Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
  • C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
  • C12N9/2462—Lysozyme (3.2.1.17)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/47—Hydrolases (3) acting on glycosyl compounds (3.2), e.g. cellulases, lactases
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
  • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
  • C12N9/503—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from viruses
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12Y—ENZYMES
  • C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
  • C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
  • C12Y302/01017—Lysozyme (3.2.1.17)
• • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
  • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
  • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
  • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
  • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

• the present invention relates to polypeptides comprising an amino acid sequence exhibiting at least about 90% sequence identity with the sequence of SEQ ID NO: 1.
• Said polypeptides preferably degrade the peptidoglycan of Gram-negative bacteria, in particular of Pseudomonas and/or Campylobacter bacteria.
• the present invention relates to nucleic acids encoding such polypeptides, vectors comprising such nucleic acids, and corresponding host cells.
• compositions comprising such polypeptides, nucleic acids, vectors, and/or host cells according to the present invention.
• the giant, lytic Myoviridae bacteriophage ⁇ (280 334 bp) infects Pseudomonas aeruginosa, an important opportunistic nosocomial pathogen resistant to many commonly used antibiotics, and is therefore the cause of considerable concern in hospital environments.
• Briers et al. (Molecular Microbiology (2007) 65(5), 1334-1344) sequenced the genome of said bacteriophage and identified the endolysin KZ144, a highly lytic peptidoglycan hydrolase.
• a fusion protein comprising the sequence of said endolysin as enzymatic element has been proposed for use in degrading the cell wall of Gram-negative bacteria.
• Fig. 1 illustrates:
• Fig. 2 illustrates:
• Fusion protein of SMAP-29 (underlined with solid line; SEQ ID NO: 76), modified KZ144 without N-terminal methionine and with T82I, A206V, S232T, I122M; A160T, C14S and C50S (underlined with semi-dotted/semi-solid line; SEQ ID NO: 33) and His-tag (underlined with dotted line; SEQ ID NO: 135).
• Fusion protein of SMAP-29 (underlined with solid line; SEQ ID NO: 76), modified KZ144 without N-terminal methionine and with T82I, A206N, S232T, I122M; A160T C14S and C50S (underlined with semi-dotted/semi-solid line; SEQ ID NO: 49) and His-tag (underlined with dotted line; SEQ ID NO: 135).
• Fusion protein of SMAP-29 (underlined with solid line; SEQ ID NO: 76), KZ144 without N-terminal methionine (underlined with semi-dotted/semi-solid line; SEQ ID NO: 2) and His-tag (underlined with dotted line; SEQ ID NO: 135).
• the present invention relates to a polypeptide comprising an amino acid sequence exhibiting at least about 90% sequence identity with the sequence of SEQ ID NO: 1, wherein SEQ ID NO: 1 is characterized by
• XI may be absent or any amino acid, in particular M,
• XI 4 may be any amino acid, preferably S, R or N, more preferably S or R
• X23 may be any amino acid, preferably S, R or N, more preferably S
• X50 may be any amino acid, preferably S, R or N, more preferably S or N
• X82 may be any amino acid, preferably T or I
• X122 may be any amino acid, preferably I or M
• XI 49 may be any amino acid, preferably M or P
• XI 54 may be any amino acid, preferably L or T
• XI 60 may be any amino acid, preferably A or T X167 may be any amino acid, preferably I or L
• X179 may be any amino acid, preferably N or F
• X180 may be any amino acid, preferably M or E
• X186 may be any amino acid, preferably V or Y
• X206 may be any amino acid, preferably A, N or V
• X212 may be any amino acid, preferably T or N
• X224 may be any amino acid, preferably P or Q
• X230 may be any amino acid, preferably N or Y
• X232 may be any amino acid, preferably S or T;
• polypeptide does neither comprise the amino acid sequence of SEQ ID NO: 2, nor of SEQ ID NO: 3, nor of SEQ ID NO: 4.
• polypeptide refers in particular to a polymer of amino acid residues linked by peptide bonds in a specific sequence.
• the amino acid residues of a polypeptide may be modified by e.g. covalent attachments of various groups such as carbohydrates and phosphate. Other substances may be more loosely associated with the polypeptide, such as heme or lipid, giving rise to conjugated polypeptides which are also comprised by the term "polypeptide” as used herein.
• the term as used herein is intended to encompass also proteins.
• the term “polypeptide” also encompasses for example complexes of two or more amino acid polymer chains.
• polypeptide does encompass embodiments of polypeptides which exhibit optionally modifications typically used in the art, e.g. biotinylation, acetylation, pegylation, chemical changes of the amino-, SH- or carboxyl-groups (e.g. protecting groups) etc..
• the polypeptide according to the present invention may also be a fusion protein, i.e. linkage of at least two amino acid sequences which do not occur in this combination in nature.
• polypeptide as used herein is not limited to a specific length of the amino acid polymer chain, but typically the polypeptide will exhibit a length of more than about 50 amino acids, more than about 100 amino acids or even more than about 150 amino acids. Usually, but not necessarily, a typical polypeptide of the present invention will not exceed about 750 amino acids in length.
• % sequence identity has to be understood as follows: Two sequences to be compared are aligned to give a maximum correlation between the sequences. This may include inserting "gaps" in either one or both sequences, to enhance the degree of alignment. A % identity may then be determined over the whole length of each of the sequences being compared (so-called global alignment), that is particularly suitable for sequences of the same or similar length, or over shorter, defined lengths (so-called local alignment), that is more suitable for sequences of unequal length.
• an amino acid sequence having a "sequence identity" of at least, for example, 95% to a query amino acid sequence is intended to mean that the sequence of the subject amino acid sequence is identical to the query sequence except that the subject amino acid sequence may include up to five amino acid alterations per each 100 amino acids of the query amino acid sequence.
• up to 5% (5 of 100) of the amino acid residues in the subject sequence may be inserted or substituted with another amino acid or deleted.
• a preferred, but not limiting, example of a mathematical algorithm which can be used is the algorithm of Karlin et a/. (1993), PNAS USA, 90:5873-5877.
• Such an algorithm is integrated in the BLAST family of programs, e.g. BLAST or NBLAST program (see also Altschul et al, 1990, J. Mol. Biol. 215, 403-410 or Altschul et al. (1 997), Nucleic Acids Res, 25:3389-3402), accessible through the home page of the NCBI at world wide web site ncbi.nlm.nih.gov) and FASTA (Pearson (1 990), Methods Enzymol. 83, 63-98; Pearson and Lipman (1988), Proc.
• Constant amino acid substitutions may occur within a group of amino acids which have sufficiently similar physicochemical properties, so that a substitution between members of the group will preserve the biological activity of the molecule (see e.g. Grantham, R. (1974), Science 185, 862-864).
• conservative amino acid substitutions are preferably substitutions in which the amino acids originate from the same class of amino acids (e.g. basic amino acids, acidic amino acids, polar amino acids, amino acids with aliphatic side chains, amino acids with positively or negatively charged side chains, amino acids with aromatic groups in the side chains, amino acids the side chains of which can enter into hydrogen bridges, e.g. side chains which have a hydroxyl function, etc.).
• Conservative substitutions are in the present case for example substituting a basic amino acid residue (Lys, Arg, His) for another basic amino acid residue (Lys, Arg, His), substituting an aliphatic amino acid residue (Gly, Ala, Val, Leu, lie) for another aliphatic amino acid residue, substituting an aromatic amino acid residue (Phe, Tyr, Trp) for another aromatic amino acid residue, substituting threonine by serine or leucine by isoleucine. Further conservative amino acid exchanges will be known to the person skilled in the art.
• deletion refers preferably to the absence of 1, 2, 3, 4, 5 (or even more than 5) continuous amino acid residues in the derivative sequence in comparison to the respective reference sequence, either intrasequentially or at the N- or C-terminus.
• insertion refers preferably to the additional intrasequential presence of 1, 2, 3, 4, 5 (or even more than 5) continuous amino acid residues in the derivative sequence in comparison to the respective reference sequence.
• addition refers preferably to the additional presence of 1, 2, 3, 4, 5 (or even more than 5) continuous amino acid residues at the N- and/or C-terminus of the derivative sequence in comparison to the respective reference sequence.
• substitution refers to the presence of an amino acid residue at a certain position of the derivative sequence which is different from the amino acid residue which is present or absent at the corresponding position in the reference sequence. As mentioned above, preferably such substitutions are conservative substitutions.
• the term “bulcell wall” as used herein refers to all components that form the outer cell enclosure of Gram-negative bacteria and thus guarantee their integrity.
• the term “apparent wall” as used herein refers to peptidoglycan, the outer membrane of the Gram-negative bacteria with the lipopolysaccharide, the bacterial cell membrane, but also to additional layers deposited on the peptidoglycan as e.g. capsules, outer protein layers or slimes.
• amino acid sequence stretch refers to a particular stretch of amino acid sequence in the amino acid sequence of the polypeptide of the invention. Said sequence refers to a sequence of a cationic peptide, a polycationic peptide, an amphiphatic peptide, a hydrophobic peptide, a sushi peptide and/or an antimicrobial peptide.
• His-tags such as His5-tags, His6-tags, His7-tags, His8-tags, His9- tags, HislO-tags, His 11 -tags, Hisl2-tags, Hisl6-tags and His20-tags, Strep-tags, Avi-tags, Myc-tags, Gst-tags, JS-tags, cystein-tags, FLAG-tags or other tags known in the art, thioredoxin or maltose binding proteins (MBP).
• MBP thioredoxin or maltose binding proteins
• cationic peptide refers preferably to a peptide having positively charged amino acid residues.
• a cationic peptide has a pKa-value of 9.0 or greater.
• at least four of the amino acid residues of the cationic peptide can be positively charged, for example, lysine or arginine.
• “Positively charged” refers to the side chains of the amino acid residues which have a net positive charge at about physiological conditions.
• cationic peptide refers also to polycationic peptides, but also includes cationic peptides which comprise for example less than 20%, preferably less than 10% positively charged amino acid residues.
• polycationic peptide refers preferably to a peptide composed of mostly positively charged amino acid residues, in particular lysine and/or arginine residues.
• a peptide is composed of mostly positively charged amino acid residues if at least about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95 or about 100 % of the amino acid residues are positively charged amino acid residues, in particular lysine and/or arginine residues.
• the amino acid residues being not positively charged amino acid residues can be neutrally charged amino acid residues and/or negatively charged amino acid residues and/or hydrophobic amino acid residues.
• the amino acid residues being not positively charged amino acid residues are neutrally charged amino acid residues, in particular serine and/or glycine.
• AMP antimicrobial peptide
• anti-bacterial peptide refers in particular to any peptide having anti-bacterial, anti-fungal, anti-mycotic, anti-parasitic, anti-protozoal, anti-viral, anti-infectious, anti-infective and/or germicidal, algicidal, amoebicidal, microbicidal, bactericidal, fungicidal, parasiticidal, protozoacidal, protozoicidal properties. Preferred are anti-bacterial peptides.
• the antimicrobial peptide may be a member of the R ase A super family, a defensin, cathelicidin, granulysin, histatin, psoriasin, dermicidine or hepcidin.
• the antimicrobial peptide may be naturally occurring in insects, fish, plants, arachnids, vertebrates or mammals.
• the antimicrobial peptide may be naturally occurring in insects, fish, plants, arachnids, vertebrates or mammals.
• the antimicrobial peptide may be naturally occurring in radish, silk moth, wolf spider, frog, preferably in Xenopus laevis, Rana frogs, more preferably in Rana catesbeiana, toad, preferably Asian toad Bufo bufo gargarizans, fly, preferably in Drosophila, more preferably in Drosophila melanogaster, in Aedes aegypti, in honey bee, bumblebee, preferably in Bombus pascuorum, flesh fly, preferably in Sarcophaga peregrine, scorpion, horseshoe crab, catfish, preferably in Parasilurus asotus, cow, pig, sheep, porcine, bovine, monkey and human.
• an "antimicrobial peptide” may in particular be a peptide which is not a cationic peptide, polycationic peptide, amphiphatic peptide, sushi peptide, defensins, and hydrophobic peptide, but nevertheless exhibits antimicrobial activity.
• sushi peptide refers to complement control proteins (CCP) having short consensus repeats.
• CCP complement control proteins
• the sushi module of sushi peptides functions as a protein-protein interaction domain in many different proteins. Peptides containing a Sushi domain have been shown to have antimicrobial activities.
• sushi peptides are naturally occurring peptides.
• amhiphatic peptide refers to synthetic peptides having both hydrophilic and hydrophobic functional groups.
• amphiphatic peptides may be e.g. alpha helical, having predominantly non polar side chains along one side of the helix and polar residues along the rest of its surface.
• hydrophobic group refers preferably to chemical groups such as amino acid side chains which are substantially water insoluble, but soluble in an oil phase, with the solubility in the oil phase being higher than that in water or in an aqueous phase.
• amino acid residues having a hydrophobic side chain interact with one another to generate a non-aqueous environment.
• Examples of amino acid residues with hydrophobic side chains are valine, isoleucine, leucine, methionine, phenylalanine, tryptophan, cysteine, alanine, tyrosine, and proline residues
• hydrophobic peptide refers to a hydrophobic peptide, which is preferably composed of mostly amino acid residues with hydrophobic groups. Such peptide is preferably composed of mostly hydrophobic amino acid residues, i.e. at least about 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95 or at least about 100 % of the amino acid residues are hydrophobic amino acid residues.
• the amino acid residues being not hydrophobic are preferably neutral and preferably not hydrophilic.
• the term "tag” refers to an amino acid sequence, which is typically in the art fused to or included in another amino acid sequence for a) improving expression of the overall amino acid sequence or polypeptide, b) facilitating purification of the overall amino acid sequence or polypeptide, c) facilitating immobilisation of the overall amino acid sequence or polypeptide, and/or d) facilitating detection of the overall amino acid sequence or polypeptide.
• tags are His tags, such as His5-tags, His6-tags, His7-tags, His8- tags, His9-tags, HislO-tags, Hisl l-tags, Hisl2-tags, Hisl6-tags and His20-tags, Strep-tags, Avi-tags, Myc-tags, GST-tags, JS-tags, cystein-tags, FLAG-tags, HA-tags, thioredoxin or maltose binding proteins (MBP), CAT, GFP, YFP, etc.
• MBP thioredoxin or maltose binding proteins
• the person skilled in the art will know a vast number of tags suitable for different technical applications.
• the tag may for example make such tagged polypeptide suitable for e.g. antibody binding in different ELISA assay formats or other technical applications.
• the polypeptide according to the present invention may exhibit in the amino acid sequence exhibiting at least about 90% sequence identity with the sequence of SEQ ID NO: 1 at least one (e.g. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16 or even all 17) of the following: XI 4 is not C; X23 is not C; X50 is not C; X82 is I; X122 is M; X149 is P; X154 is T, X160 is T; X167 is L; X179 is F; X180 is E; X186 is Y; X206 is N or V, X212 is N; X224 is Q; X230 is Y and/or X232 is T. It is understood that the number indicating the position of the respective amino acid residue indicates the relative position in the sequence corresponding to SEQ ID NO: 1 , and not to the overall amino acid sequence of the polypeptide according to the present invention, which may be longer.
• the inventive polypeptide exhibits said at least 90% sequence identity.
• the inventive polypeptide may thus for example exhibit a higher level of sequence identity, e.g. may exhibit at least about 95%, at least about 96%>, at least about 97%, at least about 98%, at least about 98,5%), at least about 99% (e.g. less than 3 amino acids deviation) , at least about 99,3% (e.g. less than 2 amino acids deviation), at least about 99,5%, at least about 99,6% or even 100% sequence identity with the sequence of SEQ ID NO: 1.
• An inventive polypeptide comprising a sequence sharing a given level of sequence identity with the sequence of SEQ ID NO: 1 (or more specific sequences thereof, see below) can for example deviate from the reference sequence by addition, substitution, insertion or deletion of one or more amino acid residues and all possible combinations thereof. Only for the sake of clarity it is pointed out that such combinations refer to distinct positions in the sequence. A “deletion” followed by “addition”, or “addition” followed by “deletion”, of one or more amino acids, at the same relative position, is not an combination of an “addition” and “deletion” (or vice versa) but falls under the term "substitution".
• the deviations in sequence from the sequence of SEQ ID NO: 1 will be of conservative nature, e.g. conservative substitutions. Even more preferably the deviation in sequence is limited to those positions in SEQ ID NO: 1 (or more specific sequences thereof, see below), which have been identified to be non-critical for the enzymatic activity, i.e. XI , X14, X23, X50, X82, X122, X149; X160, X167, X179, X180, X186; X206; X212; X224; X230 and/or X232.
• the polypeptide according to the present invention exhibits in the amino acid sequence exhibiting at least about 90% sequence identity with the sequence of SEQ ID NO: 1 a glutamic acid residue at position 1 15.
• the mutation El 15A led to a loss in activity of about 70% of the enzyme.
• an inventive polypeptide comprising said mutation will thus not be a loss of function polypeptide and may still serve various technical purposes, it is certainly preferred if such mutation is not present in the sequence stretch corresponding to SEQ ID NO: 1 within the inventive polypeptide.
• polypeptide of the present invention comprises the sequence of SEQ ID NO: 1.
• X14 is not C
• X23 is not C
• X50 is not C
• Combinations are possible, e.g. X14 and X23 are not C, X14 and X50 are not C, or X23 and X50 are not C.
• amino acid residues can be deleted or substituted by any other amino acid.
• examples for such other amino acids are S, R and N.
• X14 may for example be S, N, or R; more preferably S or R; most preferably R; X23 may for example be S, N, or R, more preferably S; and X50 may for example be S, N, or R, more preferably S or N; most preferably N.
• X14, X23 and X50 may of course exhibit different amino acid substitutions, for example X14 may be R while X23 and X50 are S; or X14 and X23 are S, while X50 is N; XI 4 may be R while X23 is S and X50 is N etc.
• XI 4 is S
• X23 is S or X50 is S.
• XI 4 and X23 are S
• XI 4 and X50 are S
• X23 and X50 are S
• X14, X23 and X50 may also all three be S. Absence of one or more or even of all of these cysteine residue has the advantage that the risk of aggregation of the polypeptide according to the present invention, e.g. by undesired disulfide bridge formation, is reduced, and is thus an preferred embodiment of the present invention.
• this second type of amino acid modifications may be combined with the above mentioned cysteine replacements in any type of combination conceivable.
• combinations are, without being limited thereto, X14S, X50S, X122M and X160T; X14S, X50S, X82I, X122M, X160T, X206V, and X232T; X14S, X50S, X82I, X122M, X160T, X206N, and X232T; X14S, X50S, X82I, X122M, X206V, and X232T; X14S, X50S, X82I, X122M, X149P, X160T, X206V, and X232T; X14S, X50S, X82I, X122M, X160T, X180E, X206V, and X232T; X14S, X14S,
• SEQ ID NO: 1 (consensus sequence of the present invention) the first amino acid residue is indicated as being either absent or any amino acid, in particular M.
• the results of the inventors, and of previous work show, that the N-terminal methionine of KZ144 is dispensable.
• the position of XI in the sequence corresponding to SEQ ID NO: 1 in the inventive polypeptide is not M.
• polypeptide of the present invention exhibits for example N-terminally of the sequence corresponding to SEQ ID NO: 1 further sequence elements, it may for instance for the purpose of effective expression in a host cell be useful, if the methionine at position 1 of SEQ ID NO: 1 is eliminated or replaced by another amino acid in order to avoid a starting codon in the corresponding nucleic acid sequence, potentially leading to parallel expression of a polypeptide lacking the further sequence elements located more N-terminally.
• XI is of course preferably methionine (e.g. for expression purposes). For the enzymatic activity XI is however never required.
• SEQ ID NO: 1 Sequences falling under the definition of SEQ ID NO: 1, which have been particularly tested by the inventors, are for instance SEQ ID NOs: 6-27 (and corresponding sequences without N-terminal methionine, SEQ ID NOs: 28-49).
• the polypeptide of the present invention may for example comprise a sequence exhibiting at least 90%> sequence identity with a sequence selected from any of SEQ ID NOs: 6-49, wherein the polypeptide does neither comprise the amino acid sequence of SEQ ID NO: 2, nor of SEQ ID NO: 3, nor of SEQ ID NO: 4.
• the polypeptide according to the present invention may comprise aside of the enzymatic amino acid sequence, e.g. the sequence exhibiting at least about 90%> sequence identity with the sequence of SEQ ID NO: 1 (or other sequences falling under these definition), further amino acid sequence stretches, e.g. as already disclosed in similar fashion in WO 2010/149792.
• the polypeptide according to the present invention may for example comprise additionally at least one amino acid sequence stretch selected from the group consisting of amphiphatic peptide, cationic peptide, polycationic peptide, hydrophobic peptide, or naturally occurring antimicrobial peptide, like sushi peptide and defensin.
• additional amino acid sequence stretches may improve the antibacterial properties of the inventive polypeptide.
• the inventive polypeptide may comprise at least two distinct amino acid sequence stretches selected from the group of amphiphatic peptide, cationic peptide, polycationic peptide, hydrophobic peptide, or naturally occurring antimicrobial peptide, like sushi peptide and defensin.
• polypeptide according to the present invention may comprise at least one additional amino acid sequence stretch selected from this group.
• additional amino acid sequence stretches see for example also WO 2010/023207, WO 2010/149792, WO 2010/149795 and WO 2012/085259.
• antimicrobial amino acid sequences which may be used in carrying out the present invention are listed in the following table.
• Cecropin A D. GWLKKIGKKIERVGQHTRDATIQGLGIPQQAANVAATA
• Lycotoxin 1 IWLTALKFLGKHAAKKLAKQQLSKL SEQ ID NO: 92
• Dermaseptin 1 ALWKTMLKKLGTMALHAGKAALGAAADTISQGTQ SEQ ID NO: 95
• the at least one additional amino acid sequence stretch may be a sushi peptide which is described by Ding JL, Li P, Ho B Cell Mol Life Sci. 2008 Apr;65(7-8): 1202-19.
• the Sushi peptides structural characterization and mode of action against Gram-negative bacteria.
• the sushi 1 peptide according to SEQ ID NO: 1 15 is particularly preferred.
• Other preferred sushi peptides are sushi peptides S I and S3 and multiples thereof; FASEB J. 2000 Sep; 14(12): 1801-13.
• Preferred hydrophobic peptides are Walmaghl having the amino acid sequence according to SEQ ID NO: 1 16 and the hydrophobic peptide having the amino acid sequence Phe-Phe-Val- Ala-Pro (SEQ ID NO: 1 17).
• Preferred amphiphatic peptides are a4-helix of T4 lysozyme according to SEQ ID NO: 1 18 and WLBU2 -Variant having the amino acid sequence according to SEQ ID NO: 1 19 and Walmagh 2 according to SEQ ID NO: 120.
• a polypeptide according to the present invention may comprise at least one additional amino acid sequence stretch selected from the group consisting of: KRK and SEQ ID NOs: 50-120.
• additional amino acid sequence stretch selected from the group consisting of: KRK and SEQ ID NOs: 50-120.
• Corresponding examples are for instance polypeptides comprising a sequence selected from the group consisting of SEQ ID NOs: 121-127 (and corresponding sequences without N-terminal methionine, SEQ ID NOs: 128-134.
• a polypeptide according to the present invention comprises an amino acid sequence exhibiting at least about 90% sequence identity with the sequence of SEQ ID NO: 1 , wherein the polypeptide does neither comprise the amino acid sequence of SEQ ID NO: 2, nor of SEQ ID NO: 3, nor of SEQ ID NO: 4.
• a polypeptide of the present invention may also comprise an amino acid sequence exhibiting at least 91 ,5 % sequence identity with an amino acid sequence selected from any of SEQ ID NOs: 121 - 134, wherein the polypeptide does neither comprise the amino acid sequence of SEQ ID NO: 2, nor of SEQ ID NO: 3, nor of SEQ ID NO: 4.
• inventive polypeptide may thus for example comprise a sequence exhibiting a higher level of sequence identity than 91 ,5% with an amino acid sequence selected from any of SEQ ID NOs: 121 - 134, e.g. may exhibit at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98,5%, at least about 98,75%, at least about 99% (e.g. less than 3 amino acids deviation) , at least about 99,5% (e.g. less than 2 amino acids deviation), at least about 99,6%> or even 100% sequence identity with an amino acid sequence selected from any of SEQ ID NOs: 121 - 134.
• the polypeptide may comprise additionally one or more tag sequences.
• tag sequence may be present N- terminally or C-terminally of the sequence exhibiting at least about 90% sequence identity with the sequence of SEQ ID NO: 1. They may for example be located at the N- or C- terminus of the inventive polypeptide.
• the one or more tag sequence is located C-terminally of the amino acid sequence exhibiting at least 90%> sequence identity with the sequence of SEQ ID NO: 1.
• the one or more tag sequences may for example be linked to the amino acid sequence exhibiting at least 90% sequence identity with the sequence of SEQ ID NO: 1 directly or via a short linker of 1 to 10 amino acid residues, preferably 1 to 5 amino acid residues, even more preferably 1 to 2 amino acids.
• Linker sequences are preferably flexible sequences, comprising one or more glycine residues.
• tags are known in the art, some of which have already been mentioned above.
• a particularly preferred tag sequence is a His-tag, preferably a His tag according to SEQ ID NO: 135.
• the length of the polypeptide according to present invention is in principle not limited, but preferably the length will not be excessively large.
• a polypeptide according to the present invention has an overall length not exceeding about 320 amino acids, preferably not exceeding about 310 amino acids.
• polypeptides according to the present invention can be selected from the group consisting of SEQ ID NOs: 136-142 (and corresponding sequences without N-terminal methionine, SEQ ID NOs: 143-149).
• a polypeptide according to the present invention comprises an amino acid sequence exhibiting at least about 90% sequence identity with the sequence of SEQ ID NO: 1, wherein the polypeptide does neither comprise the amino acid sequence of SEQ ID NO: 2, nor of SEQ ID NO: 3, nor of SEQ ID NO: 4.
• a polypeptide of the present invention may also comprise an amino acid sequence exhibiting at least 91,5 % sequence identity with an amino acid sequence selected from any of SEQ ID NOs: 136 - 149, wherein the polypeptide does neither comprise the amino acid sequence of SEQ ID NO: 2, nor of SEQ ID NO: 3, nor of SEQ ID NO: 4.
• inventive polypeptide may thus for example comprise a sequence exhibiting a higher level of sequence identity than 91,5% with an amino acid sequence selected from any of SEQ ID NOs: 136 - 149, e.g. may exhibit at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 98,5%, at least about 99%, at least about 99,25% (e.g. less than 3 amino acids deviation), at least about 99,5% (e.g. less than 2 amino acids deviation), at least about 99,6%> or even 100% sequence identity with an amino acid sequence selected from any of SEQ ID NOs: 136 - 149.
• Deviations from SEQ ID NOs: 136 - 149 may in particular occur in the two sequences linking the components SMAP29 peptide, modified KZ144 endolysin and His-tag.
• a polypeptide according to the present invention is preferably characterized by the ability to degrade the peptidoglycan of Gram-negative bacteria, in particular of Pseudomonas and/or Campylobacter bacteria.
• the polypeptide according to the present invention is preferably capable of degrading the peptidoglycan of Pseudomonas aeroginosa, in particular Pseudomonas aeroginosa PAOl, Campylobacter jejuni and/or Campylobacter coli.
• the peptidoglycan degrading activity on gram negative bacteria can be measured by assays well known in the art, e.g. by muralytic assays in which the outer membrane of gram negative bacteria is permeabilized or removed (e.g. with chloroform) to allow the putative enzyme access to the peptidoglycan layer. If the enzyme is active, degradation of the peptidoglycan layer will lead to a drop of turbidity, which can be measured photometrically ( see for example Briers et al, J. Biochem. Biophys Methods 70: 531-533, (2007).
• the present invention relates to a nucleic acid encoding a polypeptide according to the present invention.
• the present invention relates to a vector, such as an expression or cloning vector, which comprises a nucleic acid according to the present invention.
• the present invention relates to a host cell comprising a polypeptide according to the present invention, a nucleic acid according to the present invention, and/or a vector according to the present invention.
• the present invention relates to composition
• composition comprising a polypeptide according to the present invention, a nucleic acid according to the present invention, a vector according to the present invention, and/or a host cell according to the present invention.
• said composition is a pharmaceutical composition comprising a pharmaceutical acceptable diluent, excipient or carrier.
• endolysin KZ144 For identification of advantageous sites of modification in endolysin KZ144 (SEQ ID NO: 5), the inventors used in a first step targeted destabilization of the target protein. For this purpose an N-terminally truncated KZ144 was generated (SEQ ID NO: 150) into which sequence mutations were introduced via random mutagenesis (error-prone PCR) followed by subsequent fusion and selection with a chloramphenicol assay (CAT assay).
• SEQ ID NO: 150 N-terminally truncated KZ144 was generated (SEQ ID NO: 150) into which sequence mutations were introduced via random mutagenesis (error-prone PCR) followed by subsequent fusion and selection with a chloramphenicol assay (CAT assay).
• the protein melting temperature of promising candidates was determined by circular dichroism (CD). Changes of ellipticity for the proteins were recorded at 220nm as a function of temperature using Jasco J-815 CD spectrometer and fitted to a simple sigmoid unfolding model using JASCO analysis software. The protein melting temperatures (Tmelt) were determined as midpoint of unfolding transition. The spectra were recorded at protein concentrations of 5.0-5.8 ⁇ with a heating rate of l°C/min and incubation time of 3s in 410 ⁇ 1 volume in a 1mm light path Hellma quartz cuvette. Measurements were performed in 50mM NaPh buffer, 300mM NaCl at pH of 7.4, 7.0, 6.2 and 5.7.
• stabilizing mutations can and were subsequently introduced into other sequences such as full length sequences, increasing stability there as well.
• serine was used in some constructs for substitution of cysteine residues CI 4, C23 and/or C50 (position indicated with respect to SEQ ID NO: 5; conservative substitutions).
• Other substituents at said positions tested were N and R.
• Example 2 Melting temperature of some polypeptides according to the present invention and MIC for selected bacterial strains
• SMAP-29 was chosen. SMAP-29 was found in sheep leukocytes and consists of 29 amino acids (RGLRRLGRKIAHGVK YGPTVLRIIRIAG; molecular weight: 3.3 kDa, SEQ ID NO: 76). It is built up of two LPS-binding sites which are connected by a central hinge.
• the nucleic acid molecules encoding the respective peptide and endolysin were constructed with a Ndel (5 ' -CAT ATG-3 ' ) restriction site at the 5 ' -end of the nucleic acid molecule and a Xhol (5 ' -CTC GAG-3 ' ) restriction site at the 3 ' -end of the nucleic acid molecule.
• Peptide and endolysin are connected via a BamHI (5 '-GGA TCC-3 ').
• Fusion proteins were constructed by linking at least two nucleic acid sequences using standard cloning techniques as described e.g. by Sambrook et al. 2001, Molecular Cloning: A Laboratory Manual. Therefore the nucleic acid molecule encoding the peptide stretch was cleaved in a digest with the respective restriction enzymes Ndel and BamHI. Subsequently the cleaved nucleic acids encoding the peptide stretch was ligated into the pET21 b expression vector (Novagen, Darmstadt, Germany), which was also cleaved in a digest with the respective restriction enzymes Ndel and BamHI before.
• the nucleic acid molecule encoding the endolysin was cleaved in a digest with the restriction enzyme BamHI and Xhol, so that the endolysin could be ligated into the pET21b expression vector (Novagen, Darmstadt, Germany), which was also cleaved in a digest with the respective restriction enzymes BamHI and Xhol before.
• the sequence of the peptide-endolysin fusions was controlled via DNA sequencing and correct clones were transformed into E.coli BL21(DE3)pLysS (Novagen, Darmstadt, Germany) for protein expression.
• Ni 2+ affinity chromatography is performed in 4 subsequent steps, all at room temperature:
• the Hydrophobic Interaction Chromatography (HIC) is performed in 5 subsequent steps, all at room temperature:
• the elution pool of the HIC step is dialyzed (membrane: regenerated cellulose with MWCO: 6000-8000D) into storage buffer (500 mM NaCl and 20mM HEPES; pH7.4) at 4°C. Dialysis factor is 160 - 250.
• MIC Minimum inhibitory concentration
• the respective overnight culture was diluted 1 : 10.
• the bacterial culture was diluted to a concentration of 2xl0 5 to 8xl0 5 colony-forming-units per ml in Mueller-Hinton-broth (not cation-adjusted Mueller-Hinton-broth) and split in the required amount of tubes.
• the polypeptide of interest was added in different concentrations (determined as ⁇ g/ml final concentration in the Mueller-Hinton-broth). In case of Ps. aeruginosa EDTA was added to a final concentration of 2 mM. In case of Campylobacter sp no EDTA was used.
• the mixture was incubated overnight at 37°C for Ps. Aeruginosa and at 42°C for Campylobacter species. Bacterial growth was visibly determined by turbidity (in comparison to negative control). The MIC was defined as the concentration in the tube where no bacterial growth was observed. Positive (without polypeptide of interest and/or EDTA) and negative control (Mueller-Hinton-broth without bacteria) were included in the experiment.
• the mutations introduced did thus not only increase melting temperature of the polypeptides of SEQ ID NOs: 136 -142 vs the polypeptide of SEQ ID NO: 151, but did also not affect activity of the polypeptide, not even the sevenfold mutation of SEQ ID NO: 141.
• Example 3 Temperature stability of the polypeptide according to SEQ ID NO: 139 and
• the inventors exposed exemplarily the mutated polypeptides of SEQ ID NO: 139 and SEQ ID NO: 141 to temperatures clearly exceeding the melting temperature of the native, non-mutated reference polypeptide (SEQ ID NO: 151).
• the position indicated refers to the position within the sequence portion corresponding to the KZ144 sequence, SEQ ID NO: 5, and not to the position within the full- length sequence of the SEQ ID NO: indicated in the table.

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