Bacterial pilus protein complex fimgt-dsf stabilized protein complex for production of filamentous phage

Abstract

To provide an improved stabilized protein complex for production of a phagemid or a filamentous phage, and methods for production and use thereof.SOLUTION: A bacterial pilus protein complex FimGt-DsF stabilized protein complex for production of a phagemid or a filamentous phage comprises a bacterial DsF polypeptide required for binding to FimGt and a bacterial FimGt polypeptide or a bacterial homolog thereof required for binding to DsF.SELECTED DRAWING: Figure 1-A

Description

[Technical field]

[0001] The present invention relates to the bacterial pilus protein complex FimGt-DsF stabilizing protein complex for producing phagemids or filamentous phages, and methods of use thereof. [Background technology]

[0002] 2. Background of the Invention Phage display is a powerful method for selecting improved binders. Phage display studies often involve the use of the filamentous phage M13, which infects E. coli, because of the relative ease of working with M13 compared to other phages. Filamentous phages such as M13 do not lyse the host cell, but are instead released by secretion. This allows for easier and more efficient purification of the phage particles from potentially interfering cytoplasmic proteins. Display of a polypeptide of interest (POI) on M13 is most commonly achieved by fusion to the N-terminus of the minor coat protein pIII, although fusions to other coat proteins (pVI, pVII, pVIII and pIX) have also been used.

[0003] Phage display has been widely used for the successful selection of antibody and peptide libraries. However, conventional phage display shows some limitations that prevent its widespread use. One drawback is the lack of robust N-terminal display (defined as display by attachment of the N-terminus of POI) for applications that are not suitable for C-terminal display, such as display of proteins that require a free C-terminus for interaction, display of cDNA libraries, and proteins that are not tolerant of C-terminal fusions.

[0004] WO 2018 / 041740 discloses a non-covalent display system for use in immobilizing proteins on the surface of a cell, comprising a protein of interest (POI) fused to a first binding moiety, the first binding moiety comprising one partner of the FimGT / DsF interaction pair.

[0005] WO 2012 / 028697 discloses a donor strand (Ds) complementation-based system comprising a Ds tag and its cognate ligand as protein tags and affinity ligands for use in immobilization and / or affinity purification procedures.

[0006] Giese et al. (“The Most Stable Protein-Ligand Complex: Applications for One-Step Affinity Purification and Identification of Protein Assemblies, ANGEWANDTE CHEMIE INTERNATIONAL EDITION, vol. 51, no. 18, 27 April 2012, pp. 4474-4478) discloses the use of the FimGT / DsF system for one-step affinity purification and identification of protein assemblies.

[0007] N-terminal fusion of proteins to the C-terminus of pIII, pVI, pVIII and pIX has been demonstrated, but the display levels were low (Jespers et al. Bio / Technology 1995, 13, 378; Velappan et al., Nucleic Acid Res. 2010, 38, 4, e22) and have only been successfully demonstrated for peptides or small proteins (Fuh et al. FEBS Letters 2000, 480, 231; Fuh, Germaine and Sidhu, Sachde v S.(2000),Efficient phage display of polypeptides fused to the carboxy-terminus of the M13 gene-3 minor coat protein, FEBS Letters, 480, doi:10.1016 / S0014-5793(00)01946-3;25 Velappan et al. Nucleic Acid Res. 2010, 38, 4, e22), and / or required personalized optimization (Held et al., J Mol Biol. 2004, 340(3), 587). Summary of the Invention

[0008] It is therefore an object of the present invention to provide improved stabilized protein complexes for producing phagemids or filamentous phages, and methods for their production and use.

[0009] Other objects and advantages will become apparent to those skilled in the art upon review of this specification of the present invention.

[0010] In a first aspect of the present invention, the above object is achieved by providing a protein complex comprising:

[0011] a) a first polypeptide chain having, in the N-terminal to C-terminal direction, the general formula (I) X-DsF-Y-POI(I), and b) a second polypeptide chain having in the N-terminal to C-terminal direction the general formula (II) X-FimGt-Y-CPF(II), [In the formula, X is absent or represents a bacterial leader sequence or a translocation sequence, DsF represents the bacterial DsF polypeptide or a bacterial homologue thereof required for binding to FimGt; Y is absent or represents at least one of a linker sequence, a detectable peptide sequence and / or a peptide sequence for purifying the polypeptide chain, POI indicates protein of interest, FimGt denotes the bacterial FimGt polypeptide or a bacterial homolog thereof required for binding to DsF; CPF indicates filamentous phage coat protein].

[0012] In a second aspect of the present invention, the above object is achieved by providing a protein complex comprising:

[0013] a) a first polypeptide chain having, in the N-terminal to C-terminal direction, the general formula (III) X-SUB-DsF-Y(III), and b) a second polypeptide chain comprising at least one POI and having, in the N-terminal to C-terminal direction, the general formula (IV) X-FimGt(POI)-Y(POI)-CPF(IV), [In the formula, X is absent or represents a bacterial leader sequence or a translocation sequence, DsF represents the bacterial DsF polypeptide or a bacterial homologue thereof required for binding to FimGt; Y(POI) is absent or a branched linker sequence to which the POI is attached, a detectable indicating at least one peptide sequence and / or a peptide sequence for purifying said polypeptide chain, POI indicates protein of interest, FimGt(POI) denotes a bacterial FimGt polypeptide or a bacterial homologue thereof required for binding to DsF, optionally linked to a POI; SUB represents a substrate or a ligand; CPF indicates filamentous phage coat protein].

[0014] Advantageously, the protein complex according to the invention is stabilized or essentially stabilized by donor strand complementarity between the independent adjacent polypeptide sequences of formulas I and II, and formulas III and IV, respectively.

[0015] In a third aspect of the present invention, the above object is solved by providing a filamentous phage displaying at least one protein of interest (POI), including a protein complex according to the present invention. Preferred is a library of filamentous phages according to the present invention, displaying variants of the POI and / or substrate and / or ligand (SUB).

[0016] In a fourth aspect of the present invention, the above object is achieved by providing a nucleic acid encoding the first or second polypeptide chain of the protein complex of the present invention, or a nucleic acid encoding the first or second polypeptide chain of the protein complex of the present invention. A bicistronic nucleic acid encoding the first and second polypeptide chains of the protein complex according to the present invention, or a bicistronic nucleic acid encoding the first and second polypeptide chains of the protein complex according to the present invention is preferred. A library of nucleic acids according to the present invention is further preferred.

[0017] In a fifth aspect of the present invention, the above object is solved by providing a phagemid comprising a nucleic acid according to the present invention or a library of phagemids comprising a nucleic acid according to the present invention.

[0018] In a sixth aspect of the present invention, the above object is solved by providing a method for producing a phagemid according to the present invention as disclosed herein, or a method for producing a library of phagemids according to the present invention.

[0019] In a seventh aspect of the present invention, the above object is solved by providing a method for producing a filamentous phage according to the invention as disclosed herein, or a method for producing a library of filamentous phages according to the invention.

[0020] In an eighth aspect of the present invention, the above object is solved by providing a method for screening for proteins of interest (POI) that specifically interact with a substrate or ligand, comprising a) providing a phage library according to the present invention, b) contacting said substrate or ligand with said library of a), c) determining an interaction, preferably a specific interaction, between said substrate or ligand and said library, and d) identifying a POI based on said interaction, preferably said specific interaction. Preferably, said POI is an antibody or a fragment thereof, said method comprises biopanning or said POI is a polymerase or a truncated form thereof.

[0021] In a ninth aspect of the present invention, the above object is solved by the use of a protein complex according to the present invention, a nucleic acid according to the present invention or a library of nucleic acids according to the present invention for producing a phagemid or a filamentous phage as disclosed herein.

[0022] As mentioned above, in its first aspect, the present invention provides a first polypeptide chain having, in the N-terminal to C-terminal direction, the general formula (I): X-DsF-Y-POI(I), and a second polypeptide chain having the general formula (II) in the N-terminal to C-terminal direction Concerning a protein complex containing X-FimGt-Y-CPF(II).

[0023] Surprisingly, it was found that proper functional phage display of a POI (e.g., a DNA polymerase) depends on its attachment to the phage. When the POI is fused at its C-terminus, it largely loses activity. This was not observed when N-terminal fusions were used.

[0024] In formulas I and II, X is absent or represents a bacterial leader sequence or translocation sequence. Preferably, the bacterial leader sequence or translocation sequence is selected from leader sequences for secretion by secretory pathways or translocation sequences for translocation by translocation systems, such as PelB, DsbA, TorA, and PhoA, and the general secretion (Sec) pathway, twin arginine translocation (Tat) pathway, T2SS pathway, T3SS pathway, T5SS pathway, and SecA2 pathway. Suitable signal sequences and their design can be based on the respective literature and the knowledge of those skilled in the art, for example, Sec and Tat signal peptides have a similar tripartite overall structure consisting of a positively charged n region, a central hydrophobic h region, and a polar c region containing a recognition site (consensus: AXA) for signal peptidase (SPase; cleavage site is indicated by an arrow). In the Tat signal peptide, a characteristic amino acid consensus motif containing two highly conserved arginine residues (underlined) is present at the boundary between the often significantly longer n and h regions. Furthermore, the h-region of Tat signal peptides is in most cases less hydrophobic than that found in Sec signal peptides, and the c-region of Tat signal peptides often contains positively charged amino acids (so-called Sec avoidance motifs) that prevent mistargeting of Tat substrates to the Sec pathway (see, e.g., Freudl, R. Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact 17, 52 (2018). https: / / doi.org / 10.1186 / s12934-018-0901-3 and Green ER, Mecsas J. Bacterial Secretion Systems: An Overview. Microbiol Spectr. 2016; 4 (1): 10.1128 / microbiolspec.VMBF-0012-2015. doi: 10.1128 / microbiolspec.VMBF-0012-2015).Signal peptides can also be mutated and optimized for the host (see, e.g., Han S, Machhi S, Berge M, Xi G, Linke T, Schoner R. Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxin. H35L in Escherichia coli. AMB Express. 2017;7(1):93. doi:10.1186 / s13568-017-0394-1).

[0025] The present invention is 3 x 10 9 It takes advantage of the infinite kinetic stability of the bacterial pilus protein complex FimGt-DsF, which exhibits an extrapolated dissociation half-life of years (Puorger et al., Structure 2008, 16, 631). The use of FimGt-DsF for phage display in this embodiment overcomes the herein described limitations of conventional phage display by providing an extremely stable and specific linkage that allows binding of the POI via its N-terminus and independent translocation of the POI and phage protein with a combination of signal sequences for different secretion pathways.

[0026] In Formula I and Formula II, FimGt is required for binding to DsF or its bacterial homologues. wherein FimG represents the bacterial FimG polypeptide (bacterial type 1 pilus subunit FimG) and DsF (the N-terminal extension of the partner subunit FimF (called the donor strand, Ds)) represents the bacterial DsF polypeptide required for binding to FimG, FimGt or bacterial homologues thereof. Preferably, said bacterial DsF and FimGt polypeptides are selected from E. coli or from homologues of DsF and / or FimGt from Gram-negative bacteria, in particular from the family Enterobacteriaceae.

[0027] The term FimGt is intended to include the full length sequence of FimG or its bacterial homologues, as well as FimG variants, particularly variants that exhibit improved binding (see below), such as an N-terminal deletion variant in which residues 1-12 are truncated, optionally with the substitution Q134E. The sequence of FimG (E. coli) can be found in UniProtKB, Acc No: P08190.

[0028] The term DsF is intended to include the full length sequence of FimF or its bacterial homologues, as well as FimF variants, in particular variants that show improved binding (see below), such as the peptide SRIRIRGYVR (SEQ ID NO: 1, amino acids 25 to 34, T to R exchange, underlined). The sequence of FimF (E. coli) can be found in UniProtKB, Acc No: P08189.

[0029] The complex between the bacterial type 1 fimbrial subunit FimG and a peptide corresponding to the N-terminal extension (termed the donor strand, Ds) of the partner subunit FimF (DsF) is 330 m long. -1 s -1 10 for the best mutant complex, whereas the dissociation rate and 3D structure of the improved complex remained essentially unperturbed. -22 mK D values ​​were obtained, which are the lowest values ​​reported so far for a protein / ligand complex (C. Giese, J. Eras, A. Kern, M. A. Scharer, G. Capitani, R. Glockshuber, Accelerating the Association of the Most Stable Protein-Ligand Complex by More than Two Orders of Magnitude.Angew.Chem.Int.Ed.2016,55,9350).

[0030] In the context of the present invention, a "homolog" of a polypeptide means a polypeptide which performs the same or essentially the same function, preferably binding, and exhibits at least 80%, preferably at least 90%, more preferably at least 95% amino acid sequence identity compared to an earlier polypeptide (e.g., functional FimG or FimGt and / or DsF that bind to each other or length variants thereof).

[0031] In formulas I and II, Y is absent or represents at least one of a linker sequence, a detectable peptide sequence and / or a peptide sequence for purifying the polypeptide chain.

[0032] A linker is a short peptide sequence between protein domains, e.g., between fusion domains. Branched or unbranched peptide linker sequences are preferred, e.g., branched or unbranched glycine or glycine / serine-containing peptide linker sequences. Typically, linker sequences are introduced to connect functional elements of a polypeptide construct and / or to provide space between functional elements. Linkers allow adjacent protein domains to move freely relative to each other. Linkers are often composed of flexible residues such as glycine and serine. Longer linkers are used when necessary to ensure that two adjacent domains do not sterically interfere with each other. Peptide linker sequences may also include cleavable (e.g., peptidase or chemically cleavable) linkers. Suitable linkers can be designed based on the literature and the skills of those skilled in the art (see, for example, Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10): 1357-1369. doi: 10.1016 / j.addr.2012.09.039; Joshua S. Klein, Siduo Jiang, Rachel P. Galimidi, Jennifer R. Keeffe, Pamela J. Bjorkman, Design and characterization of See structured protein linkers with differing flexibilities, Protein Engineering, Design and Selection, Volume 27, Issue 10, October 2014, Pages 325-330, https: / / doi.org / 10.1093 / protein / gzu043). In one example described below, the polymerase gene is fused to the truncated pIII protein via a 23 amino acid long linker. WO 1998 / 019705 discloses branched peptide linkers. Similarly, suitable branched linkers can be designed based on the literature and the skills of those skilled in the art (see, for example, Brunetti, J, Falciani, C, Bracci, L, Pini, A. Branched peptides as bioactive molecules for drug design. Pept Sci. 2018; 110: e24089. https: / / doi.org / 10.1002 / pep2.24089).

[0033] Detectable peptide sequences (also called tags or markers) allow the identification of said peptide sequence and, consequently, of the entire fusion construct or parts thereof that contain said sequence. Some detectable sequences can also be used to (affinity) purify said fusion construct or parts thereof that contain said sequence. Examples are myc tags or Tie2 tags detected with the respective antibodies, other antigen markers (see, for example, Hopp, T., Prickett, K., Price, V. et al. A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification. Nat Biotechnol 6, 1204-1210 (1988). https: / / doi.org / 10.1038 / nbt1088-1204), chelating groups (metals, radionuclides) or fluorophore groups.

[0034] Peptide sequences for purifying the entire polypeptide chain of the fusion construct or a part thereof, which contains said sequence, are described in the literature and are known to the skilled artisan. Examples are given in Kimple ME, Brill AL, Pasker RL. Overview of the polypeptide chains. of affinity tags for protein purification. Curr Protoc Protein Sci. 2013;73:9.9.1-9.9.23. Published 2013 Sep 24. doi:10.1002 / 0471140864.ps0909s73), calmodulin binding peptides, His tags such as 6His tags, and / or maltose binding protein sequences.

[0035] In formula II, POI denotes a protein of interest, and in the polypeptide chains according to the invention, the POI can be an enzyme, an antibody and fragments thereof (e.g., scFv, Fab), a polymerase, such as a nucleic acid polymerase, a cytokine and functional fragments thereof, and libraries thereof or The substrate can be selected from the group consisting of a panel of antibodies and fragments thereof, nucleic acid polymerases or enzymes that cleave or modify a substrate or ligand, preferably a SUB (see below), either alone or in association with other proteins, e.g. to be screened (see also below). The cleaved or modified substrate can be further detected.

[0036] Finally, in formula II, CPF represents a coat protein of a filamentous phage, preferably a minor coat protein. Examples are selected from minor coat proteins of phages fd, M13, f1, and Pf1, such as pIII, pVI, pVII, pVIII, pIX, and truncated forms thereof capable of functionally replacing the respective coat proteins of filamentous phages.

[0037] In its second aspect, the present invention provides a first polypeptide chain having, in the N-terminal to C-terminal direction, the general formula (III) X-SUB-DsF-Y(III), and and a second polypeptide chain comprising at least one POI and having, in the N-terminal to C-terminal direction, the general formula (IV) Concerning a protein complex containing X-FimGt(POI)-Y(POI)-CPF(IV).

[0038] In formula III and IV, X is absent or represents a bacterial leader sequence or translocation sequence. Preferably, the bacterial leader sequence or translocation sequence is selected from leader sequences for secretion by secretory pathways or translocation sequences for translocation by translocation systems, such as PelB, DsbA, TorA, and PhoA, and the general secretion (Sec) pathway, twin arginine translocation (Tat) pathway, T2SS pathway, T3SS pathway, T5SS pathway, and SecA2 pathway. Suitable signal sequences and their design can be based on the respective literature and the knowledge of those skilled in the art, for example, Sec and Tat signal peptides have a similar tripartite overall structure consisting of a positively charged n region, a central hydrophobic h region, and a polar c region containing a recognition site (consensus: AXA) for signal peptidase (SPase; cleavage site is indicated by an arrow). In the Tat signal peptide, a characteristic amino acid consensus motif containing two highly conserved arginine residues (underlined) is present at the boundary between the often significantly longer n and h regions. Furthermore, the h-region of Tat signal peptides is in most cases less hydrophobic than that found in Sec signal peptides, and the c-region of Tat signal peptides often contains positively charged amino acids (so-called Sec avoidance motifs) that prevent mistargeting of Tat substrates to the Sec pathway (see, e.g., Freudl, R. Signal peptides for recombinant protein secretion in bacterial expression systems. Microb Cell Fact 17, 52 (2018). https: / / doi.org / 10.1186 / s12934-018-0901-3 and Green ER, Mecsas J. Bacterial Secretion Systems: An Overview. Microbiol Spectr. 2016; 4 (1): 10.1128 / microbiolspec.VMBF-0012-2015. doi: 10.1128 / microbiolspec.VMBF-0012-2015). Signal peptides can also be mutated and optimized for the host (see, e.g., Han S, Machhi S, Berge M,Xi G,Linke T,Schoner R.Novel signal peptides improve the secretion of recombinant Staphylococcus aureus Alpha toxin H35L See in Escherichia coli.AMB Express.2017;7(1):93.doi:10.1186 / s13568-017-0394-1 (This refers to.

[0039] Also, in this aspect, the present invention relates to a method for producing a 3×10 9 Taking advantage of the infinite kinetic stability of the bacterial pilus protein complex FimGt-DsF, which shows an extrapolated dissociation half-life of years (Puorger et al., Structure 2008, 16, 631). The use of FimGt-DsF in this embodiment for phage display overcomes the above-mentioned limitations of conventional phage display by providing an extremely stable and specific linkage that surprisingly allows independent translocation of POI and phage proteins with combinations of signal sequences for different secretion pathways and attachment of substrates for directed evolution of bond-forming and bond-cleaving enzymes.

[0040] In formulae III and IV, FimGt represents the bacterial FimG polypeptide (bacterial type 1 pilus subunit FimG) required for binding to DsF or its bacterial homologues, and DsF (the N-terminal extension of the partner subunit FimF (called the donor strand, Ds)) represents the bacterial DsF polypeptide required for binding to FimG, FimGt or its bacterial homologues. Preferably, said bacterial DsF and FimGt polypeptides are selected from E. coli or from DsF and / or FimGt homologues from Gram-negative bacteria, in particular from the Enterobacteriaceae family.

[0041] The term FimGt is intended to include the full length sequence of FimG or its bacterial homologues, as well as FimG variants, particularly variants that exhibit improved binding (see below), such as an N-terminal deletion variant in which residues 1-12 are truncated, optionally with the substitution Q134E. The sequence of FimG (E. coli) can be found in UniProtKB, Acc No: P08190.

[0042] FimGt(POI) denotes a bacterial FimGt polypeptide or its bacterial homologue required for binding to DsF, optionally linked to a POI. Linkage can be direct or indirect, for example by a branched or unbranched linker as described herein between the two polypeptides, e.g., FimGt-Y-(POI).

[0043] The term DsF is intended to include the full length sequence of FimF or its bacterial homologues, as well as FimF variants, in particular variants that show improved binding (see below), such as the peptide SRIRIRGYVR (SEQ ID NO: 1, amino acids 25 to 34, T to R exchange, underlined). The sequence of FimF (E. coli) can be found in UniProtKB, Acc No: P08189.

[0044] The complex between the bacterial type 1 fimbrial subunit FimG and a peptide corresponding to the N-terminal extension (termed the donor strand, Ds) of the partner subunit FimF (DsF) is 330 m long. -1 s -1 10 for the best mutant complex, whereas the dissociation rate and 3D structure of the improved complex remained essentially unperturbed. -22 mK Dvalues ​​were obtained, which are the lowest values ​​reported so far for a protein / ligand complex (C. Giese, J. Eras, A. Kern, M. A. Scharer, G. Capitani, R. Glockshuber, Accelerating the Association of the Most Stable Protein-Ligand Complex by More than Two Orders of Magnitude.An gew.Chem.Int.Ed.2016,55,9350).

[0045] Also, in this context of the present invention, a "homolog" of a polypeptide means a polypeptide which performs the same or essentially the same function, preferably binding, and exhibits at least 80%, preferably at least 90%, more preferably at least 95% amino acid sequence identity compared to an earlier polypeptide (e.g., functional FimG or FimGt and / or DsF that bind to each other or length variants thereof).

[0046] In formula III, Y is absent or represents at least one of a linker sequence, a detectable peptide sequence and / or a peptide sequence for purifying the polypeptide chain. In formula IV, Y(POI) is absent or represents at least one of a branched linker sequence to which a POI is attached, a detectable peptide sequence and / or a peptide sequence for purifying the polypeptide chain.

[0047] Linkers are short peptide sequences present between protein domains, for example between fusion domains. Branched or unbranched peptide linker sequences are preferred, for example branched or unbranched glycine or glycine / serine containing peptide linker sequences. Usually, linker sequences are introduced to connect functional elements of a polypeptide construct and / or to provide space between functional elements. Linkers are often composed of flexible residues such as glycine and serine so that adjacent protein domains can move freely relative to each other. Longer linkers are used when necessary to ensure that two adjacent domains do not sterically interfere with each other. Peptide linker sequences may also include cleavable (e.g. peptidase or chemically cleavable) linkers. Suitable linkers can be designed based on the literature and the techniques of those skilled in the art (see, for example, Chen X, Zaro JL, Shen WC. Fusion protein linkers: property, design and functionality. Adv Drug Deliv Rev. 2013; 65(10): 1357-1369. doi: 10.1016 / j.addr.2012.09.039; Joshua S. Klein, Siduo Jiang, Rachel P. Galimidi, Jennifer R. Keeffe, Pamela J. Bjorkman, Design and characterization of See structured protein linkers with differing flexibilities, Protein Engineering, Design and Selection, Volume 27, Issue 10, October 2014, Pages 325-330, https: / / doi.org / 10.1093 / protein / gzu043). In one example described below, the polymerase gene is fused to the truncated pIII protein via a 23 amino acid long linker. WO 1998 / 019705 discloses branched peptide linkers. Similarly, suitable branched linkers can be designed based on the literature and the skills of those skilled in the art (see, for example, Brunetti, J, Falciani, C, Bracci, L, Pini, A. Branched peptides as bioactive molecules for drug design. Pept Sci. 2018; 110: e24089. https: / / doi.org / 10.1002 / pep2.24089).

[0048] Detectable peptide sequences (also called tags or markers) allow the identification of said peptide sequence and, consequently, of the entire fusion construct or parts thereof that contain said sequence. Some detectable sequences can also be used to (affinity) purify said fusion construct or parts thereof that contain said sequence. Examples are myc tags or Tie2 tags, which are detected with respective antibodies, other antigen markers (e.g. Hopp, T., Prickett, K., Price, V. et al. A Short Polypeptide Marker Sequence Useful for Recombinant Protein Identification and Purification. Nat Biotechnol 6, 1204-1210 (1988). https: / / doi.org / 10.1038 / nbt1088-1204), chelating groups (metals, radionuclides), or fluorophore groups.

[0049] Peptide sequences for purifying the entire polypeptide chain of the fusion construct or a part thereof, which contains said sequence, are described in the literature and are known to the skilled artisan. Examples are given in Kimple ME, Brill AL, Pasker RL. Overview of the polypeptide chains. of affinity tags for protein purification. Curr Protoc Protein Sci. 2013;73:9.9.1-9.9.23. Published 2013 Sep 24. doi:10.1002 / 0471140864.ps0909s73), calmodulin binding peptides, His tags, such as the 6 His tag, and / or maltose binding protein sequences.

[0050] In formula IV, POI denotes a protein of interest, in a polypeptide chain according to the invention, the POI may be selected from the group consisting of enzymes, antibodies and fragments thereof (e.g. scFv, Fab), polymerases such as nucleic acid polymerases, cytokines and functional fragments thereof, and libraries or panels thereof. Preferably, it is an antibody and fragment thereof, a nucleic acid polymerase, or an enzyme which cleaves or modifies a substrate or ligand, preferably a SUB (see below), either alone or in association with other proteins, e.g. to be screened (see also below). The cleaved or modified substrate may further be detected.

[0051] In formula IV, CPF represents a coat protein of a filamentous phage, preferably a minor coat protein. Examples are selected from minor coat proteins of phages fd, M13, f1, and Pf1, such as pIII, pVI, pVII, pVIII, pIX, and truncated forms thereof that can functionally replace the respective coat proteins of filamentous phages.

[0052] Finally, in formula III, SUB denotes a substrate that can be cleaved or modified by the POI alone or in association with other proteins, such as the protein to be screened (see also below) or ligands. The cleaved or modified substrate can be further detected. The SUB can be selected from the group consisting of substrates of enzymes, such as ligands of the POI, cleavable detectable markers (including metals, fluorophores, quenchers), antigen markers, and libraries thereof.

[0053] Preferred are protein complexes according to the invention, said complexes being stabilized or essentially stabilized by donor strand complementarity between independent adjacent polypeptide sequences of formulae I and II, and III and IV, respectively.

[0054] Yet another aspect of the present invention relates to a filamentous phage displaying at least one protein of interest (POI) as disclosed herein, comprising a protein complex according to the present invention. Preferably, said filamentous phage is selected from fd, M13, f1 and Pf1. More preferably, in said filamentous phage, the CPF replaces or at least partially replaces a coat protein of said filamentous phage, preferably a minor coat protein thereof, more preferably each native minor coat protein, and most preferably is capable of functionally replacing each coat protein of said filamentous phage.

[0055] Yet another aspect of the present invention is the preparation of variants of the POI and / or substrate or ligand (SUB). The present invention also relates to a library of filamentous phages displaying the vector.

[0056] Yet another aspect of the invention relates to a bacterial host cell comprising a filamentous phage according to the invention or a bacterial host cell comprising a library of filamentous phages according to the invention. Preferred host cells are E. coli or Pseudomonas aeruginosa.

[0057] Another aspect of the invention relates to a nucleic acid encoding the first (formula I) or second (formula II) polypeptide chain of a protein complex according to the invention, or a nucleic acid encoding the first (formula III) or second (formula IV) polypeptide chain of a protein complex according to the invention. The nucleic acid may be DNA, RNA, PNA or a mixture thereof. Yet another aspect of the invention relates to a bicistronic nucleic acid encoding the first (formula I) and second (formula II) polypeptide chain of a protein complex according to the invention, or a bicistronic nucleic acid encoding the first (formula III) and second (formula IV) polypeptide chain of a protein complex according to the invention. Preferred are nucleic acids according to the invention that contain and encode variants of POI and / or SUB. Another aspect of the invention relates to a library of nucleic acids according to the invention. Also included are expression constructs and vectors that contain and express the nucleic acid(s) according to the invention.

[0058] Another aspect of the present invention is a phagemid vector comprising a nucleic acid encoding the first (formula I) and second (formula II) polypeptide chains of the protein complex according to the present invention or a nucleic acid encoding the first (formula III) and second (formula IV) polypeptide chains of the protein complex according to the present invention (see, for example, Qi H, Lu H, Qiu HJ, Petrenko V, Liu A. Phagemid vectors for phage display: properties, characteristics and construction. J Mol Biol. 2012;417(3):129-143. doi:10.1016 / j.jmb.2012.01.038). Another aspect of the invention relates to a library of phagemids comprising a nucleic acid according to the invention. Preferably, in said phagemids, the CPF replaces or at least partially replaces a coat protein of a filamentous phage, preferably a minor coat protein thereof, more preferably the respective native minor coat protein, and most preferably is capable of functionally replacing the respective coat protein of said filamentous phage.

[0059] Yet another aspect of the invention relates to a bacterial host cell comprising a phagemid according to the invention or a library of phagemids according to the invention. Preferred host cells are E. coli or Pseudomonas aeruginosa. In one embodiment, the phagemid-transformed bacterial cell produces structural proteins of a filamentous phage. In a further embodiment, the phagemid-transformed bacterial cell replicates single-stranded phagemid DNA. In yet another embodiment, the phagemid-transformed bacterial cell secretes filamentous phage together with the phagemid DNA.

[0060] Yet another aspect of the invention relates to a method for producing a phagemid according to the invention, comprising providing a suitable phagemid vector and inserting a nucleic acid according to the invention into said vector.Yet another aspect of the invention relates to a method for producing a library of phagemids according to the invention, comprising providing a suitable phagemid vector and inserting a library of nucleic acids according to the invention into said phagemid vector.

[0061] Yet another aspect of the invention relates to a method for producing a filamentous phage of the invention, comprising suitably inserting a nucleic acid according to the invention into the genome of a suitable filamentous phage and optionally expressing said genome in a suitable host bacterium. Yet another aspect of the invention relates to a method for producing a filamentous phage of the invention, comprising suitably inserting a library of nucleic acids according to the invention into the genome of a suitable filamentous phage and optionally expressing said genome in a suitable host bacterium. Preferably, in said filamentous phage or library of filamentous phages, the CPF replaces or at least partially replaces a coat protein of said filamentous phage(s), preferably a minor coat protein thereof, more preferably a respective / corresponding naturally occurring minor coat protein, and most preferably is capable of functionally replacing each coat protein of said filamentous phage.

[0062] Another important aspect of the present invention is a method for screening for a protein of interest (POI) that specifically interacts with at least one substrate or ligand (SUB), comprising the steps of: a) providing a library of filamentous phages according to the invention, b) contacting said at least one substrate or ligand with said library of a); c) determining the interaction, preferably the specific interaction, of said substrate or ligand with said library; and d) identifying a POI based on said interaction, preferably on said specific interaction.

[0063] Preferred is a method according to the invention, wherein said POI is an antibody or a fragment thereof as described herein. Preferred is a method according to the invention, wherein said POI is a polymerase or a truncated form thereof as described herein.

[0064] Phage display is a powerful technique for studying protein-ligand interactions, most frequently applied to protein-protein, protein-peptide, and protein-nucleic acid interactions. The genetic code for a protein / peptide of interest is inserted into the genome of a phage and subsequently "displayed" on the surface of the viral particle as a fusion to a native coat protein. A library of protein / peptide variants is tested against the ligand(s) of interest. Proteins / peptides that bind to specific targets are selected by 3-5 rounds of affinity-driven biopanning and subsequently identified by sequencing the genomes of the phages displaying them. Phage display is widely used for the selection of proteins / peptides with desired binding properties for a wide range of therapeutic, research, and nanotechnology related applications (see, for example, Boriana Marintcheva, Harnessing the Power of Viruses, Academic Press, 2018, Chapter 5, Pages 133-160, ISBN 9780128105146, https: / / doi.org / 10.1016 / B978-0-12-810514-6.00005-2).

[0065] Preferably, the method for screening includes biopanning. Briefly, in a preferred embodiment, biopanning is an affinity selection technique that selects peptides that bind to a given target. This technique is often used for antibody selection. Biopanning includes four main steps for peptide selection. The first step is to provide a phage display library. The next step is the capture step. It involves conjugating the phage library to the desired target. This procedure is called panning. It exploits binding interactions so that only the specific peptides displayed by the bacteriophage bind to the target. For example, select antibodies displayed by bacteriophage with antigens coated in a microtiter plate. A washing step is performed after the capture step to wash away unbound phages from the solid surface. Only bound phages with strong affinity are retained. The final step includes an elution step in which bound phages are eluted by a change in pH or other environmental conditions. The end result is that the peptides produced by the bacteriophage are specific. The resulting filamentous phages are then transferred to Gram-positive cells, again to the phage display library. Negative bacteria can be infected to produce a phage library. This cycle can occur many times with strong affinity binding peptides to the target. This process can be at least partially automated, for example using a robot.

[0066] Another aspect of the present invention relates to the use of a protein complex according to the invention, a nucleic acid(s) according to the invention or a library of nucleic acids according to the invention for producing a phagemid or a filamentous phage.

[0067] The present invention is 3 x 10 9Taking advantage of the infinite kinetic stability of the bacterial pilus protein complex FimGt-DsF, which shows an extrapolated dissociation half-life of 10 years (Puorger et al., Structure 2008, 16, 631). The use of FimGt-DsF for phage display overcomes the above limitations of conventional phage display by providing an extremely stable and specific linkage that allows 1) attachment of the POI via its N-terminus, 2) independent translocation of the POI and phage protein with a combination of signal sequences for different secretion pathways, and 3) attachment of substrates for the directed evolution of bond-forming and bond-cleaving enzymes.

[0068] The present invention relates in particular to the following items:

[0069] Item 1. A protein complex comprising: a) a first polypeptide chain having the general formula (I) in the N-terminal to C-terminal direction: X-DsF-Y-POI(I), and b) a second polypeptide chain having, in the N-terminal to C-terminal direction, the general formula (II): Contains X-FimGt-Y-CPF(II), In the formula, X is absent or represents a bacterial leader sequence or translocation sequence, DsF represents a bacterial DsF polypeptide or a bacterial homologue thereof required for binding to FimGt, Y represents at least one of a linker sequence, a detectable peptide sequence and / or a peptide sequence for purifying the polypeptide chain, POI represents a protein of interest, FimGt represents a bacterial FimGt polypeptide or a bacterial homologue thereof required for binding to DsF, and CPF represents a coat protein of a filamentous phage.

[0070] Item 2. A protein complex comprising: a) a first polypeptide chain having the general formula (III) in the N-terminal to C-terminal direction: X-SUB-DsF-Y(III), and b) a second polypeptide chain comprising at least one POI and having, in the N-terminal to C-terminal direction, the general formula (IV): Contains X-FimGt(POI)-Y(POI)-CPF(IV), In the formula, X is absent or represents a bacterial leader sequence or translocation sequence, DsF represents a bacterial DsF polypeptide or a bacterial homologue thereof required for binding to FimGt, Y(POI) represents at least one of a branched linker sequence to which a POI is attached, a detectable peptide sequence and / or a peptide sequence for purifying said polypeptide chain, POI represents a protein of interest, FimGt(POI) represents a bacterial FimGt polypeptide (optionally with a POI attached) required for binding to DsF or a bacterial homologue thereof, SUB represents a substrate, and CPF represents a coat protein of a filamentous phage.

[0071] Item 3. The protein complex according to item 1 or 2, wherein the bacterial leader sequence or translocation sequence is selected from leader sequences for secretion by a secretory pathway or translocation sequences for translocation by a translocation system, such as PelB, DsbA, TorA, and PhoA, and the general secretion (Sec) pathway, the twin arginine translocation (Tat) pathway, the T2SS pathway, the T3SS pathway, the T5SS pathway, and the SecA2 pathway.

[0072] Item 4. The protein complex according to any one of items 1 to 3, wherein the bacterial DsF and FimGt polypeptides are selected from homologs of DsF and / or FimGt derived from Escherichia coli (E. coli) or from Gram-negative bacteria, in particular from the family Enterobacteriaceae.

[0073] Item 5. The protein complex according to any one of items 1 to 4, wherein Y is selected from a branched or unbranched peptide linker sequence, such as a branched or unbranched glycine or glycine / serine peptide linker sequence, a myc tag and a Tie2 tag as detectable peptide sequences, and / or a calmodulin-binding peptide, a His tag or a maltose protein-binding sequence for purifying the polypeptide chain.

[0074] Item 6. The protein complex according to any one of items 1 to 5, wherein the complex is stabilized or essentially stabilized by donor strand complementarity between the independent adjacent polypeptide sequences of formulas I and II, and formulas III and IV, respectively.

[0075] Item 7. The protein complex according to any one of items 1 to 6, wherein the CPF is selected from minor coat proteins of phages fd, M13, f1, and Pf1, such as pIII, pVI, pVII, pVIII, pIX, and truncated forms thereof capable of functionally replacing the respective coat proteins of filamentous phages.

[0076] Item 8. The protein complex according to any one of items 1 to 7, wherein the POI is selected from the group consisting of an enzyme, an antibody, and a nucleic acid polymerase, and functional fragments thereof, and libraries thereof.

[0077] Item 9. The protein complex according to any one of items 1 to 8, wherein SUB is selected from the group consisting of a substrate for an enzyme, a cleavable detectable marker, an antigen marker, and libraries thereof.

[0078] Item 10. A filamentous phage displaying at least one protein of interest (POI), comprising the protein complex according to any one of items 1 to 9.

[0079] Item 11. The filamentous phage according to item 10, wherein the filamentous phage is selected from fd, M13, f1 and Pf1.

[0080] Item 12. A library of filamentous phages according to item 10 or 11, optionally displaying variants of the POI and / or the substrate (SUB).

[0081] Item 13. A nucleic acid encoding the first or second polypeptide chain of the protein complex according to any one of Items 1 to 9, or a nucleic acid encoding the first or second polypeptide chain of the protein complex according to any one of Items 2 to 9.

[0082] Item 14. A bicistronic nucleic acid encoding the first and second polypeptide chains of the protein complex according to any one of Items 1 to 9, or a bicistronic nucleic acid encoding the first and second polypeptide chains of the protein complex according to any one of Items 2 to 9.

[0083] Item 15. The nucleic acid according to items 13 and / or 14, comprising a variant of the POI and / or SUB.

[0084] Item 16. A library of nucleic acids according to any one of Items 13 to 15.

[0085] Item 17. A phagemid comprising the nucleic acid according to any one of Items 13 to 15, or a library of phagemids comprising the nucleic acid according to Item 16.

[0086] Item 18. A method for producing the phagemid according to Item 17, comprising providing a suitable phagemid vector and inserting the nucleic acid according to any one of Items 13 to 15 into the vector.

[0087] Item 19. A method for producing a library of phagemids according to Item 17, comprising providing a suitable phagemid vector and inserting the library of nucleic acids according to Item 16 into said vector.

[0088] Item 20. A method for producing a filamentous phage according to item 10 or 11, the method comprising inserting a nucleic acid according to any one of items 13 to 15 into the genome of a suitable filamentous phage and, optionally, expressing the genome in a suitable host bacterium.

[0089] Item 21. A method for producing a library of filamentous phages according to item 12, comprising inserting the library of nucleic acids according to item 16 into the genome of a suitable filamentous phage and, optionally, expressing said genome in a suitable host bacterium.

[0090] Item 22. A method for screening for proteins of interest (POI) that specifically interact with a substrate or ligand, comprising: a) providing a library according to item 12; b) contacting said substrate or ligand with said library of a); c) determining an interaction, preferably a specific interaction, between said substrate or ligand and said library; and d) identifying a POI based on said interaction, preferably said specific interaction.

[0091] Item 23. The method according to item 22, wherein the POI is an antibody or a fragment thereof and the method comprises biopanning.

[0092] Item 24. The method according to item 22, wherein the POI is a polymerase or a truncated form thereof.

[0093] Item 25. Use of the protein complex according to any one of Items 1 to 9, the nucleic acid according to any one of Items 13 to 15, or the nucleic acid library according to claim 16 for producing a phagemid or a filamentous phage. [Brief description of the drawings]

[0094] The present invention is further described in the following examples with reference to the accompanying drawings, but is not limited thereto. For purposes of the present invention, all references cited herein are incorporated by reference in their entirety.

[0095] [Figure 1-A] Figure 1 shows a schematic example of the use of FimGt-DsF for phage display. (A) FimGt-DsF links the POI to the phage via its N-terminus, allowing it to be translocated independently from the phage coat protein in different combinations of the secretion pathway. [Figure 1-B] (B) FimGt further provides an anchor for binding of DsF-tagged substrates, allowing evolution of bond-forming and bond-cleaving enzymes. [Figure 2-1]Figure 2 shows the phage display constructs. (Top) The Fim phagemid construct for display of DNA polymerase from Clostridium phage phiCPV4 (Pol) via N-terminal ligation contains a biscistronic arrangement of the polymerase gene fused at its N-terminus to a DsF peptide and the FimGt gene fused to the N-terminus of a truncated pIII (pIIIt, residues 250-406). A His6 tag is inserted at the N-terminus of Pol for purification and a Tie2 tag is inserted for detection. The FimGt-pIII fusion contains a myc tag between FimGt and truncated pIII for detection. Versions with six different combinations of signal sequences were constructed: for the polymerase, the PelB signal sequence of Erwinia carolovora pectate lyase B to promote translocation via the Sec pathway, the DsbA signal sequence of E. coli thiodisulfide exchange protein DsbA to promote translocation via the SRP pathway, and the TorA signal sequence of E. coli trimethylamine-N-oxide reductase to promote translocation via the Tat pathway were cloned and combined with either the PelB or DsbA signal sequence for translocation of the FimGt-pIII fusion protein. (Bottom) For the classical phagemid construct, the polymerase gene is fused to a truncated pIII via a 23 amino acid long linker. As with the Fim construct, a His6 tag is inserted at the N-terminus of Pol for purification and a Tie2 tag for detection. A version was constructed with three signal sequences, PelB, DsbA and TorA, for translocation of Pol-pIII fusion proteins via three different pathways. [Figure 2-2]Figure 2 shows the phage display constructs. (Top) The Fim phagemid construct for display of DNA polymerase from Clostridium phage phiCPV4 (Pol) via N-terminal ligation contains a biscistronic arrangement of the polymerase gene fused at its N-terminus to a DsF peptide and the FimGt gene fused to the N-terminus of a truncated pIII (pIIIt, residues 250-406). A His6 tag is inserted at the N-terminus of Pol for purification and a Tie2 tag is inserted for detection. The FimGt-pIII fusion contains a myc tag between FimGt and truncated pIII for detection. Versions with six different combinations of signal sequences were constructed: for the polymerase, the PelB signal sequence of Erwinia carolovora pectate lyase B to promote translocation via the Sec pathway, the DsbA signal sequence of E. coli thiodisulfide exchange protein DsbA to promote translocation via the SRP pathway, and the TorA signal sequence of E. coli trimethylamine-N-oxide reductase to promote translocation via the Tat pathway were cloned and combined with either the PelB or DsbA signal sequence for translocation of the FimGt-pIII fusion protein. (Bottom) For the classical phagemid construct, the polymerase gene is fused to a truncated pIII via a 23 amino acid long linker. As with the Fim construct, a His6 tag is inserted at the N-terminus of Pol for purification and a Tie2 tag for detection. A version was constructed with three signal sequences, PelB, DsbA and TorA, for translocation of Pol-pIII fusion proteins via three different pathways. [Diagram 3]Figure 3 shows a phage ELISA of polymerase-displaying phages. The display levels of phage samples with polymerase expressed under different conditions, conjugated via FimGt-DsF (dark grey, Fim) or directly fused (grey, without Fim), are compared. The signal sequences used for the Pol-fusion and FimGt-fusion proteins are shown, as well as the host strains Xl1-Blue (XL1) or TG1, and the expression temperature. For each preparation, the titer was determined and 109 phages were used per well. The signal of the helper phage M13KO7 (absorbance at 652 nm) was subtracted from the signal of all samples. Fab-displaying phages showed no signal after subtraction, indicating that the signal in this phage ELISA is specific for the polymerase-displaying phages. [Figure 4] Figure 4 shows a model selection of polymerase displaying phage for DNA binding. Phages displaying polymerase via FimGt-DsF (Fim) or direct fusion (without Fim) from the preparations listed in Table 1 were mixed in equal amounts and phage displaying 60-75% of the estimated amount of antibody Fab was added. Phage mixtures were selected for DNA binding to primer-template complexes immobilized via biotin on streptavidin magnetic beads. A control selection was performed using streptavidin beads with no DNA bound. The identity of 20 phages before (left) and after selection (middle) or control selection (right) was determined by infection of TG1 cells and sequence analysis of the phagemids. EXAMPLES

[0096] Materials and Methods material Buffers and salts were purchased from commercial suppliers. Oligonucleotides were from IDT. Restriction enzymes and DNA polymerase were from Roche, New England Biolabs or Agilent (Stratagen). Antibodies were obtained from the following sources: mouse anti-M13 pIII (NEB), rabbit anti-mouse-HRP (Invitrogen), goat anti-rabbit-HRP (Invitrogen), mouse anti-M13-HRP (GE Healthcare). Rabbit anti-Tie2 monoclonal antibody is a Roche internal research tool (not commercially available). E. coli strains Xl1-Blue and Xl10-Gold were from Agilent (Stratagene) and TG1 was from Lucigen. Helper phage M13KO7 was from GE Healthcare. Unless otherwise stated, microbiological and molecular biology methods were performed according to standard procedures (J. Sambrook, D. Roulland, Molecular Cloning: A Laboratory Manual., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0097] Cloning The inserts of the phagemids pETR-DsF-HisPol6_FimGt-pIII and pETR-HisPol6-pIII were constructed by overlap extension PCR using primers containing linkers and tags and the genes for DNA polymerase from the Clostridium phage phiCPV4 (Pol6, see WO 2017 / 148862), FimGt and the M13 minor coat protein pIII as templates. The PCR products were cloned into the pETR phagemid (vector from Roche Glycart). The PelB signal sequence was exchanged by PCR with primers containing the DsbA or TorA sequence and then cloned into the pETR phagemid.

[0098] Phage production and purification XL1-Blue and TG1 cells were transformed with the phagemid. A single colony was used to inoculate a preculture (5 ml) of 2xYT medium supplemented with 1% glucose and 100 μg / ml ampicillin and incubated overnight at 30°C with shaking. The preculture was used to inoculate a culture of 50 ml of fresh 2xYT medium containing 1% glucose and 100 μg / ml ampicillin in a 1:100 ratio. The culture was incubated at 37°C to an OD of 0.5-0.7. 600 The cells were grown to 300 rpm and then infected with 50 μl of helper phage M13KO7 (10 pfu / ml) and incubated at 37° C. for 45 min with slight agitation. Cells were harvested by centrifugation at 3320 g and 4° C. for 10 min and medium was replaced by resuspending the pellet in 50 ml of 2xYT medium containing 100 μg / ml ampicillin, 50 μg / ml kanamycin and 0.1 mM isopropyl-β-D-thiogalactoside (IPTG) for +IPTG samples. After growth at 28° C. for 16 h or at 20° C. and 250 rpm for 24 h, cells were removed by centrifugation at 4800 g and 4° C. for 30 min. The supernatant was mixed with ¼ volume of ice-cold PEG / NaCl solution (20% polyethylene glycol (PEG) 6000, 2.5 M NaCl) and incubated on ice for 1 h. The precipitated phage particles were pelleted by centrifugation at 7164 g and 4° C. for 30 min. Each pellet was washed with 40 ml of wash buffer (50 mM Tris, pH 7. The phages were resuspended in 10 ml of ice-cold PEG / NaCl solution (20% PEG 6000, 2.5 M NaCl) and 10 ml of ice-cold PEG / NaCl solution (20% PEG 6000, 2.5 M NaCl) was added. After 1 h of incubation on ice, the phages were harvested by centrifugation at 7164 g and 4° C. for 30 min and the pellets were resuspended in 1.6 ml of pol storage buffer (50 mM Tris, pH 7.5, 200 mM NaCl, 0.5% Tween 20, 25% glycerol, 5 mM DTT), respectively. To discard the bacterial debris, the phages were centrifuged at 16100 g and 4° C. for 3 min. The infectious titers of the phage samples were determined by infection of E. coli TG1 cells and titering on 2xYT agar plates containing 1% glucose and 100 μg / ml ampicillin.

[0099] Western blot analysis Purified phage samples were supplemented with SDS loading dye and reducing agent. SDS-PAGE was performed on 4-12% Bis-Tris gels (Invitrogen) followed by transfer onto nitrocellulose membranes using the iBlot dry blotting system (Invitrogen). Mouse anti-M13 pIII at 1:10000 dilution was used as the primary antibody, and horseradish peroxidase-conjugated rabbit anti-mouse IgG at 1:2000 dilution was used as the secondary antibody to detect the phage minor coat protein pIII and its fusions. Polymerase was detected by the Tie2 tag using rabbit anti-Tie2 at 1:2500 dilution as the primary antibody and horseradish peroxidase-conjugated goat anti-rabbit IgG at 1:2000 dilution as the secondary antibody. Blots were developed with Lumi-Light substrate from Roche.

[0100] Phage ELISA Rabbit anti-Tie2 was coated as a capture antibody onto Immunomaxi 96-well plates (Thermo Fisher) at a concentration of 2.5 μg / ml. After blocking and washing, 109 phage particles per well (determined by titration) were added to the wells and incubated for 1 h at room temperature. After washing, bound phage were detected with a horseradish peroxidase-conjugated mouse anti-M13 antibody (GE Healthcare) diluted 1:2500. Plates were developed using the Pierce™ TMB (3,3',5,5' tetramethylbenzidine) Substrate Kit (Thermo Fisher) and absorbance was detected at 370 nm and 652 nm.

[0101] Model Selection Phage particles (72 preparations) from nine phagemid constructs with and without FimGt-DsF produced under different conditions listed in Table 1 were mixed in equal amounts. Phage displaying antibody Fab fragments were added to the polymerase-displaying phage mixture in amounts estimated by Western blot to account for 60%-75% of the total phage in the mixture. The phage mixture was PEG precipitated once (see Phage Production and Purification section) and resuspended in pol binding buffer (50 mM Tris, pH 7.5, 55 mM glutamate, 0.1% Tween 20, 1% BSA, 5 mM DTT). Freshly centrifuged phages were added to streptavidin magnetic beads (Dynabeads M2-80 streptavidin, Invitrogen) that had been blocked with BSA and coated with biotinylated primer-template complexes, then incubated with biotin to saturate remaining free streptavidin binding sites. Control beads were not coated with biotinylated primer-template complexes but were otherwise treated identically. Phages were incubated on the beads for 20 min at RT, unbound phages were washed off, and bound phages were eluted by incubation with DNase (20 U / 100 μl beads) for 1 h at RT. Exponentially growing TG1 cells were infected with aliquots of phages and phage mixtures harvested before selection. The slightly agitated diluted cells were incubated for 30 min at 37°C, then cultured in 2xYT 2000 ng / ml containing 1% glucose and 100 μg / ml ampicillin. Colonies were obtained for titer determination on agar plates and for phage identity determination. The identity of each of the 20 phagemids was determined by sequencing.

[0102] N-terminal binding of DNA polymerase via FimGt-DsF enables functional display on filamentous phage M13 Design and cloning of phage display constructs In this example, we used FimGt-DsF to display the DNA polymerase from Clostridium phage phiCPV4 via N-terminal attachment to the phage. This polymerase loses a large amount of activity when fused at its C-terminus. Therefore, the N-terminal fusion must be crucial for the display of an active polymerase.

[0103] For N-terminal attachment of the polymerase to the phage, the polymerase was cloned biscistronically in front of a FimGt-pIII fusion protein into a phagemid vector (Figure 2, top). The DsF peptide was then ligated to the N-terminus of the polymerase, including a his tag for purification and a Tie2 tag for detection with an anti-Tie2 antibody. FimGt was fused to a truncated minor coat protein pIII and a myc tag added for detection. In addition to the standard PelB leader sequence for secretion by the Sec pathway constructs with DsbA and TorA signal sequences for translocation of the polymerase by the SRP and Tat pathways, respectively, FimGtDsF fusions were cloned and combined with either PelB or DsbA for translocation (the Tat pathway was not considered, since FimGt is naturally secreted into the periplasm where it folds and assembles). For comparison, a classical phage display construct was designed and cloned that does not contain FimGt-DsF but contains a direct fusion to the pIII coat protein via the C-terminus of the polymerase (Figure 2, bottom). As for the FimGt-DsF construct, a version with the signal sequences PelB, DsbA and TorA was constructed for translocation of the polymerase-pIII fusion through the different secretion pathways.

[0104] Phage production in several E. coli strains and under different conditions To find optimal conditions for production of the two polymerase display phage constructs, the use of E. coli strains TG1 and Xl1-Blue, as well as expression temperatures of 28°C and 20°C, and media with and without IPTG after helper phage infection, were tested.

[0105] Assessment of phage production and display by Western blot The results of phage production under the various conditions described above were evaluated by Western blot (Table 1). Total phage production is reflected by the amount of pIII protein detected by a monoclonal anti-pIII antibody. Display levels can be evaluated by the amount of pIII fusion protein (FimGt-pIII or polymerase-pIII) detected by the same monoclonal anti-pIII antibody, as well as by detection of DsF-polymerase or polymerase-pIII protein using the Tie2 tag and anti-Tie2 antibody. The amount of different proteins in the Western blot was evaluated visually and graded from undetectable (-) to strong bands (+++). The results are summarized in Table 1 and show that both Fim and classical constructs produce phage, but the Fim construct with the polymerase attached via the N-terminus generally shows higher display levels. IPTG addition to the growth medium tends to result in lower phage amounts, levels suggesting that strong induction of the polymerase and pIII fusions is unfavorable. The best host strain and expression temperature varied between constructs. The polymerase constructs showed no preference for any particular combination of signal sequences. [Table 1-1] [Table 1-2]

[0106] Demonstration of polymerase display by phage ELISA. In the case of the Fim construct, the two separate fusion proteins, DsF-polymerase and FimGt-pIII, should translocate independently to the periplasm and assemble there. Western blot detection of both fusion proteins did not prove their binding and, consequently, displaying the polymerase on the phage surface. Therefore, a phage ELISA was performed on the phage sample showing the highest display by Western blot (Figure 3). Phage titers of the different samples were determined and ranged from 5 × 1011 to 3 × 1013 (cfu / ml). Equal amounts of phages were added to the wells of an ELISA plate, and phages displaying the polymerase were captured using an anti-Tie2 antibody specific for the Tie2 tag on the polymerase. After washing, bound phages were detected with a horseradish peroxidase-conjugated anti-M13 antibody that binds to the major coat protein pVIII of the phage. Antibody Fab fragments displaying Helper phage and phages containing Fim were used as negative controls. The high ELISA signal of the Fim constructs indicates that the polymerase is indeed displayed on the phage surface. The ELISA results are in good agreement with the Western blot results, and the low ELISA signal of the classical constructs without Fim confirms that these constructs display significantly less polymerase, suggesting that N-terminal binding of the polymerase is important for display on the phage surface. As seen in the Western blot, the signal sequence does not seem to affect the display level of our polymerase.

Claims

1. A protein complex comprising: a) a first polypeptide chain having, in the N-terminal to C-terminal direction, the general formula (I) X-DsF-Y-POI(I), and b) a second polypeptide chain having, in the N-terminal to C-terminal direction, the general formula (II) X-FimGt-Y-CPF(II) Including, During the ceremony, X is absent or represents a bacterial leader sequence or a translocation sequence, DsF denotes the bacterial DsF polypeptide or a bacterial homologue thereof required for binding to FimGt; Y is absent or represents at least one of a linker sequence, a detectable peptide sequence and / or a peptide sequence for purifying said polypeptide chain, POI indicates protein of interest; FimGt denotes the bacterial FimGt polypeptide required for binding to DsF or a bacterial homologue thereof; CPF indicates filamentous phage coat protein, a protein complex.

2. A protein complex comprising: a) a first polypeptide chain having, in the N-terminal to C-terminal direction, the general formula (III) X-SUB-DsF-Y (III), and b) a second polypeptide chain comprising at least one POI and having, in the N-terminal to C-terminal direction, the general formula (IV) X-FimGt(POI)-Y(POI)-CPF(IV) Including, During the ceremony, X is absent or represents a bacterial leader sequence or a translocation sequence, DsF denotes the bacterial DsF polypeptide or a bacterial homologue thereof required for binding to FimGt; Y(POI) represents at least one of a branched linker sequence, a detectable peptide sequence and / or a peptide sequence for purifying said polypeptide chain, which is absent or to which a POI is attached, POI indicates protein of interest; FimGt(POI) denotes the bacterial FimGt polypeptide required for binding to DsF or a bacterial homologue thereof, optionally linked to a POI; SUB indicates substrate; CPF indicates filamentous phage coat protein, a protein complex.

3. 3. The protein complex of claim 1 or 2, wherein the bacterial leader sequence or translocation sequence is selected from leader sequences for secretion by a secretory pathway or translocation sequences for translocation by a translocation system, such as PelB, DsbA, TorA, and PhoA, as well as the general secretion (Sec) pathway, the twin arginine translocation (Tat) pathway, the T2SS pathway, the T3SS pathway, the T5SS pathway, and the SecA2 pathway.

4. 2. The bacterial DsF and FimGt polypeptides are selected from E. coli or from homologs of DsF and / or FimGt from Gram-negative bacteria, in particular from the family Enterobacteriaceae.

4. A protein complex according to any one of claims 1 to 3.

5. 5. The protein complex according to claim 1, wherein Y is selected from branched or unbranched peptide linker sequences, such as branched or unbranched glycine or glycine / serine peptide linker sequences, myc-tags and Tie2-tags as detectable peptide sequences, and / or calmodulin-binding peptides, His-tags or maltose protein-binding sequences for purifying the polypeptide chain.

6. 6. The protein complex of any one of claims 1 to 5, wherein the complex is stabilized or essentially stabilized by donor strand complementarity between independent adjacent polypeptide sequences of formulas I and II, and formulas III and IV, respectively.

7. 7. The protein complex of claim 1, wherein the CPF is selected from minor coat proteins of phages fd, M13, f1, and Pf1, such as pIII, pVI, pVII, pVIII, pIX, and truncated forms thereof capable of functionally replacing the respective coat proteins of filamentous phages.

8. The protein complex according to any one of claims 1 to 7, wherein the POI is selected from the group consisting of enzymes, antibodies, and nucleic acid polymerases, and functional fragments thereof, and libraries thereof.

9. The protein complex according to any one of claims 1 to 8, wherein the SUB is selected from the group consisting of a substrate for an enzyme, a cleavable detectable marker, an antigen marker, and libraries thereof.

10. 10. A filamentous phage displaying at least one protein of interest (POI) comprising a protein complex according to any one of claims 1 to 9, preferably selected from fd, M13, f1 and Pf1, or a library of said filamentous phages, optionally displaying a variant of the POI and / or the substrate (SUB).

11. A nucleic acid encoding the first or second polypeptide chain of the protein complex according to any one of claims 1 to 9, or a nucleic acid encoding the first or second polypeptide chain of the protein complex according to any one of claims 2 to 9.

12. A bicistronic nucleic acid encoding the first and second polypeptide chains of the protein complex according to any one of claims 1 to 9, or a bicistronic nucleic acid encoding the first and second polypeptide chains of the protein complex according to any one of claims 2 to 9.

13. 13. The nucleic acid of claim 11 and / or 12, comprising a variant of the POI and / or SUB.

14. A library of nucleic acids according to any one of claims 11 to 13, a phagemid comprising a nucleic acid according to any one of claims 11 to 13, or a library of phagemids comprising said nucleic acid.

15. A method for screening for a protein of interest (POI) that specifically interacts with a substrate or ligand, comprising: a) providing a library according to claim 10, b) contacting said substrate or ligand with said library of a); c) determining whether the substrate or ligand interacts with the library, preferably specifically interacting with the library; To determine the use of d) identifying a POI based on said interaction, preferably said specific interaction.

16. 16. The method of claim 15, wherein the POI is an antibody or a fragment thereof, the method comprises biopanning, or the POI is a polymerase or a truncated form thereof.

Images