Binding peptide and use thereof

Abstract

The invention relates to a binding peptide having at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 28 and its use thereof.

Description

[0001] Binding peptide and use thereof

[0002] The invention relates to a binding peptide capable to bind an unfunctionalized substrate and use thereof as an affinity tag.

[0003] Affinity tags are commonly used in recombinant protein purification to facilitate the isolation of a target protein from a complex mixture. An affinity tag is a short peptide sequence that is fused to the target protein during recombinant protein expression. The tag can be used to selectively bind to a specific ligand or matrix, allowing for efficient purification of the target protein.

[0004] Commonly used affinity tags include hexahistidine tags (His-tags), glutathione S-transferase (GST), and streptavidin binding peptide (SBP)-GST. The His-tag is one of the most widely used tags for recombinant protein affinity purification. Other affinity reagents that have been used for isolation of protein complexes include recombinant proteins, epitope-tagged proteins, antibodies, peptides, and nucleic acids.

[0005] Although affinity tags are convenient tools for protein purification, it requires functionalised substrate specific for the affinity tag.

[0006] Hence, there is a need for an affinity tag that increase simplicity, sustainability, specificity and yields of purification while remaining cost-competitive.

[0007] DESCRIPTION OF THE INVENTION

[0008] To this aim, the invention relates to binding peptide having at least 80% of identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 28.

[0009] The binding peptide of the invention is advantageously capable to bind to unfunctionalized substrate, thereby simplifying the purification of proteins and cells while preserving the specificity of the purification. Because the purification with the binding peptide of the invention does not require to functionalise a substrate before purification, it is more sustainable and cost competitive. The purification is also increased and more reproductible because it does not depend on the good functionalisation of the substrate.

[0010] In one embodiment of the invention, the biding peptide has at least 80% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11 , SEQ ID NO: 19 and SEQ ID NO: 20.

[0011] The invention also relates to an isolated nucleic acid having a sequence encoding for the amino acid sequence of the binding peptide as above defined.

[0012] In particular, the isolated nucleic acid has at least 80% sequence identity to an optimised codon sequence selected from the group consists of SEQ ID NO: 29 to SEQ ID NO: 39. The invention also relates to a recombinant vector encoding the amino acid sequence of the binding peptide of claim as above defined.

[0013] The invention also relates to a host cell comprising the recombinant vector of as above defined.

[0014] The invention also relates to a biological material comprising at least one affinity tag consisting of the biding peptide according to claim as above defined.

[0015] In an embodiment, the biological material is selected from the group consisting of a cell and a chimeric protein. In particular, the biological material is an antibody.

[0016] The invention also relates to a composition comprising the biological material according to any of claims as above defined.

[0017] The invention also relates to a method for isolating or detecting the biological material as above defined comprising the following step: a) providing an unfunctionalized substrate, b) proving the biological material as above defined, c) attaching the at least one biological material onto the unfunctionalized substrate by contacting them, and d) isolating or detecting the biological material.

[0018] A method for functionalising a substrate or for immobilising a biological material as above defined to a substrate, comprising the following step: a) providing an unfunctionalized substrate, b) providing at least one biological material as above defined, c) attaching the at least one biological material onto the unfunctionalized substrate by contacting them.

[0019] Preferably, the unfunctionalized substrate is selected from the group consisting of dextran, polymethacrylate, agarose, polyvinyl alcohol, pullulan, pectin, polystyrene, silica gel, cellulose, polyethylene glycol, cross-linked version thereof and grafted version thereof.

[0020] The invention also relates to a substrate which is bound at least one biological material as above defined.

[0021] The invention also relates to a kit for isolating or detecting a biological material as above defined, the kit comprising an unfunctionalized substrate, a washing solution or suspension, and an eluant comprising a releasing agent.

[0022] Finally, the invention relates to a kit for isolating or detecting a target comprising a substrate as above defined, a washing solution or suspension, and an eluant comprising a releasing agent, wherein the substrate comprises at least one antibody as above defined, said antibody being able to bind to the target.

[0023] DETAILED DESCRIPTION OF THE INVENTION

[0024] Biding peptide

[0025] According to a first object, the present invention relates to a biding peptide capable to bind to an unfunctionalized substrate as a target.

[0026] The biding peptide of the invention with an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 28.

[0027] The biding peptide of the invention also include peptides having one or more substitutions, additions, and / or deletions of residues relative to the sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 28 as long as the binding properties are substantially retained. Thus, the binding peptide of the invention include binding peptide with at least 80% of identity with one of the sequences set forth in the SEQ ID NO: 1 to SEQ ID NO: 28.

[0028] By “at least 80% of identity”, it is meant in the invention 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and 100% of identity with a corresponding sequence. Regarding the percentage of identity, it is defined by the percentage of amino acid residues of the sequence which align with the same amino acid in the sequence of the homologous protein. Degrees of identity between sequences can be readily calculated. The “% sequence identity” (or “% sequence similarity”) is calculated by: (1) comparing two optimally aligned sequences over a window of comparison (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window, etc.), (2) determining the number of positions containing identical (or similar) amino-acids (e.g. , identical amino acids occurs in both sequences, similar amino acid occurs in both sequences) to yield the number of matched positions, (3) dividing the number of matched positions by the total number of positions in the comparison window (e.g., the length of the longer sequence, the length of the shorter sequence, a specified window), and (4) multiplying the result by 100 to obtain the % sequence identity or percent sequence similarity.

[0029] Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2:482 (1981), by the homology alignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA 85:2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or by manual alignment and visual inspection (see, e.g., Current Protocols in Molecular Biology, Ausubel et al., eds. 1995 supplement).

[0030] Preferred examples of algorithms that are suitable for determining percent sequence identity and sequence similarity include the BLAST and BLAST 2.0 algorithms, which are described in Altschul et a / ., Nuc. Acids Res. 25:3389-3402 (1977) and Altschul et a / ., J. Mol. Biol. 215:403- 410 (1990). Polypeptide sequences also can be compared using FASTA using default or recommended parameters. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences.

[0031] A binding peptide can have an amino acid sequence consisting essentially of a sequence sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 28 or a binding peptide can have one or more different amino acid residues as a result of substituting an amino acid residue in the sequence of the exemplary binding peptide with a functionally similar amino acid residue (a “conservative substitution”); provided that the biding peptide containing the conservative substitution will substantially retain the binding activity of the biding peptide having a sequence set forth in SEQ ID NO: 1 to SEQ ID NO: 28 not containing the conservative substitution. Examples of conservative substitutions include the substitution of one non-polar (hydrophobic) residue such as alanine, isoleucine, valine, leucine, or methionine for another; the substitution between asparagine and glutamine, the substitution of one large aromatic residue such as tryptophan, tyrosine, or phenylalanine for another; the substitution of one small polar (hydrophilic) residue for another such as between glycine, threonine, serine, and proline; the substitution of one basic residue such as lysine, arginine, or histidine for another; or the substitution of one acidic residue such as aspartic acid or glutamic acid for another.

[0032] In a preferred embodiment, the biding peptide has at least 80% of identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 11 , SEQ ID NO: 19 and SEQ ID NO: 20.

[0033] In a preferred embodiment, the biding peptide has at least 80% of identity with an amino acid sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 11 , SEQ ID NO: 19 and SEQ ID NO: 20.

[0034] In one embodiment, the biding peptide has at least 80% of identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12, 14, 16-18.

[0035] In one embodiment, the biding peptide has at least 80% of identity with an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-11. The biding peptide of the invention is capable of biding by itself to an unfunctionalized substrate without requiring the need of any linker moiety. The term “linker” is used herein to refer to a compound or a chemical moiety that is optionally inserted between a binding peptide and the unfunctionalized substrate and serves the function of a linker ( / .e. to attach the binding peptide to the unfunctionalized substrate).

[0036] To reinforce the capability of the biding peptide of the invention to attach to the unfunctionalized substrate, the binding peptide according to the present invention can include one or more modifications, such as by addition of chemical moieties, or substitutions, insertions, and deletions of amino acids, where such modifications facilitate attachment to the unfunctionalized substrate and / or being bound to a linker molecule. For example, the binding peptides of the presently disclosed subject matter can comprise a functional group that is intrinsic to the binding peptide (e.g., amino groups on lysine), or the functional group can be introduced into the binding peptide by chemical modification to facilitate covalent attachment of the binding peptide to the unfunctionalized substrate.

[0037] In an embodiment of the invention, the binding peptide according to the present invention can include one or more modifications, such as by addition of chemical moieties, or substitutions, insertions, and deletions of amino acids, where such modifications improve peptide stability.

[0038] The term “unfunctionalized substrate” refers to any material that is biologically compatible with cells and / or polypeptide.

[0039] By “polypeptide”, it is meant in the invention an amino acid chain comprising at least two amino acids. When harbouring a biological function and found naturally in an organism, said polypeptide corresponds to a protein.

[0040] In a preferred embodiment of the invention, the unfunctionalized substrate is selected from the group consisting of dextran, polymethacrylate, agarose, polyvinyl alcohol, pullulan, pectin, polystyrene, silica gel, cellulose, polyethylene glycol, cross-linked version thereof and grafted version thereof.

[0041] The invention also relates to an isolated nucleic acid having a sequence encoding for the amino acid sequence of the binding peptide of sequence as set forth above.

[0042] As used herein, the term “isolated nucleic acid” refers to a nucleic acid that has been removed from its normal surrounding nucleic acid sequences in the genome or in cDNA sequences, and from which introns (if any) have been removed. The nucleic acid sequence may comprise RNA or cDNA sequence. Isolated nucleic acids consistent with the present invention may comprise additional sequences useful for promoting expression and / or purification of the encoded protein, including but not limited to polyA sequences, modified Kozak sequences, restriction sites, and sequences encoding epitope tags, export signals, and secretory signals, nuclear localization signals, and plasma membrane localization signals. It will be apparent to those of skill in the art, based on the teachings herein, what nucleic acid sequences will encode the proteins of the present disclosure.

[0043] The nucleic acid molecule according to the invention preferably corresponds to a nucleic acid sequences that has been codon-optimised for expression in a host cell, and especially for expression in E. coli. Where the host cell is a recombinant E. coli host cell, the hots cell may be any a strain of E. coli capable of expressing an exogenous nucleic acid sequence.

[0044] Especially, the isolated nucleic acid has at least 80% sequence identity to an optimised codon sequence selected in the group consisting of SEQ ID NO: 29 to SEQ ID NO: 39.

[0045] Accordingly, the nucleic acid sequence set forth in SEQ ID NO: 29 is a codon optimised sequence for encoding the amino acid sequence set forth in SEQ ID NO: 1 for expression in E. coli. The nucleic acid sequence set forth in SEQ ID NO: 30 is a codon optimised sequence for encoding the amino acid sequence set forth in SEQ ID NO: 2 for expression in E. coli. The nucleic acid sequence set forth in SEQ ID NO: 31 is a codon optimised sequence for encoding the amino acid sequence set forth in SEQ ID NO: 3 for expression in E. coli. The nucleic acid sequence set forth in SEQ ID NO: 32 is a codon optimised sequence for encoding the amino acid sequence set forth in SEQ ID NO: 4 for expression in E. coli. The nucleic acid sequence set forth in SEQ ID NO: 33 is a codon optimised sequence for encoding the amino acid sequence set forth in SEQ ID NO: 5 for expression in E. coli. The nucleic acid sequence set forth in SEQ ID NO: 34 is a codon optimised sequence for encoding the amino acid sequence set forth in SEQ ID NO: 6 for expression in E. coli. The nucleic acid sequence set forth in SEQ ID NO: 35 is a codon optimised sequence for encoding the amino acid sequence set forth in SEQ ID NO: 7 for expression in E. coli. The nucleic acid sequence set forth in SEQ ID NO: 36 is a codon optimised sequence for encoding the amino acid sequence set forth in SEQ ID NO: 8 for expression in E. coli. The nucleic acid sequence set forth in SEQ ID NO: 37 is a codon optimised sequence for encoding the amino acid sequence set forth in SEQ ID NO: 9 for expression in E. coli. The nucleic acid sequence set forth in SEQ ID NO: 38 is a codon optimised sequence for encoding the amino acid sequence set forth in SEQ ID NO: 10 for expression in E. coli. The nucleic acid sequence set forth in SEQ ID NO: 39 is a codon optimised sequence for encoding the amino acid sequence set forth in SEQ ID NO: 11 for expression in E. coli.

[0046] Biological Material The invention also relates to a biological material comprising at least one affinity tag consisting of the biding peptide as above defined.

[0047] In a preferred embodiment, the biological material is selected from the group consisting of a cell and chimeric protein.

[0048] In the invention, the term “chimeric protein" corresponds to the resulting fusion polypeptide from at least two polypeptides, wherein at least one of the polypeptides corresponds to the binding peptide as above defined ( / .e. the affinity tag). Hence, the chimeric protein may have one or more affinity tag corresponding to the binding peptide as above defined.

[0049] In the context of the chimeric protein, the one or more polypeptides other than the affinity tag(s) will be grouped under the denomination “target polypeptide”. Accordingly, the chimeric protein comprises at least one affinity tag corresponding to the binding peptide of the invention and a target polypeptide, wherein said polypeptide can be one or more polypeptides.

[0050] In an embodiment of the invention, the chimeric protein corresponds to therapeutic polypeptide, a diagnostic polypeptide, ora polypeptide of other function. The target polypeptide and the affinity tag(s) may be linked through covalent bonding including, but not limited to, covalent organic bonds (e.g., C-C, C-N, C-O, etc.), disulfide bonding, hydrogen bonding, electrostatic bonding, recombinant fusion, and / or conformational bonding. In some embodiments, the polypeptide and the affinity tag are linked by one or more linking compounds. In some embodiments, the chimeric protein exhibits the same or similar function as the polypeptide.

[0051] In some embodiments, the chimeric protein is an antibody. In the invention the term ‘antibody’ refers to a polyclonal antibody, a monoclonal antibody, a complement determining region- grafted antibody preparation, a hybrid antibody, an altered antibody, a F(ab)'2 fragment, a Fab molecule, a Fv fragment, a single domain antibody, a scFv fragment, a sdAb fragment, a VHH fragment, a chimeric antibody, or a fragment of any of the foregoing. In some embodiments, the affinity tag(s) are linked to the Fc region of the antibody, when the latter is present.

[0052] In some embodiments, the chimeric protein is green fluorescent protein (“GFP”) or a variant thereof (e.g., GFPmut2). In some embodiments, the chimeric protein is maltose-binding protein (“MBP”) or a variant thereof. In some embodiments, the chimeric protein is mCherry fluorescent protein (“mCherry”) or a variant thereof.

[0053] In some embodiments, the chimeric protein may further comprise a cleavage site, for example a protease cleavage site. In some embodiments, the affinity tag and the polypeptide of the chimeric protein are separated by the cleavage site. In some embodiments, the cleavage site is a portion of the affinity tag. In other embodiments, the cleavage site is a portion of the polypeptide. In yet other embodiments, the cleavage site comprises, consists essentially of, or consists of an amino acid sequence that is not a portion of the affinity tag or the polypeptide. In some such embodiments, treatment of the chimeric protein with a suitable protease cleaves the affinity tag, or a portion thereof, from the chimeric protein to release an amino acid sequence comprising or consisting essentially of the polypeptide. One of skill in the art will recognize that, when a protease is used to cleave the chimeric protein, the resulting cleaved polypeptide will contain at least one terminal amino acid residue in addition to the polypeptide. For example, a chimeric protein of formula (X)nRR-polypeptide, wherein (X)nRR is the affinity tag having a binding peptide as above defined with two consecutive arginine residues, may be cleaved in one embodiment by a protease that targets consecutive basic residues, such as OmpT. In such an embodiment, the chimeric protein cleavage products will be (X)nR and R- peptide. In another example, a fusion protein of formula (X)nKK-peptide, wherein (X)nKK is the affinity tag having two consecutive lysine residues, may be cleaved in one embodiment by a protease that targets consecutive basic residues, such as OmpT. In such an embodiment, the chimeric protein cleavage products will be (X)nK and K-peptide.

[0054] In an embodiment of the invention, the chimeric protein may further comprise a detectable moiety, such as an enzyme, a prosthetic group, a fluorescent material, a luminescent material, a bioluminescent material, a radioactive material, a positron emitting metal, and / or a nonradioactive paramagnetic metal ion. The specific detectable moiety used will depend on the method of detection used; non-limiting examples of detection methods for which a suitable detection moiety may be incorporated into a fusion protein of the present disclosure include: flow cytometric detection, scanning laser cytometric detection, fluorescent immunoassays, enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), bioassays (e.g., neutralization assays), and Western blotting applications. The detection moiety may be linked to the chimeric protein through covalent bonding including, but not limited to, covalent organic bonds (e.g., C-C, C-N, C-O, etc.), disulfide bonding, hydrogen bonding, electrostatic bonding, recombinant fusion, and / or conformational bonding. In some embodiments, the detection moiety and the affinity tag are linked by one or more linking compounds.

[0055] In some embodiments, the chimeric protein of the invention may further comprise a marker sequence. The marker sequence, for example, may be linked to the fusion protein through covalent bonding including, but not limited to, covalent organic bonds (e.g., C-C, C-N, C-O, etc.), disulfide bonding, hydrogen bonding, electrostatic bonding, recombinant fusion, and / or conformational bonding. In some embodiments, the marker sequence and the affinity tag are linked by one or more linking compounds. In some embodiments, the marker sequence is selected from the group consisting of a hexa-histidine tag, a myc tag, and a flag tag.

[0056] In an embodiment of the invention, the biological material may be a cell. In some embodiments, the cell displays on the surface a chimeric protein as above defined. The invention also relates to an isolated nucleic acid encoding the chimeric protein as above defined. The said isolated nucleic acid preferably corresponds to a nucleic acid molecule that has been codon-optimised for expression in a host cell, and especially for expression in E. coli.

[0057] Composition

[0058] The invention also relates to a composition comprising the biological material as above defined. The composition may be intended to be used for isolating or detecting the biological material. The composition may be in the form of a suspension or a solution.

[0059] In some embodiments, the composition may comprises any known adjuvant for the purpose of the composition, for example a buffer when the composition is intended to be used for isolating or detecting the biological material. The said buffer may be sodium phosphate. In some embodiment, the composition does not comprises sodium chloride, hydrochloric acid, isopropanol and phosphate buffered saline (PBS).

[0060] Vector and Host Cell

[0061] The invention also relates to a recombinant vector encoding the sequence of the binding peptide as above defined.

[0062] Especially, the recombinant vector comprises an insert constituted by the above defined nucleic acid encoding for the biding peptide of the invention.

[0063] The term “vector” as used herein refers to any molecule (e.g., nucleic acid, plasmid, or virus) that is used to transfer coding information to a host cell. The term “vector” includes a nucleic acid molecule that is capable of transporting another nucleic acid to which it has been linked. One type of vector is a “plasmid,” which refers to a circular double-stranded DNA molecule into which additional DNA segments may be inserted. Another type of vector is a viral vector, wherein additional DNA segments may be inserted into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host cell and thereby are replicated along with the host genome. In addition, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms “plasmid” and “vector” may be used interchangeably herein, as a plasmid is the most commonly used form of vector. However, the disclosure is intended to include other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), which serve equivalent functions.

[0064] Preferably, said recombinant vector is an expression vector in which said nucleic acid molecule is placed under the control of appropriate transcription and translation regulatory elements. In addition, said vector may comprise sequences (tags) fused in phase with the 5‘ and / or 3’ end of said insert, useful for immobilisation, and / or detection and / or purification of the protein expressed from said vector.

[0065] The invention also relates to a recombinant host cell comprising the vector as above defined.

[0066] The term “recombinant host cell” (or “host cell”) as used herein refers to a cell into which a recombinant expression vector has been introduced. A recombinant host cell or host cell is intended to refer not only to the particular subject cell, but also to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but such cells are still included within the scope of the term “host cell” as used herein. A wide variety of host cell expression systems can be used to express the binding proteins, including bacterial, yeast, baculoviral, and mammalian expression systems (as well as phage display expression systems). To express the biding peptide, a host cell is transformed or transfected with one or more recombinant expression vectors carrying DNA fragments encoding the biding peptide such that the biding peptide is expressed in the host cell and, preferably, secreted into the medium in which the host cells are cultured, from which medium the biding peptide can be recovered.

[0067] In a preferred embodiment of the invention, the host cell is E. coli.

[0068] Method for isolating or detecting

[0069] The invention also relates to a method for isolating / purifying or detecting a biological material as above defined. In the present disclosure, the term “isolating” and “purifying” will be considered as synonyms.

[0070] The method for isolating or detecting a biological material comprises the following steps: a) providing an unfunctionalized substrate, b) providing the biological material as above defined, c) attaching / immobilising the at least one biological material onto the unfunctionalized substrate by contacting them, and d) isolating or detecting the biological material.

[0071] Step a) The unfunctionalized substrate may be selected in the group consisting of dextran, polymethacrylate, agarose, polyvinyl alcohol, pullulan, pectin, polystyrene, silica gel, cellulose, polyethylene glycol, cross-linked version thereof and grafted version thereof. Especially, the unfunctionalized substrate may be selected in the group consisting of dextran, polymethacrylate, agarose, polyvinyl alcohol, pullulan, pectin, cross-linked version thereof and grafted version thereof. In particular, the unfunctionalized substrate may be selected in the group consisting of dextran, polymethacrylate, agarose, polyvinyl, cross-linked version thereof and grafted version thereof.

[0072] The unfunctionalized substrate may be plane or in the form of at least one bead.

[0073] In some embodiments, the unfunctionalized substrate is housed in a suitable vessel, such as a column, which is formed of a material other than the ones above cited to prevent any biding of the biding peptide. In some embodiments, the vessel is a plastic, silica or inert metal vessel.

[0074] Step b)

[0075] The biological material may be provided dried (especially in the form of a powder) or within a composition as above defined. Especially, the said composition comprise a buffer such as sodium phosphate. In some embodiment, the composition does not comprises sodium chloride, hydrochloric acid, isopropanol and phosphate buffered saline (PBS).

[0076] Step c)

[0077] Purpose of step c) is to obtained immobilised biological material on the substrate.

[0078] A used herein, the term “attach” and “immobilise” in reference to the biological material vis-a- vis the substrate will be taken as synonyms.

[0079] To this end, the contacting step c) may be carried out by pushing the biological material onto the unfunctionalized substrate. In case the biological material is present within a composition as above defined, this step can be carried out by pressure means, such as a syringe.

[0080] The contacting step c) may be performed at a temperature sufficient to enable efficient pouring of the biological material while avoiding denaturing the latter. Thus, a person of skill in the art will be able to select a suitable temperature based on the elements of the biological material and their sensitivities to elevated temperatures. Especially the contacting step c) is carried out at room temperature. The term “room temperature” refers in the invention to a temperature from 20°C to 30°C.

[0081] The contacting step c) may be performed for a period of time sufficient to enable complete attachment of the fusion protein to unfunctionalized substrate. Especially, said attachment occurs quickly at room temperature, usually within several minutes. Thus, the contacting step c) may be performed for a period of time of at least about 1 minute, 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 11 minutes, at least about 12 minutes, at least about 13 minutes, at least about 14 minutes, at least about 15 minutes, at least about 16 minutes, at least about 17 minutes, at least about 18 minutes, at least about 19 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, or more than 60 minutes.

[0082] The contacting step c) may further comprises a washing step of the unfunctionalized substrate. The said washing step may be performed using any suitable washing fluid capable of flushing any unattached biological material, contaminating proteins, or other undesired components of the composition comprising the biological material. The washing fluid may be for example sodium phosphate buffer.

[0083] The washing step may be performed at a temperature sufficient to enable efficient pouring and flow of the washing fluid on or through the unfunctionalized substrate, while avoiding denaturing of biological material. Thus, here again, a person of skill in the art will be able to select a suitable temperature for the washing step based on the components of the biological material and their sensitivities to elevated temperatures. Especially the washing step is carried out at room temperature.

[0084] The washing step may be performed for a period of time sufficient to enable complete flushing of non-adhered contaminants from the unfunctionalized substrate. As will be recognized by one of skill in the art, the amount of time required will depend on a number of factors including, for example, the solubility of contaminants in the washing fluid, the viscosity of the washing fluid, the amount of unfunctionalized substrate used, the amount of washing fluid to be used, and the pressure applied to the washing fluid (if any) and / or the reduced pressure applied downstream of the unfunctionalized substrate (if any). Especially, the washing step may be performed for a period of time of at least about 1 minute, 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 11 minutes, at least about 12 minutes, at least about 13 minutes, at least about 14 minutes, at least about 15 minutes, at least about 16 minutes, at least about 17 minutes, at least about 18 minutes, at least about 19 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, or more than 60 minutes. Step d)

[0085] In case the purpose of the method is the detection of the biological material, step d) may be carried out by directly detecting the immobilised biological material with any adapted detection device for the biological material, especially depending any detection tag carried out by the biological material.

[0086] In case the purpose of the method is the isolation of the immobilised biological material, step d) may comprise a releasing step of contact the unfunctionalized substrate with a realising agent to release the biological material and a subsequent step of recovering the unattached biological material.

[0087] The realising agent may be selected in the group consisting of sodium chloride, hydrochloric acid, isopropanol and phosphate buffered saline (PBS). In some embodiments, the realising agent is sodium chloride or hydrochloric acid and is in a concentration of at least 0.1 M, for example at least 0.15M, at least 0.2M, at least 0.5M, at least 1 M, at least 1.5M, at least 2M, at least 3M, at least 5M.

[0088] Alternatively, the releasing agent may comprise, consist essentially of, or consist of a protease for cleaving the biological material, for example at a cleavage site. In some embodiments, the protease is selected to cleave the biological material at a cleavage site. In some embodiments, the releasing agent comprises, consists essentially of, or consists of a protease selective for a cleavage site at or near the C-terminal or N-terminal of the affinity tag. In some embodiments, the releasing agent comprises, consists essentially of, or consists of a protease selected from the group consisting of: OmpT, TEV protease, Thrombin, Factor Xa and Enterokinase.

[0089] The releasing step may be performed at a temperature sufficient to enable efficient pouring of the releasing agent, while avoiding denaturing of biological material. Thus, here again, a person of skill in the art will be able to select a suitable temperature for the releasing step based on the components of the biological material and their sensitivities to elevated temperatures. Especially the washing step is carried out at room temperature.

[0090] The releasing step may be performed for a period of time sufficient to enable complete release of the biological material. Especially, the releasing step may be performed for a period of time of at least about 1 minute, 2 minutes, at least about 3 minutes, at least about 4 minutes, at least about 5 minutes, at least about 6 minutes, at least about 7 minutes, at least about 8 minutes, at least about 9 minutes, at least about 10 minutes, at least about 11 minutes, at least about 12 minutes, at least about 13 minutes, at least about 14 minutes, at least about 15 minutes, at least about 16 minutes, at least about 17 minutes, at least about 18 minutes, at least about 19 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 60 minutes, or more than 60 minutes.

[0091] Method for functionalizing a substrate or for immobilising a biological material onto a substrate

[0092] The invention also relates to a method for functionalizing a substrate or for immobilising a biological material onto a substrate, the comprising the following steps : a) providing an unfunctionalized substrate as above defined, b) providing at least one biological material as above defined, c) attaching / immobilising the at least one biological material onto the unfunctionalized substrate by contacting them.

[0093] Any of the above mentioned biological material may be used in this method.

[0094] In a preferred embodiment, the at least one biological material may correspond to at least one antibody as above defined.

[0095] The embodiments above recited for step a), b) and c) apply mutatis mutandis to the present step a), b) and c).

[0096] Substrates and Kits

[0097] The invention also relates to a substrate to which is attached at least one biological material as defined above.

[0098] The substrate is notably obtained by the method for functionalizing a substrate or immobilising a biological material onto a substrate as above defined.

[0099] The material of substrate to which is attached the at least one biological material may be of any kind of the ones recited for the above defined unfunctionalized substrate.

[0100] In a preferred embodiment, the biological material is an antibody as above defined.

[0101] The invention also relates to a kit for isolating or detecting a biological material as defined above comprising an unfunctionalized substrate as above defined, a washing solution or suspension, and an eluant comprising a releasing agent.

[0102] In some embodiments, the washing solution may be sodium phosphate.

[0103] In some embodiments, the releasing agent may be selected in the group consisting of sodium chloride, hydrochloric acid, isopropanol and phosphate buffered saline (PBS). Alternatively, the releasing agent may comprise, consist essentially of, or consist of a protease for cleaving the biological material, for example at a cleavage site. The invention finally relates to a kit for isolating or detecting a target comprising a substrate as defined above, a washing solution or suspension, and an eluant comprising a releasing agent, wherein the substrate comprises at least one antibody as above defined, said antibody being able to bind to the target.

[0104] BRIEF DESCRIPTION OF THE FIGURES

[0105] Figure 1 represents six biding peptides according to the invention synthesized on a cellulose substrate membrane. Spot A corresponds to binding peptide of sequence SEQ ID NO: 19. Spot B corresponds to binding peptide of sequence SEQ ID NO: 1. Spot C corresponds to binding peptide of sequence SEQ ID NO: 3. Spot D corresponds to binding peptide of sequence SEQ ID NO: 20. Spot E corresponds to binding peptide of sequence SEQ ID NO: 4. Spot F corresponds to binding peptide of sequence SEQ ID NO: 9. Spots were contacted with blue dextran. Presence of attached dextran to the spot is revealed by blue scale (appearing grey in the figure wherein a more dark grey spot indicates more dextran present and thus a better attachment to the binding peptide of the spot).

[0106] EXAMPLES

[0107] Example 1 : Biding peptide discovery

[0108] FliTrx™ peptide fusion proteins are used for displaying on the surface of a cell, and selected for their ability to attach to unfunctionalized substrate.

[0109] For this purpose, a bacterial display system utilizing the host E.coli strain GI826 was used. This bacterial display system has the following characteristics: The construct includes the cl repressor gene under the control of the trp promoter. The wild-type fliC gene, which encodes flagellin, has been deleted to ensure that only peptide fusion proteins are displayed on the cell surface. Additionally, the motB gene has been deleted to prevent flagellar rotation, which enhances the binding of cells to the unfunctionalized substrate. A Tn 10 transposon, conferring tetracycline resistance, is inserted into the eda locus, located near the motB gene. The eda locus encodes the enzyme 2-keto-3-deoxygluconate 6-phosphate aldolase.

[0110] FliTrx™ panning protocol

[0111] 50 ml IMG Medium from FliTr™ Random kit containing 100 pg / ml ampicillin (no tryptophan) was inoculated with one vial of the FliTrx™ library and grew with shaking (225-250 rpm) to saturation (Optical density at 600nm = ~3) for 15 hours (overnight) at 25°C.

[0112] Induction of the library The expression of FliTrx™ library was induced by adding 1 x 1010 cells (1 Optical density at 600nm = ~1 x 109cells for a 1 ml sample) of the overnight culture to 10 ml IMG Medium containing 100 pg / ml ampicillin and 100 pg / ml tryptophan and grown at 25°C with shaking for 6 hours.

[0113] Selection of positive clones

[0114] The induced culture was contacted with Sephacryl™ S-200 HR (Cytiva) to select for bacterial clones which show affinity to the beaded matrix. Sephacryl ™ S-200 HR is a cross-linked copolymer of allyl dextran and N,N'-methylene bisacrylamide, used for size exclusion chromatography. 1.0 ml of the matrix was equilibrated with 20 mM sodium phosphate buffer and later contacted with 10 ml of induced bacterial culture containing the FliTrx library in 125 ml Erlenmeyer flasks. After washing of unbound cells, elution of selected bacteria was accomplished by simple mechanical shearing of the flagella by vortexing. Selected cells were again inoculated into fresh IMG medium, grown overnight, induced and re-contacted with the material. This procedure was repeated 5 times (e.g., 5 panning cycles were implemented). Induction efficiency was checked at each panning cycle employing 12 % SDS PAGE electrophoresis. To select for single colonies, an overnight culture from the fifth panning was streaked onto RMG plates containing 100 pg / ml ampicillin and incubated overnight at 30°C. To the panned library glycerol was added to 20% and stored at -80°C until needed.

[0115] Plasmid isolation and sequencing

[0116] 5 ml of RM medium from FliTr™ Random kit containing single clones was used. The plasmid was then extracted using a standard miniprep procedure (Qiagen kit, Qiagen, Dusseldorf, Germany). The plasmids were sequenced at MWG Biotech (Ebersberg, Germany). 2.5 pg of each plasmid in 20 pl in 5 mM Tris-HCL, pH 8.0 - 9.0 was prepared and sent for sequencing to the mentioned service provider.

[0117] Peptide structure

[0118] Finally, peptide sequences were deduced from the results obtained from plasmid sequencing and correspond to sequences set forth in SEQ ID NO:1 to SEQ ID NO: 9 and SEQ ID NO: 12 to SEQ ID NO: 28.

[0119] Example 2: Peptide synthesis

[0120] Amounts (mEq. range) of selected natural peptide structures were produced by chemical synthesis. Solid-phase peptide synthesis was carried out on an automated peptide synthesizer (ABI-433A, Applied Biosystems, California, USA) using the standard solid phase peptide synthesis (SPPS) method. Fmoc protecting group was removed from the N-terminal of the pre- loaded resin bound amino acid by 20% piperidine in NMP, and the C-terminal of another Fmoc- amino acid was activated using HOBt / HBTU / DIEA (1 :1 :2) in DMF. Afterwards, the activated amino acid was allowed to react with the deprotected N-terminal of the resin bound amino acid. A cleavage cocktail (82.5% TFA (v / v), 5% phenol (v / v), 5% water (v / v), 5% thioanisol (v / v) and 2.5% 1 ,2-ethanedithiol (v / v)) was used to separate the desired peptide from the resin as well as to remove the acid-labile side chain protecting groups. The molecular masses of the synthesized peptides were checked by assisted laser desorption / ionization time-of-flight (MALDI-ToF) mass spectrometry (Autoflex II, Bruker Daltonics, Bremen, Germany).

[0121] Example 3: Retention and elution on Sephacryl S-300HR

[0122] Eight peptides of sequence SEQ ID NO: 1 to SEQ ID NO: 8 that were synthetised according to Example 2 were subjected to binding tests in 1.0 mL bed open columns, packed with a first unfunctionalized substrate of Sephacryl™ S-300 HR beads. Columns were pre-equilibrated in 20 mM phosphate buffer (pH = 7.4). After sample application (200 pL) of a peptide solution (1.0 mg / mL), 3 column volumes of the buffer was applied to remove any unbound peptide. Bound material was eluted in the same buffer, containing 0.75 M sodium chloride (1.5 CV). The presence of the peptides was followed spectrophotometrically (UV-1900i UV-Vis Spectrometer®), by performing measurements at 205nm-280nm.

[0123] Results are indicated in Table 1 below.

[0124] Table 1

[0125] As showed in Table 1 , each of the eight peptides shows good results in both retention properties to the substrate and elution from the substrate. Interestingly, some peptide sequences (SEQ ID NO: 3, 5, 7) displayed a superior ability to bind in a reversible manner. Example 4: Retention and elution on Toyopearl HW-55

[0126] Two peptides of sequence SEQ ID NO: 10 and 11 that were synthetised according to Example 2 were subjected to binding tests in 1.0 mL bed open columns, packed with a second unfunctionalized substrate of Toyopearl HW-55 beads. Columns were pre-equilibrated in 20 mM phosphate buffer (pH = 7.4). After sample application (1000 pL) of a peptide solution (1.0 mg / mL), 3 column volumes (CV) of the buffer was applied to remove any unbound peptide. Bound material was eluted in three different conditions:

[0127] 1 : the same buffer containing 0.15 M sodium chloride (1.5 CV);

[0128] 2: the same buffer containing 2.0 M sodium chloride (1 .5 CV);

[0129] 3: hydrochloric acid solution (1 .0 N) containing 30% isopropanol (1 .5 CV).

[0130] The presence of the peptides was followed spectrophotometrically, by performing measurements at 205nm-280nm.

[0131] Results are indicated in Table 2 below.

[0132] Table 2

[0133] As showed in Table 2, each of the two peptides shows good results in both retention properties to the substrate and elution from the substrate. The two peptide sequences showed different elution profiles depending on the elution buffer, with better elution by a particular elution buffer giving specific elution profiles for each peptide.

[0134] Example 5: Dyed-dextran onto immobilised peptides

[0135] The two peptides of Example 4 were immobilised on a matrix of Sepharose CL-4B (Cytiva®)to obtain a p1 -Sepharose and a p4-Sepharose, respectively.

[0136] NHS-activated Sepharose 4 Fast Flow was utilised for p1 peptide immobilisation. The coupling solution was prepared by dissolving the peptide in 0.2 M NaHCCh containing 0.5 M NaCI (pH 8.3). The activated material was washed with 12 volumes of cold 1 mM HCI immediately before use. Subsequently, the Sepharose beads were mixed with the coupling solution (1 :4), the pH adjusted to 8.0, and the reaction was let to proceed for 4 hours at room temperature. After the coupling is completed, any non-reacted groups were blocked with 0.1 M Tris-HCI (pH 8.5) for a 8 hours. The adsorbent was intensively washed and stored in a cold room until use.

[0137] The resulting peptide-affinity-matrix was employed to run binding experiments were a coloured version of the natural polysaccharide dextran was used as a third unfunctionalized substrate. Binding tests were run in 1.0 mL bed open columns, packed with a matrix of Sepharose CL- 4B alone (control), p1 -Sepharose and p4-Sepharose, respectively. Columns were preequilibrated in 20 mM phosphate buffer (pH = 7.4). After sample application (1000 pl) of Blue Dextran solution (0.4 mg / mL), 10 column volumes of the buffer was applied to remove any unbound polymer. Bound material was eluted in four different conditions:

[0138] 1 : the same buffer containing 2.0 M sodium chloride (3 CV);

[0139] 2: Glycine-HCI Buffer (0.1 M, pH 3.0) (3 CV);

[0140] 3: Phosphate Buffer Saline (PBS) solution comprising 20% isopropanol (3 CV). ;

[0141] 4: hydrochloric acid solution (1 .0 N) containing 30% isopropanol (3 CV).

[0142] The presence of the peptides was followed spectrophotometrically, by performing measurements at 600nm.

[0143] Results are indicated in Table 3 below.

[0144] Table 3

[0145] These results confirm that p1 -Sepharose and p4-Sepharose have a good retention properties compared to control (Sepharose CL-4B alone). The two peptide sequences showed again specific elution profiles.

[0146] Example 6: Retention of Dextran on cellulose membrane

[0147] Six peptides (A: SEQ ID NO: 19; B: SEQ ID NO: 1 ; C: SEQ ID NO: 3; D: SEQ ID NO: 20; E: SEQ ID NO: 4; F: SEQ ID NO: 9) were synthesized on a cellulose substrate membrane with a MultiPep 1 SPOT system (CEM Corporation®). After equilibration with 20 mM sodium phosphate buffer, the membrane was contacted with CB-Dextran. Visual inspection revealed binding of dextran to all of the spots, as evidenced by grey discs on Figure 1. Peptide-spots A, E and F are more intense in grey, showing a higher affinity of these peptides for the soluble polymer.

Claims

CLAIMS1 . A binding peptide having at least 80% sequence identity to an amino acid sequence selected from the group consisting of sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 28.

2. The binding peptide according to claim 1 , having at least 80% sequence identity to an amino acid sequence selected from the group consisting of sequences set forth in SEQ ID NO: 1 to SEQ ID NO: 11 , SEQ ID NO: 19 and SEQ ID NO: 20.

3. An isolated nucleic acid having a sequence encoding for the amino acid sequence of the binding peptide of claim 1 or 2.

4. The isolated nucleic acid according to claim 3, having at least 80% sequence identity to an optimised codon sequence selected from the group consists of sequences set forth in SEQ ID NO: 29 to SEQ ID NO: 39.

5. A recombinant vector encoding the amino acid sequence of the binding peptide of claim 1 or 2.

6. A host cell comprising the recombinant vector of claim 5.

7. A biological material comprising at least one affinity tag consisting of the biding peptide according to claim 1 or 2.

8. The biological material according to claim 7, wherein it is selected from the group consisting of a cell and a chimeric protein.

9. The biological material according to claim 8, which is an antibody.

10. A composition comprising the biological material according to any of claims 7 to 9.11 . A method for isolating or detecting the biological material according to any of claims 7 to 9 comprising the following step: a) providing an unfunctionalized substrate, b) proving the biological material according to any of claims 7 to 9, c) attaching the at least one biological material onto the unfunctionalized substrate by contacting them, and d) isolating or detecting the biological material.

12. A method for functionalising a substrate or for immobilising a biological material according to any of claims 7 to 9 to a substrate, comprising the following step:a) providing an unfunctionalized substrate, b) providing at least one biological material according to any of claims 7 to 9, c) attaching the at least one biological material onto the unfunctionalized substrate by contacting them.

13. The method according to claim 11 or 12, wherein the unfunctionalized substrate is selected from the group consisting of dextran, polymethacrylate, agarose, polyvinyl alcohol, pullulan, pectin, polystyrene, silica gel, cellulose, polyethylene glycol, crosslinked version thereof and grafted version thereof.

14. A substrate to which is bound at least one biological material according to any of claims 7 to 9.

15. A kit for isolating or detecting a biological material according to any of claims 7 to 9, the kit comprising an unfunctionalized substrate, a washing solution or suspension, and an eluant comprising a releasing agent.

16. A kit for isolating or detecting a target comprising a substrate according to claim 14, a washing solution or suspension, and an eluant comprising a releasing agent, wherein the substrate comprises at least one antibody according to claim 9, said antibody being able to bind to the target.

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