 colonies are stable. The growth of these colonies in a second round screening suggests that the positive colonies possess Tet and Amp resistance genes. None of the colonies sub-cultured from the control plates grew.

Example 8: Effect of Initial Incubation Time using TGI Host Cells with 6mer and pSRC-1 GPs

TGI cells were coinfected using 10 μl of 6 mer and 1 μl of pSRC-1 GPs. The GPs were incubated for 10 min, 30 min and 60 min in separate tubes. 10 μL of 6 mer and 1 μl of pSRC-1 GPs were incubated separately at the above times and pooled together before plating, to serve as controls. The results of this example are shown in Table 8.

Table 8

More colonies were observed with the control in the first plating. None of the sub- cultured control colonies grew. With the co-infection samples, the 10 min incubation time produced the highest number of co-infected cells. A subculture of 100 colonies from both sample and control plates were transferred onto new Tet+Amp plates. 48 out of 100 colonies were successfully sub-cultured from a pool of the 3 conditions, thereby showing that at least some of the cells were successfully coinfected.

Example 9: Co-infection of TGI Host Cells using 6mer or fUSE5 (control) and pSRC-1 GPs TGI host cells were infected using 10 μl of pSRC-1 and 10 μl of 6 mer GPs. The cells were incubated for 10 minutes. A co-infection control was also added which included 10 μl of fuse 5 vector and 10 μl of pSRC-1 GP incubated together for 10 min. Fuse 5 is the vector used for cloning of the 6 mer library. Fuse 5 virions express the genelll protein but not the 6 mer peptide. They are used here as negative control. The results from this experiment are shown in Table 9. The results are given in number of colonies.

Table 9

Co-infection with fUSE5 control showed significantly less colonies in the first round of cultures. In this example, 100 colonies from the sample plates were sub-cultured onto a new Tet+Amp plate. Only 3 out of 100 colonies were successfully sub-cultured, indicating successful coinfection. This low number of successfully sub-cultured colonies may be due to the colonies being too crowded in the initial culture.

Example 10: Effect of NaCl on co-infection of TGI cells using pSRC-1/ 6 mer GPs

TGI host cells were coinfected using 10 μl 6 mer and 10 μl of pSRC-1 GPs. The GPs incubation time was 10 min. NaCl concentrations of OmM, 50mM, 250mM and lOOOmM were used in a volume of 100 μl as opposed to 20 μl standard used in the other examples. The cultures were not diluted. 10 μL of 6 mer and 10 μl of pSRC-1 phage were used in a co- infection served as a control. The results from this example are shown in Table 10.

Table 10

In this example, it is shown that the "optimal" salt concentration is within the range of 50-250mM NaCl. 100 colonies from each NaCl concentration, including the control were subcultured onto a new Tet+Amp plate. From the 2^nd screen, 250mM NaCl produced the highest specificity for binding of co-infected cells. EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of the present invention and are covered by the following claims. The contents of all references, issued patents, and published patent applications cited throughout this application are hereby incorporated by reference. The appropriate components, processes, and methods of those patents, applications and other documents may be selected for the present invention and embodiments thereof.

Claims

1. A method for determining compounds which interact with each other comprising: providing a first genetic package (GP), comprising a first compound associated with the surface of said first GP, and encoded in the genetic material of said first GP, which is incapable of independently replicating in a host cell, but capable of replicating in a host cell after coinfection of said host cell with a second genetic package (GP); providing said second genetic GP, comprising a second compound associated with the surface of said second GP, and encoded in the genetic material of said second GP incapable of independently replicating in a host cell, but capable of replicating in a host cell after coinfection of said host cell with said first GP; contacting said first GP and said second GP with host cells under conditions such that coinfection occurs substantially only when the surface compound of said first GP interacts with said surface compound of said second GP; allowing for replication of said GPs; and recovering said genetic material of at least one of said GPs, thereby determining compounds which interact with each other.

2. The method of claim 1, wherein the concentration of said host cells and said GPs is such that coinfection ofa cell by two GPs without interactions between respective said surface compounds is substantially close to zero.

3. The method of claim 1, further comprising obtaining a first replicated GP and a second replicated GP; contacting said replicated first GP and said replicated second GP with host cells under conditions such that coinfection occurs substantially only when the surface compound of said first replicated GP interacts with said surface compound of said second replicated GP; and allowing for replication of said replicated GPs.

4. The method of claim 1, wherein said GP is a filamentous bacteriophage.

5. The method of claim 4 wherein said filamentous bacteriophage is selected from the group consisting of M13, fd, Ω, Ifl, Ike, Zj/2, Ff, Xf, Pfl, and Pf3.

6. The method of claim 1, wherein said GP is a lambda type phage.

7. The method of claim 1, wherein at least one of said compounds associated with the surface of a GP is encoded by a surface protein gene of the respective GP.

8. The method of claim 7 wherein said surface protein gene is gene III of the filamentous bacteriophage.

9. The method of claim 7 wherein said surface protein gene is gene NIII of the filamentous bacteriophage.

10. The method of claim 7, wherein said surface protein gene further comprises at least one cleavage site.

11 The method of claim 7, wherein said surface compounds comprise a genetically diverse collection of ligand or receptor binding peptides or proteins.

12. The method of claim 7, wherein at least one of said surface compounds is selected from the group consisting of immunoglobms, antigens, cell surface receptors, cell surface receptor binding compounds, and any fragments thereof.

13. The method of claim 1, wherein the host cells are E. coli.

14. A method for detecting peptides which bind to each other comprising: providing a first population of filamentous bacteriophage comprising a diverse library of peptides displayed on the surface of said first population of filamentous bacteriophage, wherein said peptides are encoded within gene III of said first population of filamentous bacteriophage, which are only capable of replicating in a host cell through coinfection with a filamentous bacteriophage from a second population; providing said second population of filamentous bacteriophage, comprising a diverse library of peptides displayed on the surface of said second population of filamentous bacteriophage, wherein said peptides are encoded within gene III of said second population of filamentous bacteriophage, which are only capable of replicating in a host cell through coinfection with a filamentous bacteriophage from said first population;

-20-

SUBSTITUTΕ SHEET (RULE 26) combining said first and second filamentous bacteriophage populations with host cells under conditions such that coinfection substantially occurs only when a peptide displayed on the surface of said first population of filamentous bacteriophage binds to a peptide displayed on the surface of said second population of filamentous bacteriophage; allowing for replication of said filamentous bacteriophages; and recovering at least some of the genetic material of at least one of said filamentous bacteriophages, thereby detecting for peptides which bind to each other.

15. The method of claim 14, further comprising: combining replicated first and second filamentous bacteriophage populations with host cells under conditions such that coinfection substantially occurs only when a peptide displayed on the surface of said replicated first population of filamentous bacteriophage binds to a peptide displayed on the surface of said replicated second population of filamentous bacteriophage; and allowing for replication of said replicated filamentous bacteriophages.

16. The method of claim 14, wherein said library contains about 10& peptides or more.

17. A pair of GPs which individually have insufficient genetic material for replication, but complement each other such that replication may occur after coinfection of the same cell.

18. A GP of claim 17, wherein said GP displays a surface compound which is encoded in the surface protein gene of the GP.

19. The GP of claim 18, wherein said surface protein gene further comprises at least one cleavage site.

20. The GP of claim 18, wherein said surface compounds comprise a genetically diverse collection of ligand or receptor binding peptides or proteins (LBPs).

21. A GP of claim 19, wherein said surface compound is selected from the group consisting of immunoglobins, antigens, cell surface receptors, cell surface receptor binding compounds, and any fragments thereof.

22. A GP of claim 18, wherein said surface protein gene is gene III of the filamentous bacteriophage.

23. The method of claim 18, wherein said surface protein gene is gene NIII of the filamentous bacteriophage.

24. The genetic material of a GP of claim 18.

25. The surface protein encoded by said genetic material of claim 18.

26. The method of claim 14, wherein said host cells are E. coli.