ES2355973T3 - IN VITRO PEPTID EXPRESSION BANK. - Google Patents

Abstract

A method of producing an in vitro peptide expression library comprising a plurality of peptides, where each peptide is linked to the DNA construct encoding the peptide, comprising the steps of: (a) providing a DNA construct comprising: (i) a target DNA sequence; (ii) DNA encoding a peptide member of the library; and (iii) DNA encoding a peptide capable of non-covalently binding directly or indirectly to said DNA target sequence of (i); wherein said DNA construct and encoded protein are selected to have cis (b) activity expressing a plurality of DNA constructs according to (a) where said DNA constructs encode a plurality of peptide members of the library so that each peptide expressed is non-covalently bound to the DNA from which it was produced.

Description

Field of the Invention

 The present invention generally relates to recombinant DNA technology and, more specifically, to in vitro methods for constructing and scanning DNA libraries for DNA sequences encoding biologically active molecules. 5

Background of the Invention

 Isolating an unknown gene that encodes a desired peptide from a recombinant DNA library can be a difficult task. The use of hybridization probes may facilitate the procedure, but their use generally depends on the knowledge of at least a portion of the gene sequence encoding the protein. When the sequence is not known, DNA libraries can be expressed in an expression vector, and antibodies have been used to screen plaques or colonies in search of the desired protein antigen. This procedure has been useful in the screening of small libraries, but low-occurrence sequences that are represented in less than about 1 in 105 clones can be easily overlooked, as is the case with low-level cDNA or synthetic peptide molecules. appearance, making the scrutiny of libraries greater than 106 in the best of cases laborious and difficult. The scrutiny of 15 larger libraries has required the development of methods designed to direct the isolation of sparse sequences, which are based on the simultaneous selection of molecules, together with the DNAs that encode them. In vivo methods have been developed to screen large libraries, such as phage display and "plasmid peptides" using lacI peptide fusions.

 The phage display is based on DNA libraries fused to the N-terminal end of the 20 proteins of the filamentous bacteriophage envelope and its expression in a bacterial host, resulting in the presentation of foreign peptides on the phage particle surface. with the DNA encoding the fusion protein packaged in the phage particle (Smith GP, 1985, Science 228: 1315-1317). Fusion proteins incorporated into the phage can then be selected for binding members against targets of interest (ligands). The bound phage can then be allowed to re-infect 25 Escherichia coli bacteria (E. coli) and then amplify and repeat the selection, resulting in the recruitment of binding members (Parmley, SF, and Smith, GP 1988. , Gene 73: 305-318; Barrett RW et al., 1992, Analytical Biochemistry 204: 357-364 Williamson et al., Proc. Natl. Acad. Sci. USA, 90: 4141-4145; Marks et al., 1991 , J. Mol. Biol. 222: 581-597).

 The presentation in lacI fusion plasmids is based on the DNA binding capacity of the lac 30 repressor. The random peptide libraries are fused to the C-terminal end of the lacI repressor protein. The binding of the LacI-peptide fusion to its coding DNA occurs through the lacO sequences of the plasmid, forming a stable peptide-LacI-peptide complex. These complexes are released from their host bacteria by cell lysis, and the peptides of interest are isolated by affinity purification on an immobilized receptor target. Plasmids thus isolated can be re-introduced into E. coli 35 by electroporation to amplify the selected population for additional rounds of screening (Cull, MG et al. 1992. Proc. Natl. Acad. Sci. USA 89: 1865- 1869).

 These bacterial methods are limited by the size of the library that can be created by current methods of introducing DNA into the host bacterium, the potential cellular toxicity of the expressed expressed peptides, and by the inability to introduce post-40 translational modifications, or to incorporate novel amino acids into the expressed peptide.

 A completely in vitro ribosome system based on the connection of peptides to the RNA encoding them by the ribosome has been described (document W091 / 05058). Ribosome presentation has also been used for the selection of single chain Fv antibody fragments (scFv) (Matheakis, LC et al., 1994 Proc. Natl. Acad. Sci. USA, 91: 9022-9026; Hanes, J and Pluckthun, A. 1997 Proc. 45 Natl. Acad. Sci. USA, 94: 4937-4942). This method suffers from a lower stability of the RNA genetic material and an increase in degradation under certain selection conditions in which the RNAse is likely to be present.

 The in vitro method described by Griffiths and Tawfik (WO 99/02671 and WO 00/40712) addresses some of these issues by compartmentalizing DNA before the expression of the peptides, which then modify the DNA in the compartment. The peptides susceptible to modifications, resulting from the enzymatic activity of interest, are then selected at a later stage. However, it is not possible to directly select the peptide binding activity of both the peptide and the DNA without modification of the DNA encoding that peptide, and by releasing the modified DNA from the compartment.

 Another prior art method, covalent presentation technology, or CDT, is described in document W09S37186. This method is based on the covalent bonding of the protein to the DNA to preserve the genotype's connection with the phenotype, by means of the cis action of the cross-linking protein. This method illustrates that two requirements are necessary for a successful use of this technique. First, it

they require proteins that interact in vitro with the DNA sequence that encodes them (cis action), and secondly, said proteins must establish a covalent bond with their own template DNA. This method suffers from the fact that the DNA is chemically modified, which can prevent the recovery and identification of the binding peptide of interest.

 There is still a need for more versatile in vitro methods to construct peptide libraries 5 in addition to the methods described above, which may allow direct selection of binding activity, as well as enzymatic activity, and that allow efficient production of peptide structures. complex, while still allowing the recovery of intact genetic material encoding the peptide of interest.

Compendium of the invention

 The present invention therefore provides a method for producing an in vitro peptide expression library 10 comprising a plurality of peptides, where each peptide is connected to a DNA construct encoding the peptide, comprising the steps of:

 (a) provide a DNA construct comprising:

  (i) a DNA target sequence;

  (ii) DNA encoding a peptide member of the library; and 15

  (iii) DNA encoding a peptide capable of non-covalently binding

     directly or indirectly to said DNA target sequence

     of (ii);

  where said DNA construct and encoded protein are selected

to have cis activity; twenty

 (b) express a plurality of DNA constructs according to (a),

wherein said DNA constructs encode a plurality of peptide members of the library so that each expressed peptide is non-covalently bound to the DNA from which it was produced.

 A method is also provided for producing an in vitro peptide expression library 25 comprising a plurality of peptides, where each peptide is connected to the DNA construct encoding the peptide, which comprises the steps of:

 (a) provide a DNA construct comprising:

  (i) DNA encoding a peptide member of the library; Y

  (ii) DNA encoding a peptide capable of non-covalently binding to 30

     an agent;

  where said DNA construct and encoded protein are selected

to have cis activity;

 (b) binding said agent or a DNA tag capable of binding an agent to

said DNA construct of (a), wherein said agent is capable of binding to the peptide encoded by said DNA of (ii); Y

  (c) express a plurality of DNA constructs according to (b),

wherein said DNA constructs encode a plurality of peptide members of the library so that each expressed peptide is connected by said agent to the DNA from which it was produced. 40

 The present invention is extended to the peptide libraries produced by such methods and to the DNA constructs used in such methods.

 The present invention also provides screening methods for in vitro peptide expression libraries of the invention. In one aspect there is provided a method of identification and / or purification of a peptide that shows the desired properties from an in vitro peptide expression library 45 produced in accordance with the method of any one of the preceding claims, comprising

minus the stages of (a) scrutinizing said library and (b) selecting and isolating the relevant member of the library. In a second aspect, a method is provided for identifying a specific ligand binding peptide, said method comprising at least the steps of (a) screening an in vitro peptide expression library produced in accordance with the method of the invention with molecules of ligand that are optionally bound to a solid support; (b) select and isolate a member of the library that binds to said target molecule; and (c) isolating the peptide that specifically binds to said target molecule. In a third aspect there is provided a method of identification and / or purification of a peptide that has the ability to bind to a specific DNA target sequence comprising at least the steps of (a) providing an in vitro expression library in accordance with the invention wherein said peptide or protein of (iii) is a member peptide of the library that has DNA binding activity and wherein said DNA target sequence of (i) is the target sequence of interest; (b) selecting and isolating a member of the library in which the encoded protein binds to said target sequence; (c) isolate the peptide that binds to said target sequence.

 In addition to isolating and / or identifying the specific peptides of the libraries of the invention, the screening methods of the invention can be used to isolate and / or identify the DNA encoding the specific peptides from the library. fifteen

Brief Description of the Figures

 Figure 1 provides a schematic representation of a method by which a DNA construct of the invention can be connected to the peptide encoding it.

 Figure 2 provides a schematic representation of a method of the invention whereby the DNA binding protein can be converted into a DNA binding protein that acts in cis. twenty

 Figure 3 provides a schematic representation of how a DNA binding protein specific to a target sequence can be isolated from a library of the invention.

 Figure 4 provides a schematic representation of how a library protein can be connected to its coding DNA through cis action and the use of a bi-specific binding molecule. 25

 Figure 5 demonstrates the cis: 1: 1 mixture of two different size input DNA (CK-RepA or V5-RepA) selected against any antibody. 1-DNA Marker; 2-PCR amplification after selection on human anti-CK antibody; 3-PCR amplification after selection on an anti-V5 peptide antibody.

 Figure 6 shows the specificity of the anti-V5 antibody binding clones. ELISA screening, read at 450 nM, of the seven clones (1-7) showing specific binding to the anti-V5 antibody. The bars of the group of four represent the ELISA signal of the clones screened from left to right; anti-human kappa region antibody, anti-V5 antibody, BSA, and white. There is also a negative control that does not express CK or V5 (8).

 Figure 7 shows the signals at an OD at 450 nm of the ELISA of the culture supernatant for the 35 peptides recovered after 5 rounds of selection against B. globigii spores in Example 4. A. = clone 1e; B. = clone 1f; C. = clone 1g; D. = clone 8a; E. = clone 10c; F. = clone 10e; G. = negative control.

 Figure 8 shows the OD signals at 450 nm for isolated peptides after 4 rounds of selection against anti-V5 antibody in Example 5. A. = P1C12; B. = P2H1; C. = P1B5; D. = P2B8. Phage were tested with peptides against anti-V5 and anti-ACTH peptide antibodies. 40

 Figure 9 shows signals at OD 450 nm for synthetic peptides isolated after 4 rounds of selection against ovalbumin. A. = C1; B. = C4; C. = C6; D. = C8; E. = negative control. The peptides were tested against ovalbumin, anti-V5 antibody and blocked plate (plastic).

 Figure 10 shows the PCR recoveries of scFv DNA after selection on BSA or BSA-mecoprop. A. scFv anti-mecoprop selected on BSA, 2.5 mM ox-glutathione. B. scFv anti-mecoprop 45 selected on mecoprop-BSA, 2.5 mM ox-glutathione. C. scFv anti-mecoprop selected on BSA, not ox-glutathione. D. scFv anti-mecoprop selected on mecoprop-BSA, not ox-glutathione.

Brief Description of the Sequences

 SEQ ID No 1 to 11, 19 to 23, 26 to 35 and 45 to 47 show the primers used in the Examples.

 SEQ ID NO: 12 shows the sequence of the TAC-MYC-CK-REPA-CIS-ORI construct, SEQ ID NO: 50 13 shows the sequence of the TAC-MYC-V5-REPA-CIS-ORI construct, SEQ ID NO: 24 shows the sequence of the TAC-V5-REPA-CIS-ORI-408 construct and SEQ ID NO: 25 shows the sequence of the TAC-NNB-REPA-CIS-ORI-408 construct.

 SEQ ID NO: 14 shows the recognition sequence of the estrogen receptor target.

 SEQ ID NO: 15 and 16 show the DNA and amino acid sequences of the repA gene from plasmid R1 of the incFII incompatibility group. SEQ ID Nos. 17 and 18 show the sequences of the CIS DNA element and the ori sequence of the same system.

 SEQ ID Nos. 36 to 39 show the sequences of isolated peptides after the selection of Example 5. SEQ ID Nos. 39 to 43 show the sequences of the isolated clones in Example 6. 5

Detailed description of the invention

 The present invention refers to the construction and screening of a library in search of a nucleotide sequence encoding a peptide of interest in vitro. Constructs encoding the peptide of interest are designed so that the expressed peptide shows cis activity for the construct. Cis activity is defined as the ability of the peptide to bind to the DNA from which the peptide was produced, that is from which it was transcribed and translated. In vitro expression of the construct results in the binding of the peptide to the DNA encoding that same peptide molecule through a non-covalent interaction. This differs from that illustrated in WO 98/37186, which does not have the possibility of in vitro non-covalent interaction between the protein and the DNA that encodes it, and specifically excludes in fact that such interactions have a practical use for screening. from the library. fifteen

 Non-covalent binding refers to an association that can be broken by methods well known to those skilled in the art, such as the addition of an appropriate solvent, or a change in ionic conditions, for example, the addition of pH glycine. low or high pH triethylamine. In the present case, a typical example of non-covalent binding would be the non-covalent interaction between a DNA binding protein and a DNA molecule. On the contrary, when a covalent bond is formed between the DNA and the encoded polypeptide, the presented peptide or protein will not be released from the DNA by ionic conditions and solvents that would break the DNA-binding protein: non-covalent DNA interactions. For example, the repA bacterial replication protein binds non-covalently to its oriR target DNA sequence and can be released from this target DNA sequence at salt concentrations greater than 0.2 M KCl (Giraldo R. & Diaz R. 1992 J. Mol. Biol. 228: 787-802). This salt concentration would not affect a covalent bond, which would require much more stringent conditions to release the covalent bound protein, with a higher risk of damaging the recovered DNA.

 The present invention describes cis activity and non-covalent binding that allows the encoded peptide or protein to remain associated with the DNA construct with a sufficient half-life to allow individual peptides and the associated DNA encoding that peptide with an activity of interest 30 be separated from the resulting mixture of protein-DNA complexes. For example, the association between the encoded protein and its DNA can have a half-life of up to 30 minutes, up to 45 minutes, up to one hour, up to 2 hours, up to 6 hours or up to 12 hours. The screening methods of the invention can be carried out immediately after the construction of the library, or later, for example up to one, up to two, up to six, up to twelve hours or up to twenty four hours or more than twenty four hours later . 35

 Surprisingly, therefore, the invention described herein demonstrates that such peptides or encoded proteins can be expressed in vitro and bound to the DNA encoding that peptide in the presence of other DNA sequences. The invention also demonstrates that the covalent connection between the protein and the DNA is not required to maintain said cis activity, and that a non-covalent interaction between DNA and binding protein is sufficient to allow the selection of peptides in an expression system and in vitro selection.

 In accordance with the present invention, the individual DNA library members, each of which encodes a peptide to be expressed in the peptide expression library (library member peptides), are placed in a construct of Adequate DNA. The DNA construct in which the DNA library member is placed includes all the sequences necessary to allow expression of the library member peptide from the construct and to allow the peptide encoded by the construct to bind to the construct of DNA that encoded it. Each peptide of the library will typically comprise a fusion protein comprising the member peptide of the fused library to a peptide involved in binding the fusion protein to the relevant DNA construct. Such fusion proteins may comprise additional sequences and said library peptide may be linked to said binding peptide 50 by means of a linker sequence.

 A plurality of such constructs, each encoding a plurality of different peptide members of the library, form a DNA library of the invention. The expression of such a library of DNA molecules results in the non-covalent binding of the individual encoded proteins to the DNA that encodes them and from which they have been transcribed and translated, in the presence of many other DNA molecules that encode other members of The library. The sequence encoding the member of the peptide library present in a particular protein will therefore be present in the DNA that binds to that protein. Therefore this procedure binds the peptide member of the library, in a biologically active form (usually with binding activity) to the DNA sequence member of the specific library encoding

that peptide, allowing the selection of peptides of interest, for example due to a specific binding activity, and the subsequent isolation and identification of the DNA encoding that peptide member of the library.

 For the purposes of the invention a DNA library is therefore a population of DNA constructs. Each construct comprises a DNA sequence that encodes a peptide that is to be expressed as a member peptide of a library and each contains an appropriate promoter, initiation signals and translation termination. A DNA library of the invention will contain a plurality of such DNA molecules. A plurality of DNA constructs each encoding a library member peptide is provided to provide a plurality of different library members. Preferably a DNA library will contain at least 104 discrete DNA molecules. For example, a DNA library can contain more than 106, more than 108, more than 1010, more than 1012 or more than 1014 discrete DNA molecules.

 A peptide expression library is defined as a population of peptide sequences expressed from a library of DNA molecules. A peptide expression library of the present invention therefore encompasses a library of peptides that are not covalently linked to the DNA encoding them. For example, a peptide expression library of the present invention may be a library of at least 104 discrete proteins each comprising a peptide sequence of the library, expressed from a library of DNA molecules. A peptide expression library of the invention can be any library formed by the expression of a DNA library of the present invention.

 A member of a peptide library can be defined as an amino acid chain of variable composition of at least two amino acids in length, or a part or all of a naturally occurring protein such as an enzyme, a binding molecule such as a receptor or an antibody or one of its fragments. A member of a suitable peptide library can be a peptide having a random amino acid composition. The random or variable composition peptide may be flanked by known amino acid sequences at the N and / or C end to constrict the structure. These 25 known sequences may have a variable length. The peptide of varying or random composition can be inserted in different positions in a framework of known protein, such as a receptor or antibody or another protein or one of its fragments. The peptide can be inserted into the same protein shell once or more than once, for example two or more times.

 A DNA construct according to the present invention may comprise DNA encoding a member peptide of the library and the means for the encoded peptide to bind to the coding DNA construct. In addition to the DNA encoding a peptide member of the library, a suitable DNA construct of the invention comprises at least one DNA and DNA target sequence encoding a peptide capable of directly or indirectly binding to the DNA target sequence.

 In accordance with the present invention, the DNA construct and the encoded protein are selected to have cis activity. That is, the encoded protein has the ability to specifically bind to the DNA molecule that encodes it. For example, cis activity can work by allowing the encoded DNA binding peptide to specifically (directly or indirectly) bind to the target sequence of the DNA construct that encoded it instead of the target sequence of another DNA construct.

 In some cases, cis activity may be provided due to the presence of a DNA element that directs the cis activity, that is, that allows or forces the protein encoded by the DNA construct to have cis activity, and therefore joins its coding sequence. In other cases, a DNA element separated per se may not be necessary when the nature of the coding DNA inherently confers cis activity to the encoded peptide.

 A DNA element that directs cis activity can be provided in the DNA construct 45 together with the DNA encoding a peptide that interacts with that cis element. For example, in the case of the cis element of the repA system discussed below, the DNA encoding a fragment of the repA sequence comprising at least 20 amino acids of the C-terminus of repA can be provided together with the cis DNA element. It may be possible to confer cis activity to a DNA binding peptide that normally does not act in cis including in the DNA construct such a DNA element and any additional sequence necessary for its action. For example, DNA encoding a peptide that interacts with the cis element used can be included in the DNA construct.

 Alternatively, a peptide that interacts with the cis element can be part of the DNA binding peptide. For example, the DNA binding peptide may be repA comprising the sequence that interacts with the cis element of repA. Alternatively, the DNA binding peptide can bind to its cis-encoding DNA without the need for a separate cis element.

 A suitable DNA element can be any element that allows or directs cis activity. Such a DNA element can act, for example, by interacting with the machinery involved in the translation and transcription of the DNA construct to delay the production and release of the encoded peptide.

 Any DNA element that allows the encoded peptide to specifically bind to the DNA molecule that encoded it can be used as a DNA element according to the present invention. An example of a suitable DNA element is that of a repA-cis system described in more detail below. In that system, RNA polymerase is stopped by loops in the 5 'cis sequence before the termination of rho-dependent transcription. The action of the DNA element therefore allows the encoded binding peptide to bind to the target DNA sequence in the construct from which it was produced.

 Preferably, the cis DNA element will be located 3 'in the DNA construct with respect to the member peptide of the library and with respect to the peptide or protein capable of binding to the target DNA sequence. This means that these sequences can be transcribed and translated before the RNA polymerase reaches the cis-acting sequence. 10

 In accordance with the present invention, the binding peptide can be connected to the DNA construct directly or indirectly. In the case of direct binding, the binding peptide binds directly and not covalently to the DNA target sequence. In the case of indirect binding, the connection between the binding peptide and the DNA construct is provided by an additional molecule. Such a molecule, for example a bifunctional agent as described below, will be associated with both the peptide and the DNA target sequence.

 A suitable DNA construct may comprise additional sequences, for example promoter sequences suitable to allow expression of the encoded peptide.

 An example of a system in which cis activity exists is the plasmid replication protein system of the incompatibility group that act in cis, called repA. Aspects of this system can be used in the present invention as explained below.

 Numerous plasmids include sequences encoding repA and DNA elements in cis. The repA sequence and the cis DNA element present a DNA construct of the invention can be obtained from the same plasmid strain or can be obtained from different plasmid strains.

 It is believed that the repA-cis system acts as shown in Figure 1. In summary, RNA 25 polymerase is stopped by loops in the 5'-CIS sequence before the termination of rho-dependent transcription. This allows the transient interaction of the C-terminal repA peptide with CIS, and possibly DNA bending. The RepA peptide is then linked to ori, which is at a defined distance from the terminal amino acid of the repA coding sequence (Prazkier et al. 2000 J. Bacteriology 182: 3972-3980; Praszkier and Pittard 1999 J. Bacteriol. 181: 2765 -2772; Masai and Arai. 1988 Nucleic Acids Res. 16: 6493-6514). 30

 The compatibility of a RepA sequence of a plasmid with a cis sequence of another plasmid can be easily determined by controlling the interaction of RepA with the selected cis sequence.

 Suitable repA proteins and cis DNA elements and sequences include those of the IncI complex plasmids or IncF, IncB, IncK, IncZ and IncL / M plasmids, which are distantly related at the DNA level, but which control replication. plasmid through the action of the 35 repA protein that acts in cis (Nikoletti et al. 1986 J. Bacteriol. 170: 1311-1318; Prazkier J. et al. 1991 J. Bacteriol. 173: 2393-2397). Specific plasmids that can be used to provide these sequences include the Rl plasmid of the IncII incompatibility group and the incB plasmid pMU720 (described by Praskier J. & Pittard J. 1999 Role of CIS in replication of an IncB plasmid. J. Bacteriol. 181: 2765-2772). The DNA and amino acid sequences of repA derived from plasmid R1 of IncII are given in SEQ ID NO: 15 and 16. The DNA sequence of this cis DNA element of plasmid R1 of IncIl is given in SEQ ID NO : 17. Typically, the cis element is 150 to 200 nucleotides in length. Shorter or longer sequences can be used, as long as the sequence retains the ability to interact with RepA and display cis activity. Minor variations are also contemplated, such as substitutions or deletions in the cis sequence such as modifications in 1, 5, 10 to 20 nucleotides in the wild type cis sequence. Four. Five

 The cis element is necessary for the cis activity of the repA protein (Praszkier and Pittard 1999 J. Bacteriol. 181: 2765-2772). The cis DNA element must therefore also be located 3 'in the DNA construct with respect to the DNA encoding the repA sequence. Upon reaching this cis sequence, RNA polymerase will be stopped, allowing the encoded protein to bind to the target DNA sequence.

 In one embodiment of the present invention, the DNA binding protein itself comprises RepA or one of its fragments or variants capable of binding to DNA, including at least 20 C-terminal amino acids of RepA capable of binding to the cis DNA element. In this embodiment, the DNA target sequence comprises an ori sequence, for example the oriR sequence. In alternative aspects of the present invention, the DNA binding protein is provided by an alternative protein with the relevant DNA target sequences recognized by such binding protein incorporated into the sequence. In each of these embodiments, the DNA-protein binding is direct since the peptide encoded by the DNA construct will bind directly to the DNA construct encoding. In alternative aspects of the invention, as

described in more detail below, the DNA-protein binding can be indirect through the use of a peptide-DNA tag label, bifunctional agent and / or suitable linkers.

 In one aspect, the same sequence can therefore provide both the peptide capable of binding to the DNA target sequence and the C-terminal amino acids of repA. Such a sequence may be or may comprise a complete repA sequence, or a fragment or variant of a repA sequence that retains the ability to bind to the DNA target sequence used. When repA acts as a DNA binding protein, both cis and ori sequences (Praszkier and Pittard 1999 J. Bacteriol. 181: 2765-2772) are required for cis (cis) activity and DNA binding (ori). In this respect, therefore, the DNA target sequence is an ori sequence and the peptide or protein capable of binding to said target is a repA protein. The position of ori in the DNA constructs of the invention may vary. As described above, suitable repA, 10 cis and ori sequences can be provided by one or more plasmids. For example, suitable sequences can be provided from complex IncI plasmids or IncF, IncB, IncK, IncZ and IncL / M plasmids. The DNA sequence of the ori from the RI plasmid of IncII is provided in SEQ ID NO: 18. This sequence, or one of its fragments can be included in a DNA construct of the invention. A DNA construct of the invention may include a complete ori sequence or may include one of its 15 fragments that is capable of being bound by the repA protein being used.

 The RepA protein used in accordance with the present invention may also comprise a fragment or variant of RepA, provided such a variant or fragment of RepA retains the ability to bind to the selected ori sequence. Such a variant or fragment of RepA may include substitutions, for example, in 1, 2, 3 to 20 amino acids in the RepA sequence as long as such variants retain the ability to bind to the ori sequence. A suitable fragment of RepA is an Ori binding sequence of RepA. Ori sequences include those that are present in the wild type plasmids described above. Typically, such an ori sequence is 170 to 220 nucleotides in length. Fragments and variants of wild-type ori sequences can also be used, provided that such ori sequences retain the ability to be recognized by RepA. Additionally, the 25 cis-acting members of the RepA protein family can be used. For example, the RepA family of proteins is found in plasmids with a wide range of hosts, that is, a RepA plasmid can be found in different bacterial species. Isolation of a plasmid from the repA family (for example) from a thermophilic, sulphophilic, halophilic or acidophilic bacteria would provide repA-cis-ori DNA that could be used in the present invention at elevated temperatures or extreme salt concentrations, pH or sulfur. Such 30 members of the RepA family would be advantageous in isolating members of the library that can bind to target molecules under such extreme conditions. Such ori sequences suitable for use in combination with selected RepA proteins can be readily determined by controlling the interaction of RepA with said ori sequence.

 The basic principle of the invention can therefore be described with reference to the repA / cis / ori system, as shown in Figure 1. This shows an example of a DNA construct of the invention. This construct comprises, from 5 'to 3', a promoter sequence, a sequence encoding a member peptide of the library, a sequence encoding a repA protein, a cis DNA element and an ori sequence. In summary, the DNA sequence is transcribed from the promoter by means of RNA polymerase to RNA. The rho-dependent termination function present in the cis DNA element causes the RNA polymerase to stop at this step in the sequence. This allows the repA protein and the peptide library to be translated. Then the repA protein is able to bind to the ori sequence, connecting the encoded protein to the coding DNA construct.

 In a preferred embodiment, the library DNA member sequences are fused to the repA, cis and ori DNA of the IncFII Rl plasmid (Masai H et al. 1983 Proc Natl Acad Sci USA 80: 6814-6818). In this embodiment, the DNA sequences members of the library of interest may be linked by a region of DNA encoding a flexible amino acid linker, to the 5 'end of the repA DNA, under the control of an appropriate promoter and sequences of translation for transcription / translation in vitro. Many suitable promoters are known to those skilled in the art, such as araB, the tac promoter and promoters of T7, T3 or SP6, among others. The promoter must be upstream of the polypeptide sequence to be expressed.

 The repA family of proteins is used herein by way of example, not limitation. Other DNA-binding proteins that act in cis that bind non-covalently unbound could be used in this invention.

 In a further embodiment, DNA-binding proteins that do not act in cis can be converted to have cis activity (see Figure 2). This can be achieved using such proteins, capable of binding to the DNA target sequence, either directly or indirectly, combined with sequences that can confer cis activity thereto. The cis activity can be conferred on a binding protein that normally does not act in cis including in the DNA construct a DNA element that directs the cis activity such as the cis element of the repA system. Such an element can be included to ensure that DNA binding by DNA binding protein is cis, that is, an encoded DNA binding protein will bind to the DNA construct from which it has been transcribed and translated.

 In one embodiment, a suitable DNA construct may therefore comprise the DNA element that directs the cis activity (the cis DNA element) of the repA system. Such an element may additionally comprise DNA encoding a portion of the C-terminal end of RepA, preferably at least 20 amino acids, more preferably 30 amino acids, up to 40, 50, 60 or 70 amino acids of the C-terminal portion of repA, wherein said fragment RepA is able to interact with the DNA element of the construct. In a further example, proteins such as transposases acting on cis, Tn5 and IS903, among others, could be used in the present invention (McFall E. J Bacteriol. 1986 Aug 167: 429-432; Derbyshire KM & GrindleyND. Mol Microbiol. 1996 Sep 1: 1261-72.). The DNA coding sequences of the present invention may comprise wild-type sequences encoding the desired RepA fragment, degenerate sequences encoding wild-type RepA fragments or sequences encoding 10 variants of such RepA fragments that retain the ability to interact. with the cis element incorporated in the DNA construct. Such variants may include the substitution of 1, 2, 3 or 4 amino acids of the 20 amino acids of the C-terminal end of RepA.

 The repA family of proteins is used herein by way of example, not limitation. Any DNA element capable of conferring cis activity to a protein that does not act in 15 cis could be used.

 In this way any protein that does not act in cis can be converted. By way of example, not exclusive, the estrogen receptor DNA binding domain (DBD) can be converted into a cis-binding DNA binding protein. The estrogen receptor DNA binding domain fragment (amino acids 176-282) has been expressed in E. coli and has been shown to bind to the 20 nucleotide sequence HRE target of the specific double stranded DNA estrogen receptor , with a similar affinity (0.5 nM) to that of the parental molecule (Murdoch et al. 1990, Biochemistry 29: 8377-8385; Mader et al., 1993, DNAs Research 21: 1125-1132). In one embodiment, the DNA encoding this sequence is fused, preferably at the 3 'end, to the DNA encoding at least the last 20 amino acids of repA, the cis DNA element, and the DNA to the sequence ori followed by the sequence recognition target of the estrogen receptor (5'-TCAGG TCAGA GTGAC CTGAG CTAAA ATAAC ACATT CAG-3 ', SEQ ID NO: 14) that replaces the repAori recognition sequence. The DNA sequences of interest can then be spliced by means of a DNA region encoding a flexible amino acid linker, at the 5 'end of the estrogen receptor DNA fragment, under the control of an appropriate promoter and translation sequences for In vitro transcription / translation. The expression of this polypeptide directs the 30 DBD of the estrogen receptor to its target sequence, present instead of the normal ori sequence, in the DNA encoding that polypeptide. The protein-DNA complexes can then be isolated by capture on a target protein. Unbound protein-DNA complexes can be washed, allowing enrichment in DNA encoding the peptides or proteins of interest, which can be recovered by PCR, and further enriched by performing several additional cycles of in vitro expression and capture of complex 35. protein-DNA using the methods described previously.

 It will be clear that this approach will be applied to other DNA binding proteins simply using the cis DNA element and a sequence encoding at least the 20 C-terminal amino acids of repA, or equivalent elements of a different cis-acting system in the DNA constructs

 In another embodiment, libraries of randomized DNA binding proteins, such as zinc finger proteins 40, helix-loop-helix proteins or helix-spin-helix proteins by way of example, can be screened for binding specific to a target sequence of interest (see Figure 3). In this embodiment, the ori recognition sequence of repA can be replaced by a target sequence of interest, and the majority of the repA coding sequence by a randomized zinc finger protein library. The DNA binding proteins thus act as peptide members of the library and 45 as proteins capable of binding to the DNA target sequence in this regard. The DNA encoding each zinc finger protein can be additionally attached, at the 5 'end, to a sequence of a peptide tag that can be recognized by another capture protein such as an antibody, and at the 3' end, to the DNA encoding at least the last 20 amino acids of repA, the cis DNA element, and the DNA to the ori sequence followed by the target sequence of interest. The expression of this polypeptide directs the zinc finger protein to the target sequence of interest, present instead of the normal ori sequence, in the DNA encoding that polypeptide. Binding to the target sequence will only occur if the randomized zinc finger domain is capable of binding to the sequence of interest. The protein-DNA complexes can then be isolated by capture with a binding protein that recognizes the peptide tag at the N-terminus of the polypeptide-fusion protein. Unbound DNA can be washed, allowing enrichment in DNA encoding zinc finger proteins capable of binding to the target sequence, which can be recovered by PCR, and further enriched by performing several additional cycles of in vitro expression and capture. of the protein-DNA complex.

 As explained above, the binding peptide can be directly linked to the DNA target sequence, for example in the case of a DNA-binding protein-target sequence pair, or can be indirectly linked to the target sequence. of DNA, for example by means of a bifunctional agent and optionally a DNA tag (see Figure 4).

 In one embodiment, the DNA encoding a peptide tag that is not capable of binding directly to the DNA target sequence is spliced to the 5 'end of the sequence or member sequences of the libraries of interest, optionally via a region of DNA encoding a flexible amino acid linker, under the control of an appropriate promoter and translation sequences for in vitro transcription / translation. This forms the DNA encoding the binding peptide, since the encoded peptide 5 is indirectly connected to the DNA target sequence. Optionally at the 3 'end of the DNA member sequence of the library is the DNA encoding at least the last 20 amino acids of repA and the cis DNA element, but not the target sequence ori of repA. The DNA target sequence can be or can comprise a DNA tag. Such a DNA tag can be a single modified base. For example, when the DNA construct of the library containing the elements described is prepared, the DNA can be labeled at the 3 'end, by way of example but not limitation, with molecules such as fluorescein or biotin.

 Before in vitro expression, the DNA fragments of the library can be mixed with a bifunctional agent, one of whose functions is to recognize and bind to the target sequence that may be at the 5 'end of the DNA, at a rate of one DNA fragment: a bifunctional molecule. The other functional element 15 of this bifunctional agent is a binding agent that can recognize and bind to the peptide tag that can be encoded at the 5 'end of the DNA fragment. By way of non-exclusive example, the bifunctional agent may be composed of a Fab fragment that recognizes the fluorescein or biotin tag of DNA, and another Fab fragment that recognizes the peptide tag encoded in the DNA. It is clear to those skilled in the art that this bifunctional agent can be made by many different methods such as chemical cross-linking of the two elements, or expression of the two elements as a fusion protein, or as a bi-specific antibody. Such methods for creating a bifunctional agent are provided by way of non-exclusive example.

 The bifunctional agent can be attached to the DNA construct before expression of the encoded peptide or can be provided during expression. 25

 The fusion protein is transcribed and then translated from the DNA construct while binding to the bifunctional agent. The peptide tag is translated first, and can be linked by the second element of the bifunctional agent, before the release of messenger RNA or RNA polymerase from DNA. This creates a functional protein-DNA complex where both the expressed polypeptide and the DNA encoding that peptide are connected through the bifunctional agent. The peptide tag molecule is therefore indirectly connected, but specifically, to the DNA target (tag). By connecting the protein to the DNA construct in this way, it is possible to screen for a protein that has specific properties, as described below, and then identify the coding DNA that is connected to that protein. Using a bifunctional agent instead of the covalent bond between the protein and the DNA, the DNA construct can be more easily separated from the protein without the risk of damaging the DNA. 35

 The protein-DNA complexes can then be isolated by capturing a target protein. Unbound DNA-protein complexes can be washed, allowing enrichment in DNA encoding peptides or proteins of interest, which can then be recovered by PCR, and further enriched by performing several additional cycles of in vitro expression and protein complex capture. -DNA using the methods described previously. 40

 Additionally, in this embodiment, the DNA can be directly linked, for example by covalent binding, to a bifunctional agent such as a polymer. Such a polymer may contain more than one binding element that could recognize the peptide tag, allowing multivalent presentation of a molecule from the peptide expression library in a unit containing the DNA encoding the presented peptide. By way of example, not limitation, said polymers may be composed of polyethylene 45 as well as other polymeric compounds, capable of being fused to DNA. The DNA construct of the invention can be provided attached to said bifunctional agent, or attached to a DNA tag as described above that is capable of being bound by such a bifunctional agent.

 In all embodiments of the invention, DNA constructs include an appropriate promoter and translation sequences for in vitro transcription / translation. Any suitable promoter 50 can be used, such as the ara B, tac, or T7, T3 or SP6 promoters among others. The promoter is positioned so that it is operably connected to the DNA sequences of the invention so that such sequences are expressed.

 The DNA encoding the peptide members of the library can be produced by any available method. In particular, such DNA can comprise DNA isolated from cDNA, obtained by shuffling DNA, and synthetic DNA.

 The DNA construct can also encode amino acid connectors in the expressed fusion protein. In particular, a flexible amino acid linker can be included to splice the DNA / RepA binding peptide to the library member peptide.

 According to the invention, with reference to this preferred embodiment, the peptide or protein expression libraries, connected to the DNA encoding them, can be generated and the peptides with the desired activity selected by means of the following steps:

Construction of a fusion protein library.

 A DNA library of peptides or proteins can be fused to a DNA encoding a peptide 5 capable of binding to the DNA target sequence, such as a DNA of a cis-binding DNA-binding protein, through a region of DNA encoding a flexible amino acid linker, under the control of an appropriate promoter and with a translation site, or ribosome binding, initiation and termination codons, in a manner suitable for in vitro expression of the members of the library of peptides and binding proteins. In the example of the repA protein, the DNA (members of the DNA library (such as DNA) 10 are fused to the DNA of the repA DNA binding protein, or one of its fragments. The cis and ori sequences they can be included in the construct downstream of the other elements.In the case of a DNA library, such constructs are designed to be suitable for transcription and translation in vitro.

Expression and cis binding of DNA library fusion proteins

 In order to allow cis activity, a coupled bacterial transcription / translation environment 15 such as the S30 extract system (Zubay, G. 1973. Arm. Rev. Genet. 7: 267) can be used. Expression of the peptide, such as the peptide fusion protein member of the DNA-repA library, in this environment, will result in the binding of the fusion protein to the DNA encoding that fusion protein, provided that both cis sequences and ori be present. When the peptide-repA fusion protein libraries are expressed in this manner, this procedure results in the production of libraries of protein-DNA complexes where the DNA-anchored protein is encoded by that DNA fragment from which It was expressed, thereby allowing the subsequent selection of both peptides or proteins of interest, and the DNA encoding said peptides. The complexity of these libraries increases due to the in vitro nature of the method, libraries of at least 1010-1014 DNA fragments can be easily generated, if not even larger libraries. 25

 Compounds that prevent nuclease activity, or reduce non-specific DNA-protein or protein-protein interactions can be added during this transcription / translation reaction and cis binding. Examples of suitable compounds include detergents and blocking agents such as bovine serum albumin (BSA).

Selection of the peptide of interest. 30

 An in vitro peptide expression library produced by a method of the present invention can be used to screen specific members of the library. For example, the library can be screened for peptides with a specific activity or a specific binding affinity. The protein-DNA complexes of interest can be selected from a library, for example, by means of affinity or activity enrichment techniques. This can be achieved by means of a specific ligand for the protein of interest, such as an antigen if the protein of interest is an antibody. The ligand can be presented on a solid surface such as the surface of a well of an ELISA plate, or in solution, for example, with biotinylated ligand followed by capture on a surface coated with streptavidin or magnetic beads, after having incubated a library of protein-DNA complexes with the ligand to allow ligand-ligand interaction. After solid phase or solution incubation, unbound complexes are removed by washing, and bound complexes are isolated by disrupting ligand-ligand interactions by altering the pH of the well, or by other methods known to those skilled in the art. such as digestion with proteases, or release of DNA directly from the complexes by heating or extraction with phenol chloroform to denature the repA-DNA ori junction. The DNA can also be released by one of the above methods, directly in PCR buffer, and amplified. Alternatively, the DNA can be amplified by PCR directly, without complex release. Optionally, unbound DNA, for example by the repA binding protein, can be protected from degradation by non-specific DNA binding proteins such as histones, by way of example. It will be apparent to one skilled in the art that other non-specific DNA binding proteins could be used for this purpose. In addition, compounds that prevent nuclease activity, or reduce non-specific DNA-protein or protein-protein interactions may be present during the selection procedure. Examples of suitable compounds include detergents, blocking proteins such as those found in powdered milk or bovine serum albumin (BSA), heparin or aurintricarboxylic acid.

 The recovery of bound complexes, the re-amplification of the bound DNA, and the repetition of the selection procedure provide enrichment of the clones encoding the desired sequences, which can be isolated for sequencing, additional cloning and / or expression. . For example, the DNA encoding the peptide of interest can be isolated and amplified, for example, by PCR. In one embodiment, repeated rounds of DNA selection and recovery can be facilitated by the use of sequential nesting of PCR primers. DNA ends are usually damaged after 60

Multi-stage PCR. To recover the DNA from such damaged molecules it was necessary that the primers be annealed from the ends of the DNA construct, thereby sequentially shortening the construct with each round of selection.

 In one aspect, the DNA construct and / or the encoded protein can be configured to include a tag. Such a peptide or DNA tag, for example as described above, can be used in the separation and isolation of a member of the library of interest. Such a tag can also be used to hold the members of the library, for example, on a solid support for use in the screening methods described herein.

 Therefore it can be seen that the screening methods of the present invention may include the additional step of selecting and isolating the peptide member of the relevant library, allowing the peptide to show the desired properties, and also the DNA encoding that peptide , which will be identified and purified.

 Therefore the invention encompasses peptides and DNA that have been identified by the method of the invention. These peptides and DNA can be isolated and / or purified. The peptides or DNA isolated by the method of the invention can be modified, for example by deletion, addition or substitution of 15 amino acids or nucleotides. Suitable modified peptides or DNA may show an amino acid or nucleotide sequence identity of at least 50%, at least 75%, at least 90%, at least 95% or more with the peptide or DNA isolated by the method of the invention. . The peptides identified by the method of the invention can be modified for the purpose of release and / or stability. For example, such peptides can be pegylated (anchored to polyethylene glycol) to prolong serum half-life or to prevent attack by proteases. The peptides identified by the method of the invention can be modified in other presentation systems such as phage display or by synthesizing and screening peptide variants. A collection of such modified sequences may form a new library that can be incorporated into constructs of the invention and further screened to find, for example, a variant sequence that shows improved binding to a particular ligand. Thus in one embodiment, a peptide library for use in the methods of the invention can be a structurally related peptide library.

 Alternatively, a library of essentially random peptide sequences can be used. Numerous types of peptide libraries fused to the DNA binding protein that acts in cis can be screened in this embodiment including:

 (i) Random peptide sequences encoded by synthetic DNA of variable length.

 (ii) Antibodies or antibody fragments, for example single chain Fv antibody fragments. These consist of domains of the variable region of the heavy and light chains of the antibody bound by a flexible linker peptide to create a single chain antigen binding molecule.

 (iii) Random cDNA fragments of naturally occurring proteins isolated from a population of cells that contain an activity of interest.

 (iv) Sequences of random peptides inserted into, or that replace a region of a known protein, where the sequence of the known protein acts as a framework, which constrains the sequence of random peptides. Many such frames have been described, CTLA-4 (WO 00/60070), as a framework for peptide libraries, has been used by way of example, without exclusion. 40

 In another embodiment the invention relates to methods for screening a DNA library whose members require more than one chain for activity, as required, for example, by Fab antibody fragments for ligand binding. In this embodiment, the DNA of the heavy or light chain antibody binds to a nucleotide sequence encoding a DNA binding domain, for example, of repA. Typically the DNA library sequences of antibodies unknown to heavy (VH and CH1) or light chain (VL and CL) genes are inserted into the 5 'region of the repA DNA, behind an appropriate promoter and The translation sequences. Thus, repA fused to a protein encoded by a member of the DNA library is produced bound to the DNA encoding that protein. The second known chain, which encodes the light or heavy chain protein, is expressed separately:

 (a) from the same DNA fragment that contains the repA fusion protein library and the first polypeptide, or

 (b) from a separate DNA fragment present in the in vitro transcription / translation reaction.

 The known chain is associated with the library of unknown fusion proteins that are fused to the repA protein and thereby bound to the DNA for the unknown chain. The functional Fab 55 library can then be selected by means of a specific ligand for the antibody.

 The DNA identified by a screening method of the invention, e.g. ex. The DNA encoding the member peptide of the selected library can be cloned into a vector. In one embodiment, the DNA identified by a method of the invention is operably connected to a control sequence that is capable of providing expression of the coding sequence by the host cell, that is, the vector is an expression vector. The term "operably connected" refers to a juxtaposition 5 in which the components described are in a relationship that allows them to function in the intended manner. A regulatory sequence, such as a promoter, "operably connected" to a coding sequence is positioned such that expression of the coding sequence is achieved under conditions compatible with the regulatory sequence.

 Such expression vectors are routinely constructed in the technique of molecular biology and may involve, for example, the use of plasmid DNA and appropriate initiators, promoters, enhancers and other elements, such as for example polyadenylation signals that may be necessary, and that they are located in the correct orientation, in order to allow the expression of the protein. Other suitable vectors would be apparent to those skilled in the art. As a further example in this regard, the authors of the invention refer to Sambrook et al. 1989. 15

 The vectors can be, for example, plasmid, viral or phage vectors with an origin of replication, optionally a promoter for the expression of said DNA and optionally a promoter regulator. The vectors may contain one or more selectable marker genes, for example an ampicillin resistance gene in the case of a bacterial plasmid or a fungal vector resistance gene. The vectors can be used in vitro, for example for the production of DNA or RNA or used for the production of DNA or RNA or used to transfect or transform a host cell, for example, a mammalian host cell. Vectors can also be adapted for use in vivo, for example in a method of gene therapy.

 Promoters and other expression regulation signals may be selected to be compatible with the host cell for which the expression is designed. For example, yeast promoters include S. cerevisiae GAL4 and ADH promoters, and S. pombe nmt1 and adh promoter. Mammalian promoters include the metallothionein promoter that can be induced in response to heavy metals such as cadmium. Viral promoters such as the SV40 large T antigen promoter or adenovirus promoters can also be used. All these promoters are easily available in the art. 30

 Mammalian promoters such as p-actin promoters can be used. Tissue specific promoters are especially preferred. Viral promoters can also be used, for example the long terminal repeat of Moloney murine leukemia virus (MMLV LTR), the LTR promoter of the Rous Sarcoma virus (RSV), the SV40 promoter, the human cytomegalovirus IE promoter (CMV), adenovirus, HSV promoters (such as HSV IE promoters), or HPV promoters, specifically the upstream regulatory region (URR) of HPV. Viral promoters are easily available in the art.

 The vector may additionally include sequences flanking the polynucleotide of interest resulting in polynucleotides comprising sequences homologous to eukaryotic genomic sequences, preferably mammalian genomic sequences, or viral genomic sequences. This will allow the introduction of polynucleotides of the invention into the genome of eukaryotic cells or viruses by homologous recombination. In particular, a plasmid vector comprising the expression cassette flanked by viral sequences can be used to prepare a viral vector suitable for releasing the polynucleotides of the invention in a mammalian cell. Other examples of viral vectors include viral vectors of herpes simplex and retroviruses, including lentiviruses, adenoviruses, adeno-associated viruses and HPV viruses. The gene transfer techniques that use these viruses are known to those skilled in the art. For example, retrovirus vectors can be used to stably integrate the polynucleotide giving rise to the polynucleotide in the host genome. Defective replication adenovirus vectors, on the other hand, remain episomes and therefore allow transient expression.

 Such expression vectors may be used to identify ligands of interest, that is, molecules that bind to the peptide library member by means of conventional binding analyzes such as ELISA, or enzymatic analyzes where appropriate substrates provide, for example, a color change, light emission or fluorescence. Other functional analyzes could be used, when available.

 In an alternative embodiment, a DNA identified by a method of the invention can be cloned into a non-expression vector. Such a vector can be used to further characterize the DNA, for example, by sequencing.

 Alternatively, ligands of interest can be identified without cloning. Examples of suitable methods include in vitro expression of individual DNA sequences recovered from a screening method of the invention, and sequencing of individual DNAs recovered from such a screening method. Such individual DNA sequences can be optionally amplified. 60

 The invention also includes cells that have been modified to express a peptide identified by a method of the invention, for example by introducing an expression vector as described above into the cell. Such cells include transient, or preferably stable, upper eukaryotic cell lines, such as mammalian cells or insect cells, using for example a baculovirus expression system, lower eukaryotic cells, such as yeast or prokaryotic cells as bacterial cells. Concrete examples of cells that can be modified by insertion of vectors encoding a peptide identified by a method of the invention include mammalian HEK293T, CHO, HeLa and COS cells. Preferably the selected cell line will be one that is not only stable, but also allows mature glycosylation and expression of the peptide on the cell surface. Expression can be achieved in transformed oocytes. A peptide identified by a method of the invention can be expressed in cells of transgenic non-human animals, preferably a mouse. A peptide identified by a method of the invention can also be expressed in oocytes or melanophores of Xenopus laevis.

 In order to more fully understand the invention, embodiments will now be described in greater detail by way of examples and not by way of limitations with reference to the following figures. fifteen

 Examples of some embodiments of the invention are given below:

Materials and methods.

 The following procedures used by the author of the present application are described by Sambrook, J., et al., 1989 above citation: analysis of restriction enzyme digestion products on agarose gels, DNA purification using phenol / chloroform stock solutions, preparation of phosphate buffered Saline twenty

 General purpose reagents were purchased from SIGMA-Aldrich Ltd (Poole, Dorset, U.K.). Oligonucleotides were obtained from SIGMA-Genosys Ltd (Cambridgeshire, United Kingdom). Amino acids, S30 extracts were obtained from Promega Ltd (Southampton, Hampshire, U. K.). Deep Vent and Taq DNA polymerase were obtained from New England Biolabs (Cambridgeshire, U.K.). Taqplus DNA polymerase was obtained from Stratagene Inc. (Amsterdam, The Netherlands). GeneClean DNA gel purification kits were obtained from BIO 101 (La Jolla, California, USA), the anti-IgKhumana antibodies of Immunologicals Direct Ltd (Oxfordshire, UK), the polyclonal anti-c-myc of Vector Labs Inc (Cambridgeshire UK), and the anti-V5 antibody from Abcam Ltd (Cambridgeshire UK). The blocking agent Superblock was obtained from Perbio Science (Cheshire, U. K.).

Example 1. Isolation of protein-DNA complexes that act in specific cis 30

 In vitro expression constructs were prepared by successively adding the TAC promoter, the c-myc epitope, the human kappa constant region or the V5 epitope to the RepA-CIS-ORI region, by PCR amplification. Such constructs can be prepared by many methods known to those skilled in the art, for example, by amplifying different DNA fragments followed by assembly PCR. In this example, the initial amplification template was plasmid R1 containing the RepA-35 CIS-ORI region (Masai, H. and Arai, K. (1988). DNAs Res. 16, 6493-6514).

 (a) Primary amplification. The RepA-CIS-ORI region was amplified by PCR from a single colony of strain ECO K12 that harbored plasmid R1 using 12.5 pmoles of each of the REPAFOR primers (SEQ ID 01) and ORIREV (SEQ ID 02 ) in 50 μl of reaction containing 0.25 mM dNTP, 2.5 units of Taqplus Precision DNA polymerase, 1x PCR reaction buffer (Stratagene Inc., 40 Amsterdam, The Netherlands). The REPAFOR primer hybridizes to the 5 'end of the RepA coding region. The ORIREV primer hybridizes with the 3 'end of the non-coding ORI region.

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 4 minutes and 15 seconds at 94 ° C followed by 30 cycles of 94 ° C, 45 seconds; 60 ° C, 45 seconds; 72 ° C, 45 seconds, followed by a single 10 minute cycle at 72 ° C. The reaction products were subjected to 45 electrophoresis on an agarose gel, excised and the products were purified from the gel in 40 μl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla , California, USA).

 (b) Secondary amplification. One μl (500 pg) of purified gel primary reaction product diluted 100 times was re-amplified using 12.5 pmoles of each of the primers CKREPFOR (SEQ ID 50 03) and ORIREV (SEQ ID 02) in a 50 μl reaction containing 0.25mM dNTP, 2.5 units of Taqplus Precision DNA polymerase, and 1x PCR reaction buffer (Stratagene Inc, Amsterdam, The Netherlands). The CKREPFOR primer hybridizes with the 5 'end of the primary reaction product and is added to the 3' portion of the kappa constant region DNA. The ORIREV primer hybridizes with the 3 'end of the secondary reaction product. 55

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94 ° C followed by 30 cycles of 94 ° C, 45 seconds; 60 ° C, 45 seconds; 72 ° C, 2 minutes, followed by a single 10 minute cycle at 72 ° C. The reaction products were subjected to

electrophoresis on an agarose gel, were cleaved and the products were purified from the gel in 40 µl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla, California, U.S.A.).

 (c) Third amplification. 1 µl (500 pg) of gel purified primary reaction product diluted 100 times was re-amplified using 12.5 pmoles of each of the primers V5REPFOR (SEQ ID 5 04) and ORIREV (SEQ ID 02) in 50 µl of reaction containing 0.25mM dNTP, 2.5 units of Taqplus Precision DNA polymerase, and 1x PCR reaction buffer (Stratagene Inc, Amsterdam, The Netherlands). The V5REPFOR primer hybridizes to the 5 'end of the primary reaction product and is added to the 3' portion of the V5 epitope DNA. The ORIREV primer hybridizes with the 3 'end of the primary reaction product.

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 2 10 minutes and 15 seconds at 94 ° C followed by 30 cycles of 94 ° C, 45 seconds; 60 ° C, 45 seconds; 72 ° C, 2 minutes, followed by a single 10 minute cycle at 72 ° C. The reaction products were electrophoresed on an agarose gel, excised and the products were purified from the gel in 40 μl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla, California, USA). fifteen

 (d) Fourth amplification. 1 μl (500 pg) of plasmid pCKV5 diluted 100 times was again amplified using 12.5 pmoles of each of the MYCCKFOR (SEQ ID 05) and CKREV (SEQ ID 06) primers in 50 μl of reaction containing dNTP 0, 25 mM, 2.5 units of Taqplus Precision DNA polymerase, and 1x PCR reaction buffer (Stratagene Inc, Amsterdam, The Netherlands). Plasmid pCKV5 contains the cDNA of the human kappa constant region (McGregor DP, Molloy PE, Cunningham C, and Harris WJ. 1994 Mol. Immunol. 20 31: 219-26) and the epitope DNA V5 (Southern JA, Young DF , Heaney F, Baumgartner WK, Randall RE. 1991 J. Gen. Virol. 72: 1551-7). The MYCCIsFOR primer hybridizes with the 5 'end of the DNA of the human kappa constant region and is added to the 3' portion of the MYC epitope DNA. The CKREV primer hybridizes with the 3 'end of the kappa constant region DNA.

 The PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 2 25 minutes and 15 seconds at 94 ° C followed by 30 cycles of 94 ° C, 45 seconds; 60 ° C, 45 seconds; 72 ° C, 2 minutes, followed by a single 10 minute cycle at 72 ° C. The reaction products were electrophoresed on an agarose gel, excised and the products were purified from the gel in 40 μl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio 101, La Jolla , California, USA). 30

 (e) Fifth amplification. 1 μl (500 pg) of plasmid pCKV5 diluted 100 times was reactivated using 12.5 pmoles of each of the MYCV5FOR (SEQ ID 07) and V5REV (SEQ ID 08) primers in 50 μl of reaction containing 0.25 mM dNTP , 2.5 units of Taqplus Precision DNA polymerase, and 1 x PCR reaction buffer (Stratagene Inc, Amsterdam, The Netherlands). The MYCV5FOR primer hybridizes with the 5 'end of the V5 epitope DNA and is added to the 3' portion of the MYC epitope DNA. The V5REV primer hybridizes with the 3 'end of the V5 epitope DNA.

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94 ° C followed by 30 cycles of 94 ° C, 45 seconds; 60 ° C, 45 seconds; 72 ° C, 30 seconds, followed by a single 10 minute cycle at 72 ° C. The reaction products were electrophoresed on an agarose gel, excised and the products were purified from the gel in 40 µl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla , California, USA).

 (f) Sixth amplification. 1 μl (500 pg) of plasmid pTACP2A diluted 100 times (ref) was reamplified using 12.5 pmoles of each of the primers TAC3 (SEQ ID 09) and MYCTACREV (SEQ ID 10) in 50 μl of reaction containing dNTP 0 , 25 mM, 2.5 units of Taqplus Precision DNA polymerase, and 1x PCR reaction buffer (Stratagene Inc, Amsterdam, The Netherlands). The TAC3 primer hybridizes to the 5 'end of the TAC promoter DNA. The MYCTACREV primer hybridizes with the 3 'end of the TAC promoter DNA and is added to the 5' portion of the MYC epitope DNA.

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94 ° C followed by 30 cycles of 94 ° C, 45 seconds; 60 ° C, 45 seconds; 72 ° C, 30 50 seconds, followed by a single 10 minute cycle at 72 ° C. The reaction products were electrophoresed on an agarose gel, excised and the products were purified from the gel in 40 μl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla, California, USA).

 (g) First assembly PCR. 1 μl (50 ng) of each of the reaction products 55 of (f) and (d) was amplified using 50 pmoles of each of the primers TAC5 (SEQ ID 11) and CKREV (SEQ ID 06) in 50 μl reaction containing 0.25 mM dNTP, 2.5 units of TaqDeepVent DNA polymerase mixture (20: 1), and 1x PCR reaction buffer (New England Biolabs, Beverly, MA, USA). The hybrid TAC5 primer

with the 5 'end of the reaction product (f) and add 20 nucleotides. The CKREV primer hybridizes with the 3 'end of the reaction product (d).

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94 ° C followed by 30 cycles of 94 ° C, 45 seconds; 60 ° C, 45 seconds; 72 ° C, 45 seconds, followed by a single 10 minute cycle at 72 ° C. The reaction products were subjected to 5 electrophoresis on an agarose gel, excised and the products were purified from the gel in 40 ml of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla , California, USA).

 (h) Second assembly PCR. 1 μl (50 ng) of each of the reaction products of (f) and (e) was amplified using 50 pmoles of each of the primers TAC5 (SEQ ID 11) and V5REV (SEQ ID 08) in 50 10 μl reaction containing 0.25 mM dNTP, 2.5 units of TaqDeepVent DNA polymerase mixture (20: 1), 1x PCR reaction buffer (New England Biolabs, Beverly, MA, USA). The TAC5 primer hybridizes with the 5 'end of the reaction product (f) and adds 20 nucleotides. The V5REV primer hybridizes with the 3 'end of the reaction product (e).

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 2 15 minutes and 15 seconds at 94 ° C followed by 30 cycles of 94 ° C, 45 seconds; 60 ° C, 45 seconds; 72 ° C, 45 seconds, followed by a single 10 minute cycle at 72 ° C. The reaction products were electrophoresed on an agarose gel, excised and the products were purified from the gel in 40 μl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla, California, USA). twenty

 (i) Third assembly PCR. 1 μl (50 ng) of each of the reaction products of (b) and (g) was amplified or using 50 pmoles of each of the primers TAC3 (SEQ ID 09) and ORIREV (SEQ ID 02) in 50 μl reaction containing 0.25 mM dNTP, 2.5 units of TaqDeepVent DNA polymerase mixture (20: 1), and 1x PCR reaction buffer (New England Biolabs, Beverly, MA, USA). The TAC3 primer hybridizes 20 nucleotides downstream with respect to the 5 'end of the reaction product (g). The ORIREV 25 primer hybridizes with the 3 'end of the reaction product (b). The reaction product of (i) is called TAC-MYC-CK-REPA-CIS-ORI (SEQ ID 12).

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94 ° C followed by 30 cycles of 94 ° C, 45 seconds; 60 ° C, 45 seconds; 72 ° C, 1 minute, followed by a single 10 minute cycle at 72 ° C. The reaction products were subjected to 30 electrophoresis on an agarose gel, excised and the products were purified from the gel in 40 μl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla , California, USA).

 (j) Fourth assembly PCR. 1 μl (50 ng) of each of the reaction products of (b) and (h) was amplified using 50 pmoles of each of the primers TAC3 (SEQ ID 09) and ORIREV (SEQ ID 02) in 50 35 μl reaction containing 0.25 mM dNTP, 2.5 DNA polymerase mixing units TaqDeepVent (20: 1), and 1x PCR reaction buffer (New England Biolabs, Beverly, MA, USA). The TAC3 primer hybridizes 20 nucleotides downstream with respect to the 5 'end of the reaction product (g). The ORIREV primer hybridizes with the 3 'end of the reaction product (b). The reaction product of (i) is called TAC-MYC-V5-REPA-CIS-ORI (SEQ ID 13). 40

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 2 minutes and 15 seconds at 94 ° C followed by 30 cycles of 94 ° C, 45 seconds; 60 ° C, 45 seconds; 72 ° C, 1 minute, followed by a single 10 minute cycle at 72 ° C. The reaction products were electrophoresed on an agarose gel, excised and the products were purified from the gel in 40 μl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla, 45 California, USA).

 Preparation of the transcription / translation reaction in vitro. The reaction was placed on ice, using a Promega S30 bacterial linear template coupled to the in vitro transcription / translation reaction kit as follows:

20 μl of TAC-MYC-CK-REPA-CIS-ORI template (0.5 μg of final construct DNA SEQ ID 012 above); 20 50 μl of TAC-MYC-V5-REPA-CIS-ORI template (0.5 μg of final construct DNA SEQ ID 013 above); 20 μl of complete amino acid mixture (Promega); 80 μl of Premix S30; 60 μl of S30 mixture;

and the reaction was allowed to continue at 25 ° C for 30 minutes and placed on ice, then diluted 10 times with blocking buffer (Superblock (Perbio Ltd), 0.1% Tween 20, 200 µg / ml DNA of herring sperm). 55

 DNA-protein complex capture. NUNC star immunotubes were coated with 10 μg / ml of anti-c antibody, anti-V5 antibody, or anti-human kappa chain antibody, in 500 μl of PBS per tube overnight at 4 ° C. An additional tube was left blank as a negative control. The tubes were washed 2x

PBS and were blocked for 1 hour at room temperature with Superblock / PBS / 0.1 mg / ml 0.1% herring sperm / Tween 20 DNA and then washed with 2x PBS. 500 µl of diluted transcription / translation reaction was added to each tube and incubated at room temperature for 1 hour. The tubes were washed 5x PBS / 0.1% Tween 20, then 1x 30 minutes with 2 ml Superblock / PBS / 0.1 mg / ml 0.1% herring sperm / Tween 20 DNA, then with 5x PBS. The DNA was recovered with 300 µl of 5 T.E. plus 300 µl of phenol / chloroform for 5 minutes with oscillatory movement. This was centrifuged at 13,200 g for 5 minutes and the DNA was precipitated with 0.5 volumes of 7.5 M ammonium acetate, 20 μg of glycogen and three volumes of absolute ethanol. After centrifugation, the sediments were washed with 70% ethanol, dried under vacuum and resuspended in 20 µl of water. 10 µl of the recovered DNA was re-amplified with 50 µl reactions with primers TAC3 (SEQ ID 09) and ORIREV (SEQ ID 02). The reaction products 10 were electrophoresed on an agarose gel / 1% APR (Figure 5).

Example 2. Separation of the RepA-DNA complex

 The two in vitro expression constructs (SEQ ID 12 and SEQ ID 13) already described in Example 1 were used in a screening experiment against human anti-kappa C antibody as described in Example 1, except that the DNA was recovered and released from RepA using any of the following 15 methods; Glycine, Triethylamine, Phenol / Chloroform, Proteinase K, and EDTA. These methods are described below.

 Glycine: The tube was incubated with 500 μl of 200 mM Glycine, 150 mM NaCl (pH 2.0) for 10 minutes. The eluted glycine product was then transferred to a new eppendorf tube and 50 µl of 2M Tris (pH 8.5) was added. twenty

 Triethylamine: The tube was incubated with 500 µl of 0.1 M Triethylamine for 10 minutes and the eluted product of triethylamine was then transferred to a new eppendorf tube and 250 µl of 1M Tris (pH 7.4) was added.

 Phenol / Chloroform: as in example 1 above.

 Proteinase K: The tube was incubated with 500 μl of 100 mM Tris (pH 8.0), 10 mM EDTA (pH 8.0), 25% SDS for 30 minutes at 37 ° C. The eluted proteinase K product was then transferred to a new eppendorf tube.

 EDTA: The tube was incubated with 250 µl of 10 mM Tris (pH 8.0), 1 mM EDTA, 500 mM NaCl and 250 µl of Phenol / Chloroform for 5 minutes. The product eluted with EDTA was transferred to a new eppendorf tube.

 After DNA recovery, DNA was extracted with Phenol / Chloroform, when appropriate, followed by ethanol precipitation as described in Example 1. 10 µl of resuspended DNA was again amplified in 50 µl reactions with TAC3 primers ( SEQ ID 09) and CISREV (SEQ ID 019). The CISREV primer hybridizes 196 bases upstream of the ORIREV binding site (SEQ ID 02). The reaction products were electrophoresed on a 1% agarose gel / APR (data not shown). Only the construct containing CK-DNA (SEQ ID 12) was amplified in approximately equivalent amounts. 35

 This not only tells us that any of the methods described above can be used to recover and release RepA DNA, but this result also suggests that RepA interacts noncovalently with its cognate DNA.

Example 3. Detection of specific anti-V5 binders in a V5 addition experiment using CIS presentation technology. 40

 In vitro expression constructs were prepared by adding the TAC promoter and the V5 epitope or a 12-unit NNB library to the RepA-CIS-ORI region, by PCR amplification. Such constructs can be prepared by many methods known to those skilled in the art, for example, by amplifying different DNA fragments followed by assembly PCR. In this example, the initial amplification template was plasmid R1 containing the RepA-CIS-ORI region (Masai, H. and Arai, K. (1988) .45 Nucleic Acids Res. 16, 6493-6514).

 (to). Primary amplification The RepA-CIS-ORI region was amplified by PCR from a single colony of strain ECO K12 that harbored plasmid R1 using 12.5 pmoles of each of the REPAFOR primers (SEQ ID 01) and ORIREV408 (SEQ ID 20 ) in a 50 μl reaction containing 0.25 mM dNTP, 2.5 units of TaqDeepVent DNA polymerase mixture (20: 1), and 1x PCR 50 reaction buffer (New England Biolabs, Beverly, MA, USA) . The REPAFOR primer hybridizes the 5 'end of the RepA coding region. The ORIREV408 primer hybridizes downstream of the 3 'end of the non-coding ORI region.

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 4 minutes and 30 seconds of 94 ° C followed by 25 cycles 94 ° C, 30 seconds; 60 ° C, 45 seconds; 72 ° C, 1 minute, followed by a single 10 minute cycle at 72 ° C. The reaction products were electrophoresed on an agarose gel, excised and cleaved and the products purified from the gel in 40 μl.

of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla, California, U.S.A.).

 (b). Secondary amplification. 1 µl (500 pg) of gel purified primary reaction product diluted 100 times was re-amplified using 12.5 pmoles of each of the primers V5 (NNB) REPFOR (SEQ ID 21) and ORIREV408 (SEQ ID 20) in a 50 μl reaction containing 0.25 mM dNTP, 2.5 units of TaqDeepVent DNA polymerase mixture (20: 1), and 1x PCR reaction buffer (New England Biolabs, Beverly, MA, USA). The V5 (NNB) REPFOR primer hybridizes with the 5 'end of the primary reaction product and is added to the V5 epitope DNA. The ORIREV408 primer hybridizes with the 3 'end of the primary reaction product.

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 4 10 minutes and 30 seconds of 94 ° C followed by 25 cycles of 94 ° C, 30 seconds; 60 ° C, 45 seconds; 72 ° C, 1 minute, followed by a single 10 minute cycle at 72 ° C. The reaction products were electrophoresed on an agarose gel, excised and the products were purified from the gel in 40 μl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla, California, USA). fifteen

 (C). Third amplification 1 µl (500 pg) of gel purified primary reaction product diluted 100 times was re-amplified using 12.5 pmoles of each of the primers NNBREPFOR (SEQ ID 22) and ORIREV408 (SEQ ID 20) in a reaction of 50 µl containing 0.25 mM dNTP, 2.5 units of TaqDeepVent DNA polymerase mixture (20: 1), and 1x PCR reaction buffer (New England Biolabs, Beverly, MA, USA). The NNBREPFOR primer hybridizes with the 5 'end of the primary reaction product and is added to a DNA from the NNB library of 12 random amino acid units. The ORIREV408 primer hybridizes with the 3 'end of the primary reaction product.

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 4 minutes and 30 seconds of 94 ° C followed by 25 cycles of 94 ° C, 30 seconds; 60 ° C, 45 seconds; 72 ° C, 1 minute, followed by a single 10 minute cycle at 72 ° C. The reaction products were subjected to 25 electrophoresis on an agarose gel, excised and the products were purified from the gel in 40 μl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla , California, USA).

 (d). Fourth amplification 1 μl (500 pg) of plasmid pTACP2A (ref) diluted 100 times was again amplified using 12.5 pmoles of each of the primers TACFARUP (SEQ ID 23) and TACREV (SEQ ID 27) 30 in a 50 μl reaction containing 0.25 mM dNTP, 2.5 units of TaqDeepVent DNA polymerase mixture (20: 1), and 1x PCR reaction buffer (New England Biolabs, Beverly, MA, USA). The TACFARUP primer hybridizes with the 5 'end of the TAC promoter DNA. The TACREV primer hybridizes with the 3 'end of the TAC promoter DNA.

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 1 minute 35 and 45 seconds of 94 ° C followed by 25 cycles of 94 ° C, 15 seconds; 60 ° C, 30 seconds; 72 ° C, 30 seconds, followed by a single 10 minute cycle at 72 ° C. The reaction products were electrophoresed on an agarose gel, cleaved and the products purified from the gel in completely sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla, California, USES). 40

 (and). First assembly PCR. 1 μl (50 ng) of each of the reaction products of (b) and (d) was amplified using 50 pmoles of each of the primers TACFARUP (SEQ ID 23) and ORIREV408 (SEQ ID 20) in a reaction of 50 μl containing 0.25 mM dNTP, 2.5 units of TaqDeepVent DNA polymerase mixture (20: 1), and 1x PCR reaction buffer (New England Biolabs, Beverly, MA, USA). The TACFARUP primer hybridizes with the 5 'end of the reaction product (d). The ORIREV480 primer hybridizes with the 3 'end of the reaction product (b). The reaction product of (e) is called TAC-V5-REPA-CIS-ORI-408 (SEQ ID 24).

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 1 minute and 45 seconds of 94 ° C followed by 25 cycles of 94 ° C, 15 seconds; 60 ° C, 30 seconds; 72 ° C, 1 minute and 30 seconds, followed by a single 10 minute cycle at 72 ° C. The reaction products were subjected to 50 electrophoresis on an agarose gel, excised and the products were purified from the gel in 40 μl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla , California, USA).

 (f) Second assembly PCR. 1 μl (50 ng) of each of the reaction products of (c) and (d) was amplified using 50 pmoles of each of the TACFARUP primers (SEQ ID 23) and ORIREV408 (SEQ ID 20) in one reaction 50 µl containing 0.25 mM dNTP, 2.5 units of TaqDeepVent DNA polymerase mixture (20: 1), and 1x PCR reaction buffer (New England Biolabs, Beverly, MA, USA). The TACFARUP primer hybridizes with the 5 'end of the reaction product (d). The ORIREV480 primer hybridizes with the 3 'end of the reaction product (c).

 PCR reactions were carried out in an Eppendorf Master Cycler for 1 cycle of 1 minute and 45 seconds of 94 ° C followed by 25 cycles of 94 ° C, 15 seconds; 60 ° C, 30 seconds; 72 ° C, 1 minute and 30 seconds, followed by a single 10 minute cycle at 72 ° C. The reaction products were electrophoresed on an agarose gel, excised and the products were purified from the gel in 40 μl of sterile water using a Geneclean II kit according to the manufacturers instructions (Bio101, La Jolla, 5 California, USA). The reaction product of (f) is called TAC-NNB-REPA-CIS-ORI-408 (SEQ ID 25).

 Preparation of the in vitro transcription / translation reaction: The reaction set was placed on ice, using a Promega S30 bacterial linear template coupled to an in vitro transcription / translation reaction kit as follows:

20 μl of TAC-V5-REPA-CIS-ORI-408 template diluted 5,000 times (0.1 ng of the DNA of the final construct SEQ ID 10 24 above)

20 μl of TAC-NNB-REPA-CIS-ORI-408 template (0.5 μg of the DNA of the final construct SEQ ID 25 above)

20 μl of complete amino acid mixture (Promega)

80 μl of Premix S30

60 μl of mixture S30 15

 and the reaction was allowed to continue at 25 ° C for 30 minutes and placed on ice, then diluted 10 times with Marvel / 2% PBS.

 DNA-protein complex capture. NUNC star immunotubes were coated with 10 µg / ml anti-V5 antibody in 500 µl PBS overnight at 4 ° C. An additional tube was left blank as a negative control. The tubes were washed 2x PBS and blocked for 1 hour at room temperature with 20 blocking buffer (2% Marvel, 0.1% Tween 20, 0.1 mg / ml herring sperm DNA) and then 2x PBS was washed. 1 ml of diluted transcription / translation reaction was added to each tube and incubated at room temperature for 1 hour. The tubes were washed 5x PBS / 0.1% Tween 20 and then 5x PBS. The DNA was recovered with 500 µl of TE buffer plus 500 µl of phenol / chloroform. This was centrifuged at 13,200g for 5 minutes and the DNA was precipitated with 1/10 volume of 3M sodium acetate, 50 µg / ml of glycogen 25 and two volumes of absolute ethanol. After centrifugation, the sediments were washed with 70% ethanol, dried under vacuum and resuspended in 40 µl of water. 20 μl of the recovered DNA was re-amplified in 50 μl reactions with the biotinylated primers bTAC6 (SEQ ID 26) and bCISREV (SEQ ID 19). The reaction products were electrophoresed on an agarose gel / 1% APR.

 Cloning of the DNA recovered in the expression vector pDMG-K (SEQ ID 27). The reaction products were gel purified and eluted with 50 µl of sterile water using a QIAquick Gel extraction kit according to the manufacturers instructions (QIAGEN Ltd West Sussex, U.K.). Both the purified reaction product and the plasmid pDMG-K were digested with 20 units of NcoI and NotI (New England Biolabs, Beverly, MA, U.S.A.). The cut plasmid was gel purified using a QIAquick Gel extraction kit according to the manufacturers instructions (QIAGEN Ltd West Sussex, 35 UK), then reacted with 0.01 units of Veal Intestinal Alkaline Phosphatase (Promega , Southampton, UK) followed by extraction with phenol / chloroform and ethanol precipitation as described above. The precipitated DNA was dissolved in 20 µl of water. The PCR cut product was transferred to Streptavidin-coated strips (Roche Diagnostics Ltd, East Sussex, UK) in 1x TBS, 0.3 mg / ml BSA, 0.1% Tween 20 and incubated for 30 minutes at the room temperature, with oscillatory movement. 40 This approach removes adjoining biotinylated DNA upstream and downstream from the NcoI and NotI site of the PCR product and allows recovery of the small DNA fragment that contains the selected peptide sequence. The supernatant was extracted with phenol / chloroform and precipitated with ethanol as described above. The precipitated DNA was dissolved in 10 µl of water. The cut plasmid and the isolated small DNA fragment containing the sequence of the selected peptide, which both had NcoI and 45 NotI protrusions, were ligated using the Quick ligation kit according to the manufacturers instructions (New England Biolabs, Beverly, MA, USA) followed by extraction with phenol / chloroform and ethanol precipitation as described above. The precipitated DNA was dissolved in 10 μl of water and subjected to electroporation in electrocompetent TG1 cells according to the manufacturers instructions (Stratagene, USA) and was selected on plates with 2xTY, 100 μg / ml ampicillin, and glucose at 2%. fifty

 ELISA screening with anti-V5 antibody of the selected clones. 88 colonies were selected in 400 µl of 2x TY, 2% glucose, and 100 µg / ml ampicillin and grown overnight at 37 ° C, with oscillatory motion at 300 rpm. 50 µl of the cultures performed overnight were transferred to 1 ml of 2x TY, 2% glucose, and 100 μg / ml of ampicillin and grown at 37 °, with oscillatory motion at 300 rpm to an OD of 0, 5. The cells were then centrifuged at 1000x g for 10 minutes. The 55 supernatants were discarded and the sediments were resuspended in 600 µl of 2x TY, 0.4 M sucrose, 100 µg / ml ampicillin, and 1 mM IPTG and grown for 4 hours at 37 ° C, 300 rpm. After induction the cells were centrifuged at 1000xg for 10 minutes. 150 μl of the supernatants were used

in the ELISA test. NUNC Maxisorp plates were coated with 100 µl of 1 µg / ml in 1x PBS of anti-human kappa region antibody or anti-V5 antibody or 50 µg / ml of BSA for 7 hours at room temperature. An additional plate was left blank, coated only with PBS. The wells were rinsed 2x PBS followed by blocking for 1 hour at room temperature with 300 µl of 4% Marvel, 0.1% Tween in 1x PBS. The wells were rinsed 2x PBS, then 150 µl of supernatant and 150 µl of Marvel 5 4%, 0.1% Tween 20 in 1x PBS were added to the wells and incubated for 1 hour at room temperature. Then the wells were washed in 2xPBS, 0.1% Tween 20 and 2x PBS. The secondary anti-human kappa region antibody conjugated to horseradish peroxidase (HRP) (final concentration 1.6 μg / ml) was diluted 500 times in 4% Marvel, 0.1% Tween 20, 1x PBS and added to the wells and incubated for 1 hour at room temperature. Then the 4x PBS, 0.1% Tween 20 and 2x PBS wells were washed. The 10 HRP signal was detected by adding 200 μl of TMB substrate. The reaction was stopped with 100 µl of 0.5 M sulfuric acid. The absorbance was read at 450 nm. Thirty-five of eighty-eight clones were well expressed judging by the HRP signal of the clones screened against the anti-human kappa region antibody. Seven of these thirty-five clones showed a specific binding to the anti-V5 antibody, thereby enriching V5 peptides of 1 by 5,000 to 1 by 5, that is, an enrichment factor of 1,000 (Figure 6). fifteen

Example 4: Construction, selection and scrutiny of the CIS Presentation Library against Bacillus globigii

Construction of the Library

 To generate the DNA from the library, a DNA fragment from the promoter library and the RepA-CIS-ori fragment must be generated, then linked together by digestion-ligation. In this example, the tac promoter of a P2A-HA vector was used, but many available promoters could be used, and are well known to those skilled in the art. The initial PCR of the Rep-CIS-ori and TAC fragments adds a Bsp120I site and the random library / NotI site, respectively. Two master mixtures were prepared:

 10 μl of P2A-HA diluted 1:50 plasmid DNA (25 ng / reaction) was amplified by PCR in a 20x 50 μl reaction volume containing 200 mM dNTP, 1xNEB 25 polymerase amplification buffer (10 mM KCl, (NH4 ) 10 mM 2SO4, 20 mM Tris-HCl pH 8.8, 2 mM MgSO4, 0.1% Triton X-100) with 10 pmoles of each of the TACFARUP primers (SEQ ID 23) and NTERM18MER (SEQ ID28) and 2 units of 20: 1 mixture of Taq DNA polymerase: Deep Vent DNA polymerase (NEB) for 25 cycles of 94 ° C, 40 seconds; 60 ° C, 40 seconds; 72 ° C, 60 seconds; followed by an extension of 5 minutes at 72 ° C. 20 µl of reaction product was electrophoresed on a 1% agarose gel / APR and photographed, while the rest was purified on a Qiagen column in 200 µl of water.

 10 μl of Rep-CIS-ori DNA corrected with Bsp120I (50ng / reaction) was amplified by PCR in a reaction volume of 10x50 μl containing 200 μM dNTP, 1x NEB polymerase amplification buffer (10 mM KCl, (NH4) 10 mM 2SO4, 20 mM Tris-HCl pH 8.8, 2mM MgSO4, 0.1% Triton X-100) with 10 pmoles of each of the BSPREPAFOR primers (SEQ ID 29) and ORIREV (SEQ ID 02) and 2 units of 35: 20: 1 mixture of Taq DNA polymerase: Deep Vent DNA polymerase (NEB) for 30 cycles of 94 ° C, 40 seconds; 60 ° C, 40 seconds; 72 ° C, 90 seconds; followed by an extension of 5 minutes at 72 ° C. 20 μl of reaction product were electrophoresed on a 1% agarose gel / APR and photographed, while the rest was purified on a Qiagen column in 120 μl of water.

 The TAC library product was then digested with 10 µl of NotI (NEB) (100 u) for 1 hour at 40 37 ° C in a reaction volume of 300 µl, then it was purified on a Qiagen column in a volume of 120 µl of water. The two products were then combined by restriction-ligation as follows:

 10x buffer NEB 4

 17 μl

 100 mM ATP (SIGMA)

 15 μl

 10 mg / ml acetylated BSA (NEB)

 1 μl

 RepA DNA

 40 μl

 TAC library DNA

 40 μl

 Bsp 120I (10 u / μl Fermentas)

 5 μl

 NotI (10 u / μl NEB)

 5 μl

 T4 DNA ligase (400 u / μl NEB)

 5 μl

 Water

 39 μl

 The reaction was carried out at 37 ° C for two hours. 20 μl were evaluated by gel electrophoresis, amplified by 30 μl PCR directly in 10x 50 μl reactions, and the rest was gel purified and the library band was cleaved, purified on Qiagen column and amplified by PCR in reactions 20x50 μl with primers TACFAR4 (SEQ ID 30) and ORIREV (SEQ ID 02) for 30 cycles of 94 ° C, 40 seconds; 60 ° C, 40 seconds; 72 ° C, 90 seconds; followed by an extension of 5 minutes at 5 72 ° C. The DNA was gel purified on 4 Qiagen columns and the 200 μl of eluted product was pooled for the ITT reaction / selection.

Round 1 Selection

 A reaction of 2 x 200 µl of ITT was arranged and incubated at room temperature for 1 hour as follows:

 REACTION

 l

 DNA Library 2.5x methionine buffer 100 nM Extract S30

 56 μl / 7 μg) 80 μl 2 μl 60 μl

 1 ml of blocking buffer was added to each reaction (the blocking buffer is 4% Marvel, 100 µg / ml of sperm-submitted salmon sperm DNA, 0.1% Tween 20, 2.5 mg / ml of heparin, in TBS), centrifuged at 10,000g for 2 minutes, transferred to a fresh tube, then placed on ice.

 100 µl of Bacillus globigii spore suspension (Bg) was washed twice with 1 ml of 0.1% TBS / Tween 20 and resuspended in 100 µl of Blocking buffer. This was then added to the Blocking buffer and allowed to bind at room temperature for 1 hour while mixing.

 The mixture was then centrifuged at 16,100 g for 1 minute and the spore sediment was washed six times with 1 ml of 0.1% TBS / Tween 20 mixing with a pipette and vortexed before centrifugation. Finally the sediment was washed in 1 ml of TBS and the supernatant was discarded.

 The DNA was eluted by incubation of the spores in 120 μl of 0.5 M sodium acetate pH 5.5 for 10 minutes in a mixer. The spores were centrifuged at 16,100 g for 1 minute and the supernatant was neutralized by the addition of 120 µl of Tris pH 8.0 and then extracted with phenol / CHCl3 for 5 minutes at 16,100 g. The DNA was precipitated with 20 μg of carrier glycogen and two and a half volumes of ethanol. The DNA was sedimented at 16,100 g for 20 minutes and the sediment was washed three times with 0.75 ml of 70% ethanol, centrifuging for 3 minutes at 16,100 g between each wash, then air dried and resuspended in 20 μl of water.

 10 μl of DNA recovered in 10 x 50 μl reactions were amplified by PCR with primers CISREV (SEQ ID 19) and TACFAR5 (SEQ ID 31) and 2 20: 1 mixing units of Taq DNA polymerase: DNA 30 Deep Vent polymerase (NEB) for 30 cycles of 94 ° C, 40 seconds; 60 ° C, 40 seconds; 72 ° C, 90 seconds; followed by an extension of 5 minutes at 72 ° C. The DNA was purified, precipitated in ethanol and re-suspended in 10 µl of water. 5 μl were further amplified by PCR using the above conditions but using primers NOTRECREV2 (SEQ ID 32) and TACFAR5 (SEQ ID 31) for 10 cycles. The product was purified using a Qiagen PCR purification kit and eluted in 50 µl of 35 mM Tris 5 pH 8.0.

Restriction-Ligation

 This was carried out in a 30 μl reaction for 1 hour at 37 ° C to re-anchor the RepA-CIS-ori DNA to the recovered peptides for an additional round of selection.

 10x buffer NEB 4

 3 μl

 100 mM ATP (SIGMA)

 1.5 μl

 10 mg / ml acetylated BSA (NEB)

 0.3 μl

 RepA DNA

 2 μl

 TAC library DNA

 10 μl

 Bsp 120I (10 u / μl Fermentas)

 1.5 μl

 NotI (10 u / μl NEB)

 1.5 μl

 T4 DNA ligase (400 u / μl NEB)

 1.5 μl

 Water

 8.7 μl

 20 µl of 10x 50 µl reactions were amplified by PCR with primers TACFAR5.1 (SEQ ID 33) and ORIREV (SEQ ID 02) for 20 cycles of 94 ° C, 40 seconds; 60 ° C, 40 seconds; 72 ° C, 90 seconds; followed by an extension of 5 minutes at 72 ° C. The DNA was gel purified on a Qiagen column and the eluted product was used for Round 2 of ITT reactions / selection (58 µl used in R2).

Round 2

 The second round of selection was carried out as round 1, with the following changes: Approximately 3 μg of input DNA was used. The blocking buffer used was 2% bovine serum albumin 10%, 1% gelatin, 100 µg / ml of sperm-sheared salmon sperm DNA, 2.5 mg / ml heparin, in TBS. In each selection, 10 μg of washed spores were used. Recovery PCR used primers TACFAR5.2 (SEQ ID 34) and NOTRECREV2 (SEQ ID 32). Finally, the "pull through" PCR used primers TACFAR5.2 (SEQ ID 34) and ORIREV (SEQ ID 02) for 10 cycles.

Round 3 15

 The third round of selection was carried out as round 2, with the following changes: Approximately 2.5 µg of input DNA was used. Recovery PCR used primers TACFAR6 (SEQ ID 35) and NOTRECREV2 (SEQ ID 32). Finally, the "pull through" PCR used primers TACFAR6 (SEQ ID 35) and ORIREV (SEQ ID 02) for 10 cycles.

Round 4 20

 Round 4 was carried out as round 3, except that approximately 2 µg of input DNA was used for selection. Recovery PCR used primers TAC3 (SEQ ID 09) and NOTRECREV2 (SEQ ID 32). Finally, the "pull through" PCR used primers TAC3 (SEQ ID 09) and ORIREV (SEQ ID 02) for 10 cycles.

Round 5 25

 Round 5 was carried out as round 4.

 For cloning in the form of NcoI-NotI fragments, the stored DNA recovered from round 5 was amplified by PCR with primer TAC6 (SEQ ID 26) and NOTRECREV2 (SEQ ID 32) biotinylated. Digestion with NotI was followed by purification using the Qiagen PCR purification kit, digestion with NcoI followed by incubation on a streptavidin coated plate. After purification with phenol / CHCl3 and precipitation with ethanol, the digested DNA was then ligated into a phagemid vector digested in a similar manner and transformed into E. coli ER2738, then plated on 2% glucose, 2xTY, 100 µg / ml ampicillin and incubated overnight at 37 ° C.

 Individual colonies were chosen in 200 μl of medium with 2% glucose, 2xTY, 100 μg / ml of ampicillin in 96-well plates, and grown at 37 ° C / 200 rpm for 6 hours. 10 μl 35 were transferred to a plate of deep wells containing 100 μl of 2% glucose, 2xTY, 100 μg / ml of ampicillin plus 10 μl of M13K07 adjuvant phage / wells and incubated for 1 hour without oscillatory movement at 37 ° C. 500 μl per well of medium with 2xTY, 100 μg / ml of ampicillin / 25 μg / ml of kanamycin / 20 μM of IPTG was added and incubation was carried out overnight at 37 ° C / 200 rpm.

ELISA scrutiny

 Round bottom 96-well plates were blocked with 4% Marvel in 0.1% TBS / Tween 20 in PBS for 1 hour at room temperature. The phage cultures chosen were centrifuged at 3000g for 5 minutes and the phage supernatant was analyzed in an ELISA. In each well, 50 μl of phage supernatant was mixed with 5 μl of Bg spores in 50 μl of 4% bovine serum albumin, 1% gelatin in 5 TBS and incubated while shaking at room temperature for 1 hour. The wells were washed 5 x with 200 µl of 0.1% TBS / Tween 20 by centrifugation at 3000g for 5 minutes between each wash before incubation with anti-M13 antibody conjugated to horseradish peroxidase, 0.2 µg / ml in 4% bovine serum albumin, 1% gelatin in TBS. The spores were incubated at room temperature for 1 hour while shaking. The wells were then washed 5 x with 0.1% TBS / Tween 20 and the 10 spores were transferred to a new plate. The spores were then washed once with TBS as described above before development with the TMB substrate. The development was stopped with 0.5 M H2SO4 and the solution was transferred to a new flat bottom plate for reading at 450 nm. Binding data for the selected peptides are shown in Figure 7.

Example 5. Construction, selection and screening of the CIS Presentation Library against the anti-V5 antibody

 The construction of the library was carried out as described in Example 4.

Round 1 Selection

 An in vitro transcription / translation (ITT) reaction of 1x 200 µl was established and incubated at room temperature for 1 hour as described in Example 4. 1 ml of blocking buffer was added to each reaction (buffer of Blocking is 5% skimmed milk powder, 100 μg / ml of sperm salmon DNA sheared, 2.5 mg / ml heparin, in TBS), then placed on ice.

 For the first round of selection of the library, a 70x11 mm Nunc Maxisorb Immunotube (Life Technologies, Paisley, Scotland UK) was coated with 1 ml of 10μg / ml of polyclonal V5 anti-peptide antibody (Harlan-Seralab) in PBS for 1 hour at 37 ° C. The tube was rinsed three times with PBS (full and empty) and blocked with 3 ml of blocking buffer for 1 hour at 37 ° C and washed as before. DNA-protein library complexes were added in blocking buffer, and incubated for 1 hour, leaving room temperature. The tube was washed five times with 0.1% PBS / Tween 20, then five more times with PBS only.

 The DNA was eluted in 500 µl of 1 M sodium acetate pH 5.2 for 10 minutes on the blood mixer, neutralized with 100 µM Tris-HCl pH 8.0, then extracted with phenol / CHCl3 for 5 minutes at 16,100g. The DNA was precipitated with 20 g of carrier glycogen, 1/2 volume of 7.5 M ammonium acetate, and three volumes of ethanol. The DNA was pelleted at 16,100 g for 20 minutes and the pellet was washed with 0.5 ml of 70% ethanol for 5 minutes at 16,100 g then dried under vacuum, and re-suspended in 20 µl of water. 35

 The recovered DNA was amplified by PCR in a 1x50 μl reaction with primers NOT1RECREV2 (SEQ ID 32) and TACFAR4 (SEQ ID 30) and 2 20: 1 mixing units of DNA polymerase Taq: Deep Vent DNA polymerase (NEB) for 30 94 ° C cycles, 40 seconds; 60 ° C, 40 seconds; 72 ° C, 90 seconds; followed by an extension of 5 minutes at 72 ° C. 50 µl of reaction product was electrophoresed on a 1% agarose gel / APR and photographed, then purified by GeneClean in 10 µl of water. The DNA was re-anchored to RepA DNA and re-amplified for round two as described in example 4 using primers TACFAR5 (SEQ ID31) and ORIREV (SEQ ID 02).

 The second round of selection was carried out as round 1, using the same primer pairs described in example 4, with the following changes: The coating concentration with anti-V5 antibody was reduced to 5 μg / ml. The input DNA was approximately 4 μg. The third round of selection was carried out as round 2, with the following changes: Approximately 4 μg of input DNA was used. Recovery PCR used primers TACFAR5.1 (SEQ ID 33) and NOTRECREV2 (SEQ ID 32). Finally, the "pull through" PCR used primers TACFAR6 (SEQ ID 35) and ORIREV (SEQ ID 02) for 10 cycles. Round 4 was carried out as round 3. 50

 To clone in the form of NcoI-NotI fragments, the stored DNA recovered from round 4 was amplified by PCR with the biotinylated TAC6 primers (SEQ ID 26) and NOTIREPRECREV2 (SEQ ID 32) and cloned into the phagemid vector VIII and subjected electroporation in electrocompetent E. coli TG-1 as described in example 4.

 Individual colonies were chosen in 200 µl of medium with 2% glucose, 2xTY, 100 µg / ml of 55 ampicillin in 96-well plates, and grown at 37 ° C / 200rpm for 6 hours. 100 μl was transferred to a plate of deep wells containing 100 μl of 2% glucose, 2xTY, 100 μg / ml of ampicillin plus 109 kru adjuvant phages Mru K13 / well and incubated for 1 hour without oscillatory movement at 37 ° C.

400 µl per well of 2xTY medium, 100 µg / ml ampicillin / 25 µg / ml kanamycin / 30 µM IPTG was added and phage amplification continued for 16 hours at 37 ° C while shaking at 200 rpm. Bacterial cultures were centrifuged in microtiter plate carriers at 2000 g for 10 minutes at 4 ° C in a tabletop centrifuge to sediment the bacteria and the culture supernatant was used for the ELISA.

 A NUNC Maxisorp ELISA plate was coated with 100 ng / well of anti-V5 peptide antibody in 5 100 µl / well for one hour at 37 ° C. The plate was washed 2x200 µl / well and blocked for 1 hour at 37 ° C with 200 µl / well of 2% BSA / PBS and then washed with 2x200 µl / well of PBS. 50 µl of phage culture supernatant was added to each well containing 50 µg / well of 4% BSA / PBS, and allowed to bind for 1 hour at room temperature. The plate was washed twice with 200 µl / well of PBS / 0.1% Tween 20, then twice with 200 µl / well of PBS. The bound phages were detected with 10 100 µl / well, anti-M13-HRP conjugate product diluted 1: 5,000 (Amersham-Pharmacia) in 2% BSA / PBS for 1 hour at room temperature and the plate washed four times as before. The plate was developed for 5 minutes at room temperature with 100 µl / well of TMB substrate buffer (3,3 ', 5,5'-Tetramethylbenzidine). The reaction was stopped with 100 µl / well of 0.5 N H2SO4 and read at 450 nm. The phagemid DNA of the ELISA positive clones were then sequenced with conventional direct and reverse pUC sequencing primers. The amino acid sequence of these isolated clones is shown below. Four positive ELISA clones were grown in culture volumes of 10 ml and phage particles were precipitated with PEG-NaCl and resuspended in 1 ml of PBS and retested in 50 μl ELISA as tested. described above. OD signals at 450 nm against anti-V5 antibody and anti-ACTH control are shown in Figure 8. 20

Isolated peptide sequences after selection:

P1C12 (SEQ ID 36) CGCPTMAARVRPVLNSKH

P2H1 (SEQ ID 37) MTTVPVLMISV

P1B5 (SEQ ID 38) TLSTRHHNVIDRFNLRNF

P2B8 (SEQ ID 39) SIRTLTGSTPAQFDATAD 25

Example 6. Selection of ovalbumin binding peptides from a CIS presentation library

 For any selection methodology it is important that the selected entities are able to bind to the selected target, regardless of the carrier molecule associated with it during selection and screening. In this example, the peptides are selected and synthesized to allow confirmation of target binding. The construction of a 1230 random unit peptide library was carried out as described in Example 3. Four rounds of selection were carried out as described in example 4 with 100 μg / ml ovalbumin ( SIGMA, Dorset, UK applied as a coating on immunotubes.

 To clone in the form of NcoI-NotI fragments, the DNA recovered from round 4 was amplified by PCR with the biotinylated TAC6 primers (SEQ ID 26) and NOTIREPRECREV2 (SEQ ID 32) and cloned 35 in a pVIII phagemid vector and electroporated in E. coli electrocompetent TG-1, as described in example 4.

 The individual colonies were chosen in 200 μl of medium with 2% glucose, 2xTY, 100 μg / ml of ampicillin in 96-well plates, and grown at 37 ° C / 200 rpm for 6 hours. 100 µl was transferred to a deep well plate containing 100 µl of 2% glucose, 2xTY, 100 µg / ml of ampicillin 40 plus 109 kru adjuvant phages M13K07 / well and incubated for 1 hour without oscillatory movement at 37 ° C . 400 µl per well of medium with 2xTY, 100 µg / ml ampicillin / 25 µg / ml kanamycin / 20 µM IPTG was added and phage amplification continued for 16 hours at 37 ° C while shaking at 200 rpm. Bacterial cultures were centrifuged in carriers for microtiter plates at 2,000 g for 10 minutes at 4 ° C in a tabletop centrifuge to sediment the bacteria and the culture supernatant was used for an ELISA.

 A NUNC Maxisorp ELISA plate was coated with 100 μg / well of ovalbumin in 100 μl / well of PBS overnight at 4 ° C. The plate was washed with 2x200 µl / well and blocked for 1 hour at 37 ° C with 200 µl / well of 2% BSA / PBS and then washed with 2x200 µl / well of PBS. 50 µl of phage culture supernatant was added to each well containing 50 µl / well of 4% BSA / PBS, and 50 were allowed to bind for 1 hour at room temperature. The plate was washed twice with 200 μl / well of PBS / 0.1% Tween 20, then twice with 200 μl / well of PBS. The bound phages were detected with 100 µl / well of anti-M13-HRP conjugate product diluted 1: 5000 (Amersham-Pharmacia) in BSA // 2% PBS for 1 hour at room temperature and the plate washed four times like before. The plate was developed for 5 minutes at room temperature with 100 µl / well of TMB 55 substrate buffer (3,3 ', 5,5'-Tetramethylbenzidine). The reaction was stopped with 100 µl / well of 0.5 N H2SO4 and read at 450 nm. The phagemid DNA of the ELISA positive clones was then sequenced with the M13REV primer. The amino acid sequence of these isolated clones is shown below.

C1 (SEQ ID 40) ANLWRIVLHGWW

C4 (SEQ ID 41) VSFMLLGPHRHR

C6 (SEQ ID 42) LVLHWLSLGSR

C8 (SEQ ID 43) SNQVVLILHLRP

Control (SEQ ID 44) AESWLHQSWIHL 5

 Peptide sequences of four representative positive ELISA clones (SIGMA-Genosys Ltd) were synthesized with biotin added to the C-terminus to aid detection in the ELISA. These peptides were tested in an ELISA against ovalbumin, together with a control peptide previously isolated by selection by phage display against B. globigii spores. A NUNC Maxisorp ELISA plate was coated with 100 µg / well of ovalbumin in 100 µl / well of PBS, or, 200 ng / well 10 of polyclonal anti-V5 antibody in PBS, overnight at 4 ° C. The plate was washed with 2x200 µl / well of PBS and blocked for 1 hour at 37 ° C with 200 µl / well of 2% skimmed milk powder / PBS and then washed with 2x200 µl / well of PBS. 1 µg of diluted peptides was added to each well in 100 µl / well of 2% BSA / PBS, and allowed to bind for 1 hour at room temperature. The plate was washed twice with 200 µl / well of PBS / 0.1% Tween 20, then twice with 200 µl / well of PBS. The bound peptides were detected with 100 µl / well of streptavidin-HRP conjugate product diluted 1: 2000 (Pierce) in 2% BSA / PBS for 1 hour at room temperature and the plate was washed four times as before. The plate was developed for 5 minutes at room temperature with 100 µl / well of TMB substrate cap (3,3 ', 5,5'-Tetramethylbenzidine). The reaction was stopped with 100 µl / well H2SO4 0.5 N and read at 450 nm (Figure 9). twenty

Example 7. Presentation of single chain Fv antibody (scFv) fragments in a CIS presentation system

 A tac-scFv-RepA-CIS-ori construct was constructed by prolongation of the PCR overlap essentially as previously described in Example 1. Anti-mecoprop scFv DNA (Haptogen Ltd, Aberdeen, UK) was amplified in a volume of 50 μl reaction containing 200 μM dNTP, 1x 25 amp buffer with NEB polymerase (10 mM KCl, 10 mM (NH4) 2SO4, 20 mM Tris-HCl pH 8.8, 2 mM MgS04, Triton X-100 at 0, 1%) with 10 pmoles of each of the primers TACMECOFOR (SEQ ID 45) and REPAMECOBAK (SEQ ID 46) and 2 20: 1 mixing units of Taq DNA polymerase: Deep Vent DNA polymerase (NEB) for 30 cycles of 94 ° C, 40 seconds; 60 ° C, 40 seconds; 72 ° C, 80 seconds; followed by an extension of 5 minutes at 72 ° C. The products were electrophoresed on a 30% agarose gel / APR and purified with a Geneclean II kit in 20 µl of water. This was assembled with RepA-CIS-ori DNA generated with ORIREV408 (SEQ ID 20) and MECOREPAFOR (SEQ ID 47), and Tac promoter DNA generated with TACFARUP (SEQ ID 23) and MECOTACBAK (SEQ ID 48) in a volume of 50 µl reaction containing 200 µM dNTP, 1x Amplification buffer with NEB polymerase (10 mM KCl, 10 mM (NH4) 2SO4, 20 mM Tris-HCl pH 8.8, 2 mM MgSO4, 0.1% TritonX-100 ) with 10 pmoles of each of the primers TAC3 (SEQ ID 35 09) and ORIREV (SEQ ID 02) and 2 20: 1 mixing units of Taq DNA polymerase: Deep Vent DNA polymerase (NEB) for 30 cycles of 94 ° C, 40 seconds; 60 ° C, 40 seconds; 72 ° C, 80 seconds; followed by an extension of 5 minutes at 72 ° C. The products were electrophoresed on a 1% agarose gel / APR and purified with a Geneclean II kit in 20 µl of water.

 DNA was reamplified in 10x 50 µl reactions containing 200 µM dNTP, 1x NEB 40 polymerase amplification buffer (10 mM KCl, 10 mM (NH4) 2SO4, 20 mM Tris-HCl pH 8.8, 2 mM MgSO4, Triton 0.1% X-100) with 10 pmoles of each of the primers TAC3 (SEQ ID 09) and ORIREV (SEQ ID 02) and 2 20: 1 mixing units of Taq DNA polymerase: Deep Vent DNA polymerase (NEB ) for 30 cycles of 94 ° C, 40 seconds; 60 ° C, 40 seconds; 72 ° C, 80 seconds; followed by an extension of 5 minutes at 72 ° C. The products were electrophoresed on a 1% agarose gel / APR and purified with a Geneclean II kit in 100 µl of water.

 The ScFv DNAs were then translated under the following two reaction conditions:

 REACTION

 l 2

 Tac-scFv-RepA DNA 2.5x 10 mM methionine buffer H2O Extract S30

 28 μl (1 μg) 40 μl 1 μl 1 μl 30 μl 28 μl (1 μg) 40 μl 1 μl - 30 μl

 The reactions were incubated at 30 ° C for 30 minutes after 1 µl of 0.25 M oxyglutathione was added to reaction 2 and incubation at 30 ° C continued for a further 30 minutes. 1 ml of blocking buffer was added to each reaction (The blocking buffer is 1% gelatin, 100 µg / ml of sperm-treated salmon sperm DNA, 2.5 mg / ml of heparin, in TBS), centrifuged at 10,000 g for 2 minutes, then placed on ice.

 NUNC star immunotubes were coated with 0.5 ml of 10 µg / ml BSA-mecoprop conjugate product, or 10 µg / ml of BSA in PBS for 1 hour at 37 ° C. The tubes were washed 2x PBS, then blocked for 1 hour at room temperature with 3 ml of blocking buffer in a blood mixer, then the 2x PBS tubes were washed. 10

 0.5 ml of each diluted ITT was added to a tube coated with BSA blocked or coated with BSA-mecoprop and incubated at room temperature for 1 hour. The tubes were washed 5x TBS / 0.1% Tween 20, 5x TBS.

 The bound DNA was eluted for 10 minutes at room temperature with 0.5 ml of 0.5 M NaCl / 10 mM Tris pH 8, 1 mM EDTA, then extracted with 0.5 mL of phenol / chloroform and made precipitate with 15 20 μg of carrier glycogen, 1/2 volume of 7.5 M ammonium acetate, and three volumes of ethanol. The DNA was pelleted at 14,000 g for 20 minutes and then the sediment was washed with 0.5 ml of 70% ethanol for 5 minutes at 14,000 g then dried under vacuum, and resuspended in 20 µl of water.

 10 µl of DNA recovered by PCR was amplified in a 50 µl reaction volume containing 200 µM dNTP, 1x amplification buffer with NEB polymerase (10 mM KCl, 10 mM (NH4) 2SO4, 20 mM Tris-20 HCl pH 8, 8, 2 mM MgSO4, 0.1% Triton X-100) with 10 pmoles of each of the TACMECOFOR primers (SEQ ID 45) and REPAMECOBAK (SEQ ID 46) and 2 20: 1 mixing units of Taq DNA polymerase : Deep Vent DNA polymerase (NEB) for 30 cycles of 94 ° C, 40 seconds; 60 ° C, 40 seconds; 72 ° C, 80 seconds; followed by an extension of 5 minutes at 72 ° C. The products were electrophoresed on a 1% agarose gel / APR and photographed. Higher amounts of DNA were observed from the 25 selections on antibody target than with the recovery of the BSA coated tubes, indicating that scFv-RepA-functional DNA complexes were being selected (Figure 10).

SEQUENCE LIST

<110> ISOGENICA LIMITED

 5

<120> METHOD OF PRESENTATION OF A BANK OF PEPTIDES

<130> N.86234C SER

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gctacgttga atccagtcct aggagag 27

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catattgtcg ttagaacgcg gc 22 10

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cgtaagccgg tactgattga 20

<210> 21 5

<211> 110

<212> DNA

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<220> 10

<223> Primer

<400> 21

 fifteen

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<211> 20

<212> DNA

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<220>

<223> Primer

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<221> miscellaneous characteristic 25

<222> (29) .. (30)

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<221> miscellaneous feature 30

<222> (32) .. (33)

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<221> miscellaneous feature 35

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<223> n = a, g, c or t

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<221> miscellaneous feature

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<220>

<221> miscellaneous feature

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<220>

<221> miscellaneous feature

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<220>

<221> miscellaneous feature

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<223> n = a, g, c or t

<220>

<221> miscellaneous feature

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<220>

<221> miscellaneous feature

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<223> n = a, g, c or t

<220>

<221> miscellaneous feature

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<223> n = a, g, c or t

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<220>

<221> miscellaneous feature

<222> (62) .. (63)

<223> n = a. g, c or t

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<212> DNA

<213> Artificial sequence

<220>

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<211> 2390

<212> DNA

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<220>

<221> miscellaneous feature

<222> (698) .. (699)

<223> n = a, g, c or t 10

<220>

<221> miscellaneous feature

<222> (701) .. (702)

<223> n = a, g, c or t 15

<220>

<221> miscellaneous feature

<222> (704) .. (705)

<223> n = a, g, c or t 20

<220>

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<223> n = a, g, c or t 25

<220>

<221> miscellaneous feature

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<223> n = a, g, c or t 30

<220>

<221> miscellaneous feature

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<220>

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<223> n = a, g, c or t

<220> 5

<221> miscellaneous feature

<222> (719) .. (720)

<223> n = a, g, c or t

<220> 10

<221> miscellaneous feature

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<223> n = a, g, c or t

<220> 15

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<222> (725) .. (726)

<223> n = a, g, c or t

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<212> DNA

<213> Artificial sequence

<220> 10

<223> Primer

<400> 27

ggtagatcct gtttcctgtg tg 22

 fifteen

<210> 28

<211> 110

<212> DNA

<213> Artificial sequence

 twenty

<220>

<223> Primer

<220>

<221> miscellaneous characteristic 25

<222> (37) .. (38)

<223> n = a, g, c or t

<220>

<221> miscellaneous feature 30

<222> (40) .. (41)

<223> n = a, g, c or t

<220>

<221> miscellaneous feature 35

<222> (43) .. (44)

<223> n = a, g, c or t

<220>

<221> miscellaneous feature

<222> (46) .. (47)

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<220>

<221> miscellaneous feature

<222> (49) .. (50)

<223> n = a, g, c or t 10

<220>

<221> miscellaneous feature

<222> (52) .. (53)

<223> n = a, g, c or t 15

<220>

<221> miscellaneous feature

<222> (55) .. (56)

<223> n = a, g, c or t 20

<220>

<221> miscellaneous feature

<222> (58) .. (59)

<223> n = a, g, c or t 25

<220>

<221> miscellaneous feature

<222> (61) .. (62)

<223> n = a, g, c or t 30

<220>

<221> miscellaneous feature

<222> (64) .. (65)

<223> n = a, g, c or t 35

<220>

<221> miscellaneous feature

<222> (67) .. (68)

<223> n = a, g, c or t

<220> 5

<221> miscellaneous feature

<222> (70) .. (71)

<223> n = a, g, c or t

<220> 10

<221> miscellaneous feature

<222> (73) .. (74)

<223> n = a, g, c or t

<220> 15

<221> miscellaneous feature

<222> (76) .. (77)

<223> n = a, g, c or t

<220> 20

<221> miscellaneous feature

<222> (79) .. (80)

<223> n = a, g, c or t

<220> 25

<221> miscellaneous feature

<222> (82) .. (83)

<223> n = a, g, c or t

<220> 30

<221> miscellaneous feature

<222> (85) .. (86)

<223> n = a, g, c or t

<220> 35

<221> miscellaneous feature

<222> (88) .. (89)

<223> n = a, g, c or t

<400> 28

 5

<210> 29

<211> 49

<212> DNA

<213> Artificial sequence

 10

<220>

<223> Primer

<400> 29

ctggagatgg catcaagggc cccaactgat cttcaccaaa cgtattacc 49 15

<210> 30

<211> 20

<212> DNA

<213> Artificial sequence 20

<220>

<223> Primer

<400> 30 25

ggcgctatca tgccataccg 20

<210> 31

<211> 20

<212> DNA 30

<213> Artificial sequence

<220>

<223> Primer

 35

<400> 31

accattcggc tagcgatgac 20

<210> 32

<211> 33

<212> DNA 5

<213> Artificial sequence

<220>

<223> Primer

 10

<400> 32

ggtgaagatc agttgcggcc gctgatcctc ctc 33

<210> 33

<211> 22 15

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 20

<400> 33

gattggttcg ctgaccattt cc 22

<210> 34 25

<211> 21

<212> DNA

<213> Artificial sequence

<220> 30

<223> Primer

<400> 34

cggcgttacc agaaactcag a 21

 35

<210> 35

<211> 20

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 5

<400> 35

aaccgactct gacggcagtt 20

<210> 36 10

<211> 18

<212> PRT

<213> Artificial sequence

<220> 15

<223> Peptide

<400> 36

 twenty

<210> 37

<211> 11

<212> PRT

<213> Artificial sequence

 25

<220>

<223> Peptide

<400> 37

 30

<210> 38

<211> 18

<212> PRT

<213> Artificial sequence

<220>

<223> Peptide

 5

<400> 38

<210> 39

<211> 18 10

<212> PRT

<213> Artificial sequence

<220>

<223> Peptide 15

<400> 39

<210> 40 20

<211> 12

<212> PRT

<213> Artificial sequence

<220> 25

<223> Peptide

<400> 40

<210> 41

<211> 12

<212> PRT

<213> Artificial sequence

 5

<220>

<223> Peptide

<400> 41

 10

<210> 42

<211> 11

<212> PRT

<213> Artificial sequence 15

<220>

<223> Peptide

<400> 42 20

<210> 43

<211> 12

<212> PRT 25

<213> Artificial sequence

<220>

<223> Peptide

 30

<400> 43

<210> 44

<211> 12

<212> PRT

<213> Artificial sequence

 5

<220>

<223> Peptide

<400> 44

 10

<210> 45

<211> 42

<212> DNA

<213> Artificial sequence

 fifteen

<220>

<223> Primer

<400> 45

caggaaacag gatctaccat gctgcaggag tcaggacctg ag 42 20

<210> 46

<211> 53

<212> DNA

<213> Artificial sequence 25

<220>

<223> Primer

<400> 46 30

gtttggtgaa gatcagttga tcctcctccc ccccgctcga ggaagatgga tac 53

<210> 47

<211> 53

<212> DNA 35

<213> Artificial sequence

<220>

<223> Primer

 5

<400> 47

gtatccactct tcctcgagcg ggggggagga ggatcaactg atcttcacca aac 53

<210> 48

<211> 42 10

<212> DNA

<213> Artificial sequence

<220>

<223> Primer 15

<400> 48

ctaggtcct gactcctgca gcatggtaga tcctgtttcc tg 42

Claims (41)

 1. A method for producing an in vitro peptide expression library comprising a plurality of peptides, where each peptide is attached to the DNA construct encoding the peptide, which comprises the steps of:  (a) provide a DNA construct comprising:   (i) a target DNA sequence;   (ii) DNA encoding a peptide member of the library; Y   (iii) DNA encoding a peptide capable of non-covalently binding directly or indirectly to said DNA target sequence of (i); where said DNA construct and encoded protein are selected to have cis activity  (b) express a plurality of DNA constructs according to (a) wherein said DNA constructs encode a plurality of peptide members of the library so that each expressed peptide is non-covalently bound to the DNA from which it was produced.  2. A method according to claim 1, wherein said DNA construct further comprises:   (iv) a DNA element that directs cis activity.  3. A method according to claim 2, wherein said DNA construct of (a) further comprises   (v) DNA encoding a fragment comprising at least 20 20 C-terminal amino acids of a repA protein where said fragment is capable of interacting with said DNA element of (iv); wherein said DNA element of (iv) is located 3 'with respect to said DNA of (ii), (iii) and (v).  4. A method according to any one of the preceding claims, wherein the peptide encoded by said DNA of (iii) is capable of directly recognizing and binding to said DNA target sequence of (i).   5. A method according to claim 4, wherein the peptide encoded by said DNA from (iii) is a repA protein and wherein said DNA target sequence from (i) is ori.  6. A method according to claim 4 or 5, wherein said DNA from (ii) is connected to said DNA from (i) and (iii) by digestion with restriction enzymes and ligation. 30  7. A method according to any one of claims 3 to 6, wherein said repA is selected from repA of the complex plasmids IncI and repA of the plasmids IncF, IncB, IncK, IncZ and IncL / M.  8. A method according to claim 5, wherein said DNA construct comprises the sequence encoding repA, the cis DNA element and the ori DNA of the Rl plasmid of IncFII.  9. A method according to any one of the preceding claims wherein said repA protein has the sequence given in SEQ ID NO: 16 and wherein said cis DNA element has the sequence given in SEQ ID NO: 17.  10. A method according to any one of the preceding claims wherein the DNA not bound by the peptide encoded by said DNA of (iii) is linked by a non-specific DNA binding protein.  11. A method according to claim 4, wherein the peptide encoded by said DNA of (iii) 40 is an estrogen receptor DNA binding domain and wherein said DNA target sequence of (i) is a target sequence of the estrogen receptor  12. A method according to claim 9, wherein said DNA binding domain comprises amino acids 176 to 282 of the estrogen receptor DNA binding fragment and wherein said DNA target sequence comprises the given estrogen receptor target sequence in SEQ ID NO: 14. 45  13. A method according to claim 1, 2 or 3, wherein the peptide encoded by said DNA of (iii) is indirectly linked to said DNA target sequence of (i) by means of an agent, wherein said agent binds to said DNA target sequence of (i) and wherein said agent also binds to the peptide encoded by said DNA of (iii).  14. A method according to claim 13, wherein said DNA target sequence comprises a DNA tag capable of being bound by said agent, said tag being optionally selected from biotin and fluorescein. 5  15. A method according to claim 13 or 14, wherein the binding activities of said agent are conferred by means of two antibodies or their fragments.  16. A method according to claim 15, wherein one or both of said binding activities are conferred by means of a Fab fragment.  17. A method according to any one of claims 13 to 16, wherein said agent is provided before step (b).  18. A method according to claim 13, wherein said agent is linked to said DNA target sequence of (i) and is capable of binding to the peptide encoded by said DNA of (iii).  19. A method according to claim 18, wherein said agent is a polymer.  20. A method according to any one of the preceding claims, wherein said DNA is under the control of promoter and translation sequences suitable to allow transcription and translation in vitro.  21. A method according to any one of the preceding claims, wherein said peptide member of the library is an antibody or one of its fragments.  22. A method according to any one of the preceding claims, wherein said library comprises at least 104 molecules.  23. A method according to any one of the preceding claims, wherein said expression is carried out in the presence of a compound that avoids nuclease activity, or reduces non-specific DNA-protein or protein-protein interactions.  24. A method according to any one of the preceding claims, wherein said expression is carried out in a coupled bacterial transcription / translation environment.  25. A method according to claim 24, wherein said coupled bacterial transcription / translation environment is the S30 extract system.  26. A method for producing an in vitro peptide expression library comprising a plurality of peptides, where each peptide is connected to the DNA construct encoding the peptide, which comprises the steps of:  (a) provide a DNA construct comprising:   (i) DNA encoding a peptide member of the library; Y   (ii) DNA encoding a peptide capable of binding to an agent; wherein said DNA construct and encoded protein are selected to have cis activity;  (b) binding said agent or a DNA tag capable of binding an agent to said DNA construct of (a), wherein said agent is capable of binding to the peptide encoded by said DNA of (ii); Y  (c) express a plurality of DNA constructs according to (b), 40 wherein said DNA constructs encode a plurality of peptide members of the library so that each expressed peptide is connected by said agent to the DNA from which it was produced.  27. A method of identifying and / or purifying a peptide that shows the desired properties from an in vitro peptide expression library produced in accordance with the method of any one of the preceding claims, comprising at least the steps of (a) scrutinizing said library and (b) selecting and isolating the member of the relevant library.  28. A method of identifying a specific ligand binding peptide, said method comprising at least the steps of (a) screening an in vitro peptide expression library produced in accordance with the method of any one of claims 1 to 26 with ligand molecules that are optionally bound to a solid support; (b) select and isolate a member of the library that binds to said ligand molecule; and (c) isolating the peptide that specifically binds to said ligand molecule. 5  29. A method according to claim 27 or 28, wherein said library member peptides are antibodies or their fragments.  30. A method of identifying and / or purifying a peptide that has the ability to bind to a specific DNA target sequence comprising at least the steps of  (a) provide an in vitro expression library in accordance with a any one of claims 1 to 26, wherein the peptide encoded by the DNA of (iii) is a library member peptide that has DNA binding activity and wherein said DNA target sequence of (i) is the target sequence of interest ;  (b) selecting and isolating a member of the library in which the encoded protein binds to said target sequence; and 15  (c) isolate the peptide that binds to said target sequence.  31. A method according to claim 30, wherein said library member peptides are zinc finger proteins, helix-loop-helix proteins or helix-spin-helix proteins.   32. A method according to any one of claims 27 to 31, wherein said screening and / or selection step is carried out in the presence of a compound that avoids nuclease activity or reduces DNA-protein or protein-protein interactions. non-specific protein  33. A method according to claim 32, wherein said compound is heparin.  34. A method according to any one of claims 27 to 33, wherein additionally the DNA expressing said isolated peptide is isolated.  35. A method according to claim 34, further comprising cloning said DNA 25 into an expression vector.  36. A method according to claim 35, further comprising introducing said expression vector into an in vitro cell.  37. A method according to claim 35 or 36, further comprising expressing the peptide encoded by said DNA. 30  38. An in vitro peptide expression library produced according to the method of any one of claims 1 to 26.  39. A DNA construct as described in any one of claims 1 to 26.  40. A method according to any one of claims 1 to 30, wherein said peptides or peptides members of the library are anchored to polyethylene glycol. 35