Method and means for enrichment removal and detection of
The present invention relates to polypeptide fragments of endolysin Ply511, which recognize and bind listeria irrespective of the serotype but which have no cell wall hydrolysing enzymatic activity. The invention further relates to methods for enrichment, removal, and detection of listeria.
1. A polypeptide that binds Listeria , but does not exhibit any cell wall hydrolysing enzymatic activity, wherein the polypeptide comprises an endolysin Ply511 fragment including SEQ ID NO: 2 residues 180-341, but no contains more of SEQ ID NO: 2 than residues 116-341.
2. The polypeptide according to claim 1 , comprising an amino acid sequence according to SEQ ID NO:4, 6, or 8.
3. The polypeptide according to claim 1 , wherein the polypeptide fragment further comprises an affinity tag or a spacer molecule.
4. The polypeptide according to claim 3 , wherein the affinity tag is a His-Tag, Trp-Ser-His-Pro-Gly-Phe-Lys (SEQ ID NO:9), Avi-Tag, or a biotinylation domain.
5. The polypeptide according to claim 3 , wherein the spacer molecule is green fluorescent protein, maltose binding protein or a biotinylation domain.
6. A nucleic acid molecule comprising a sequence coding for a polypeptide fragment according to claim 1 .
7. The acid molecule according to claim 6 , comprising a sequence according to SEQ ID NO:3, 5, or 7.
8. A method for enrichment and/or removal of Listeria from a sample-comprising the steps of:
a) incubating or contacting a sample with a polypeptide according to claim 1 , which is unspecifically or directedly immobilised to a solid carrier, and
b) separating the carrier-polypeptide Listeria -complex from the sample.
9. The method according to claim 8 , further comprising after step b) the step of:
c) washing away of sample components unspecifically adhering to the carrier-polypeptide Listeria -complex.
10. The method according to claim 8 , wherein the steps a) and b) are performed in a chromatography column flow through method.
11. The method according to claim 8 , wherein the solid carrier is cellulose, filtration media, glass particles, magnet particles, centrifugation-materials, sedimentation-materials or filling materials for chromatography columns.
12. A method for the enrichment and/or removal of Listeria from a sample comprising the steps of:
a) incubating or contacting a sample with a polypeptide according to claim 3 ,
b) contacting and incubating of Listeria -polypeptide-complex with a carrier, which is coated with the respective binding partner of the polypeptide or the affinity tag, and
c) separating the carrier-polypeptide Listeria -complex from the sample.
13. The method according to claim 12 , further comprising after step c) the step of:
d) washing away of sample components unspecifically adhering to the carrier-polypeptide Listeria -complex.
14. The method according to claim 8 , further comprising after step b) a step of detecting the Listeria.
15. A kit comprising (i) a carrier onto which a polypeptide fragment according to claim 1 is immobilised, and (ii) washing buffer, detaching buffer and/or cell cracking buffer.
16. A kit comprising (i) a polypeptide fragment according to claim 3 , (ii) a carrier coated with the respective binding partner of the affinity tag or the spacer molecule, and (iii) washing buffer, detaching buffer and/or cell cracking buffer.
17. The method according to claim 12 , further comprising after step c) a step of detecting the Listeria.
18. The polypeptide fragment of claim 1 , wherein the polypeptide comprises the amino acid sequence of residues 116-341 of SEQ ID NO: 2.
19. The polypeptide fragment of claim 3 , wherein the spacer molecule comprises an affinity tag or a biotin.