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Recombinant lytic and GFP-CBD fusion proteins based on the Listeria phage endolysin Ply511

Recombinant lytic and GFP-CBD fusion proteins based on the Listeria phage endolysin Ply511. Diploma Thesis Lisa Conza Supervised by Mathias Schmelcher. Introduction. Materials & Methods. Results. Overview. Discussion & Outlook. Recombinant endolysins. Introduction Methods & Results

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Recombinant lytic and GFP-CBD fusion proteins based on the Listeria phage endolysin Ply511

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  1. Recombinant lytic and GFP-CBD fusion proteins based on the Listeria phage endolysin Ply511 Diploma Thesis Lisa Conza Supervised by Mathias Schmelcher

  2. Introduction Materials & Methods Results Overview Discussion & Outlook Recombinant endolysins • Introduction • Methods & Results • Conclusions • Outlook

  3. Introduction Materials & Methods Results Overview Discussion & Outlook Listeria • Listeria are Gram-positive, non-spore forming, facultative anaerobic, short, rods • tolerates temperature between -0.4 - 50°C • pH optimum at pH 6-8 food borne pathogen causes epidemic or sporadic illness • motility performed by peritrichous flagella Listeria (Loessner, 2005)

  4. Introduction Materials & Methods Results Overview Discussion & Outlook Bacteriophages • is a broad-host-range bacteriophage • is virulent, it can lyse approximately 95% of Listeria strains 1/2 and 4 • 96% of phages are tailed • classified in the order of the Caudovirales • subdivided into three families: Siphoviridae, Podoviridae, Myoviridae (A511) A511 Bacteriophage A511, the black bar is 50 nm (Ackermann, 2006)

  5. Introduction Materials & Methods Results Overview Discussion & Outlook (Korndörfer et al., 2006) Endolysins • endolysins (bacteriophage lysins) are proteins which work as cell wall-hydrolyzing enzymes • produced in bacteriophage’s lytic cycle • with modular organization N C EAD CBD

  6. Introduction Materials & Methods Results Overview Discussion & Outlook Dualistic model • The phage mediated cell lysis works in a dualistic system: -holin is inserted in the cytoplasmic membrane ->form hole (membrane disruption) ->endolysins can pass through (access the peptidoglycan layer) Loessner, 2005, modified.

  7. Introduction Materials & Methods Results Overview Discussion & Outlook Alignment of three ply genes C Ply006 N C Ply118 N C Ply511 N • CBD118 and CBD006 have very similar binding range for serovars (1/2) and have high sequence homology at C-terminus • CBD511 and CBD118 are similar in sequence at N-terminus • Affinity to the cells of CBD006 is 100-fold lower then that of CBD118

  8. Introduction Materials & Methods Results Overview Discussion & Outlook KPSTPAPKPSTPSTNLDKLGLVDYMNAKKMDSSYSNRDKLAK QYGIANYSSGTASQNTTLLSKIKGGAPKPSTPAPKPST EAD CBD Ply511 • Ply511 is an N-acetylmuramoyl-L-alanine amidase • molecular mass of 36.5kDa Loessner, 2005, modified.

  9. Introduction Materials & Methods Results Overview Discussion & Outlook Aim of my work test for binding properties 1) HGFP_CBD constructs test for lytic activity 2) HEAD_CBD constructs

  10. Introduction Materials & Methods Results Overview Discussion & Outlook Methods • Construction of pHGFP_CBD plasmids -fragments amplification and ligation in pHGFP -protein overexpression and purification -protein binding assays • Construction of pHEAD_CBD plasmids -EAD fragment instead of the GFP gene -proteins overexpression and purification -protein photometric lysis assays and overlay assays

  11. Introduction Materials & Methods Results Overview Discussion & Outlook EAD S S CBD2 CBD1 S CBD1 S CBD2 S CBD2 CBD1 CBD1 S CBD1 S CBD1 CBD1 S S CBD2 S CBD2 CBD amplification

  12. Introduction Materials & Methods Results Overview Discussion & Outlook 250 CBD fragment amplification M CBDS1 CBD2 Size [bp] • PCR amplifications • -line 1 M • -line 2 CBDS1 • -line 3 CBD2 500

  13. Introduction Materials & Methods Results Overview Discussion & Outlook BamHI SphI SacI KpnI SmaI XmaI SalI PstI HindIII PT5 - lac O - lac O – RBS – ATG - 6xHis – MCS - Stop Codons pQE-30 3.4 kb Ampicillin Col E1 pQE-30 and pHGFP • pHGFP derived from pQE-30 vector (Loessner et al. 2002) • 4.159 kb cloning and expression vector • GFP gene inserted between BamHI and SacI • CBD fragments ligated downstream the GFP gene (Sacl/SalI)

  14. Introduction Materials & Methods Results Overview Discussion & Outlook CBD1 CBD2 GFP SacI BamHI SalI MCS Ampicillin pHGFP Col E1 Construction of HGFP_CBD fusion proteins • pHGFP extraction from E.coli JM109 • SacI and SalI digestion of pHGFP and the CBD fragments • ligation of plasmids with fragmens • E.coli XL1-Blue electroporation • colony PCR

  15. Introduction Materials & Methods Results Overview Discussion & Outlook CBD1 CBD2 GFP S S CBD1 CBD2 GFP S CBD1 GFP S CBD1 GFP CBD1 GFP S CBD1 GFP S S CBD2 GFP S CBD2 GFP HGFP_CBD constructs

  16. Introduction Materials & Methods Results Overview Discussion & Outlook M 1 2 3 4 5 6 Size [bp] Size [bp] 750 1000 250 GFP_CBD plasmids CBD2 M CBD2 M pHGFP 4000 500 • Colony PCR of pHGFP_CBDS2 250 • SacI and SalIdigestion of pHGFP and of CBD2

  17. Introduction Materials & Methods Results Overview Discussion & Outlook EAD HEAD_CBD constructs • PCR amplification of EAD511 coding region (BamHI/SacI sites) • excision of GFP fragment from HGFP_CBD constructs • insertion of EAD fragment CBD1 CBD2 GFP S S

  18. Introduction Materials & Methods Results Overview Discussion & Outlook CBD2 CBD1 S S EAD S EAD CBD1 EAD CBD1 S CBD1 EAD S CBD1 S EAD CBD2 S EAD CBD2 EAD EAD HEAD_CBD constructs

  19. Introduction Materials & Methods Results Overview Discussion & Outlook Size [bp] M EAD Size [bp] 4000 3500 2000 750 GFP 750 550 500 EAD amplified with PCR HEAD_CBD constructs pHGFP_CBD1S digestion with BamHI/SacI. For purification the plasmids are cut out from the gel

  20. Introduction Materials & Methods Results Overview Discussion & Outlook tested in binding assay with Listeria strains tested in lysis assays with Listeria WSLC 1001 and in overlay assays with Listeria WSLC 1001 and WSLC 1020 Protein preparations • control of overexpression and purification • -activity test protein purification with IMAC (Ni-NTA gravity flow columns) E.coli overexpression cell disruption (French Press) HGFP_CBD fusion proteins HEAD constructs

  21. Introduction Materials & Methods Results Overview Discussion & Outlook Results • HGFP_CBD fusion proteins • HEAD, HEAD_CBD fusion proteins

  22. Introduction Materials & Methods Results Overview Discussion & Outlook M 0h 1h 2h 4h CE Flow Wash Elute c.E. Size [kDa] 116.0 66.2 37.5 kDa HGFP_CBDS1S 45.0 35.0 25.0 18.4 14.4 • SDS-PAGE analysis of 6x his-tagged HGFP_CBDS1S • Overexpression was performed in E.coli XL-1 Blue MRF’ and induced with IPTG SDS-PAGE

  23. Introduction Materials & Methods Results Overview Discussion & Outlook Binding assay • ON cultures of Listeria cells • 100 µl of the centrifuged cells resuspended in 1 ml PBST were incubated with excess of fusion protein • 2 wash step with 500 µl PBST

  24. Introduction Materials & Methods Results Overview Discussion & Outlook -binding assay of fusion protein HGFP_CBDS1S2to Listeria strain WSLC 1033. Binding assay

  25. Introduction Materials & Methods Results Overview Discussion & Outlook Binding properties of different HGFP_CBD constructs Interpretation of binding properties: ++ cells displayed an intensive green fluorescence + cells visible with lower intensity (+)very weak signal - no signal

  26. Introduction Materials & Methods Results Overview Discussion & Outlook Binding assay SacI CBDS1 Linker PSA GFP H kpnI SalI • HGFP_LPSA_CBD constructs with a PSA linker are tested in binding assays • results obtained are very similar of those displayed by the HGFP_CBD proteins

  27. Introduction Materials & Methods Results Overview Discussion & Outlook Binding assay • HGFP_CBD “1” constructs don’t bind Listeria cells • HGFP_CBD “2” constructs bind the different Listeria cells • Confirmation of the idea that give the alignments of the three endolysins

  28. Introduction Materials & Methods Results Overview Discussion & Outlook HEAD_CBD constructs • HEAD_CBD tested in -lysis assays -overlay assays

  29. Introduction Materials & Methods Results Overview Discussion & Outlook Lysis assay • Equimolar amounts of all HEAD constucts • Positive control: HPL511 (Loessner et al., 1996) • Listeria WSLC 1001 • OD600 measurement in photometer after addition of enzyme • Steepest slope of the lysis curve corresponds to relative activity

  30. Introduction Materials & Methods Results Overview Discussion & Outlook Lysis assay • Lysis curve of EAD_CBD1S is representative for all the CBD1 constructs • Lysis curve of EADS2 represent the all the CBD2 constructs • HPL511 used as positive control OD600 Time[seconds]

  31. Introduction Materials & Methods Results Overview Discussion & Outlook Lysis assay • Lysis curves of HEAD_CBD“2“ constructs showed strong lytic activity • HEAD_CBD“1“ constructs: low activity • Lytic properties correspond to the binding properties of the CBD constructs

  32. Introduction Materials & Methods Results Overview Discussion & Outlook Overlay assays • E.coli plated on IPTG plates • incubation 4 h • disruption of the cell membrane with chloroform • Plate overlayed with Listeria cells in soft agar

  33. Introduction Materials & Methods Results Overview Discussion & Outlook Overlay assays -left: Listeria WSLC 1001; right:Listeria WSLC 1020

  34. Introduction Materials & Methods Results Overview Discussion & Outlook Overlay assays -left: Listeria WSLC 1001; right:Listeria WSLC 1020

  35. Introduction Materials & Methods Results Overview Discussion & Outlook Overlay assays -left: Listeria WSLC 1020 HEAD_CBD“1“ -right:Listeria WSLC1020 HEAD_CBD“2“

  36. Introduction Materials & Methods Results Overview Discussion & Outlook Overlay assays • HEAD_CBD“1“: big lysis zone, unclear boundaries > protein is able to diffuse • HEAD_CBD“2“: smaller, well defined boundaries > higher affinity

  37. Introduction Materials & Methods Results Overview Discussion & Outlook Conclusions 1 • The HGFP-CBD”1” fusion proteins don’t bind the different Listeria strains • Binding properties seem to be mainly located in the CBD”2” part • The isolated CBD”1” part seems not to play any role in binding • The HGFP-CBD proteins containing the PlyPSA linker show the same binding pattern of the proteins without linker

  38. Introduction Materials & Methods Results Overview Discussion & Outlook Conclusions 2 • The lytic activity of HEAD_CBD”1” constructs is very low when compared with Ply511, HEAD_CBD”2” proteins show a stronger lytic activity • The lysis zones in overlay assays of HEAD_CBD”1” are big and diffuse, the ones of HEAD_CBD”2” and Ply511 are smaller and very definite -> difference in diffusion • The isolated CBD”1” part seems not to play any role in binding

  39. Introduction Materials & Methods Results Overview Discussion & Outlook C Ply006 N C Ply118 N C Ply511 N Conclusions 3 • Both CBD subdomains are necessary for full functionality • Presence or absence of the repeated sequences does not seem to play a big role • Results obtained so far correspond to the situation in CBD118 and CBD006.

  40. Introduction Materials & Methods Results Overview Discussion & Outlook Outlook • Biacore measurent of the affinity of binding of HGFP_CBD2 to the listerial cell wall compared to the complete CBD construct HGFP_CBD511 • New pictures of binding assays

  41. Thank you to your attention! • I want to thank Mathias for the assistance and Professor Loessner to give me the possibility to perform my diploma thesis in its group.

  42. Questions?

  43. Introduction Materials & Methods Results Overview Discussion & Outlook HGFP_LPSA_CBD constructs • LPSA linker cloned between the GFP and the CBDs • Linker produced with overlapping primer (KpnI-SacI restriction site at their ends) • GFP gene amplifiedwith primer (BamHI-KpnI restriction site at their ends) SacI GFP H CBDS1 Linker PSA SalI kpnI • The two products were KpnI digested and ligated • Product was PCR amplified with a primers pair annealing to the linker sequence and the GFP gene • Sequencing for linker presence confirmation

  44. Introduction Materials & Methods Results Overview Discussion & Outlook pH_CBD GFP_LPSA digestion BamHI-SacI digestion BamHI-SacI purification purification GFP SacI BamHI SalI ligation MCS E.coli electrophoration Ampicillin pH_GFP Colony PCR Col E1 GFP_LPSA_CBD plasmids already digested for EAD clonation

  45. Introduction Materials & Methods Results Overview Discussion & Outlook BamHI SacI Size [bp] GFP Linker PSA kpnI 2000 750 GFP_LPSA Amplification of the GFP_LPSA Linker and GFP gene amplification Schema GFP_LPSA with restriction site

  46. Introduction Materials & Methods Results Overview Discussion & Outlook Listeria strains and H_GFP_LPSA_CBDs tested in binding assay Interpretation of binding properties: ++ cells displayed an intensive green fluorescence + cells can see but with less intensity, difficult to see (+) very low signal, weak binding - any cells could be seen red: results different to there obtained by the GFP_CBD constructs

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