Oct 2010 sci pres
- 1. A new platform for the high-density display of proteins on biopolyester beads
- 2. Overview PolyBa'cs is a privately held development stage biotechnology company based in Palmerston North, New Zealand. We have developed a patented technology for the high densty display of proteins/pep'des on the surface of bioplas'c beads. To date we have demonstrated proof-‐of-‐concept in a number of applica'ons including separa'ons science, diagnos'cs, vaccine delivery and biocatalysts. R&D is managed at Massey Univ, Palmerston North and the Univ of Canterbury (Christchurch)
- 3. Vision To help create a cleaner, healthier world through the applica3on of our technology.
- 4. Our Business… Our goal is to make the plaPorm widely available to researchers who can benefit from the plaPorm’s versa'lity and poten'al. Copyright PolyBa'cs Ltd 2009
- 5. The Science… • high-density display of proteins on the surface of granules (nanoparticles or biobeads). • the bioplastic protein complex is produced in vivo. • grown at high cell density (HCD) to keep production costs down. • the system is versatile, i.e. Polybatics has shown that polypeptides with many different functionalities can be displayed simultaneously on the granules in a variety of hosts. • functionalities of expressed proteins are very well maintained Copyright PolyBa'cs Ltd 2009
- 6. Polyester Beads in Nature 50% of all microbes in nature produce polyester inclusions in response to nutrient stress as a means of carbon storage. Bacterial cell Several bioplastics (polyhydroxyalkanoates) can be produced in different bacteria, but the most common is comprised of (R)-3-hydroxybutyric acid. The enzyme:polyester complexes self-assemble into granules which accumulate in the cytosol of the cells and range in size from 50-3000nm. 100 nm The enzyme polyester synthase polymerizes (R)-3- hydroxyacyl-CoA thioester monomers into polyester with the release of coenzyme A. Grage, K, et al, Biomacromolecule.s 2009, 10, 660-‐669 Copyright PolyBa'cs Ltd 2009
- 7. Polyester synthases and bead assembly Proposed reac'on mechanism Granule self assembly Hydroxyacyl-‐ CoA Soluble Amphipathic Polyester granule polyester synthase polyester synthase Copyright PolyBa'cs Ltd 2009
- 8. Polyester granule Polyester Synthase Structural protein Regulatory protein Depolymerase Phospholipids 100-‐3000 nm
- 9. Only the polyester synthase is required for granule formation Polyester Synthase 100-‐3000 nm
- 10. A natural chemical cross-link Bead surface Polymer core O O Cys S Cys O n > 1000 Polyester PhaC synthase Polyester bead Copyright PolyBa'cs Ltd 2009
- 11. Control of biopolyester bead size in vivo Gene'c engineering of E. coli 200 nm TEM 200 nm 1 µm TEM 20-‐3000 nm FM Rehm, B.H.A. (2007) Curr. Issues Mol. Biol. 9: 41-62. Copyright PolyBa'cs Ltd 2009
- 12. Bead Properties Density 1.06 g /ml Size distribu'on 100-‐600 nm pH stability 2.0 – 12.5 Temperature stability 160oC (for polymer) Electron backscaaering micrograph of lyophilized beads Copyright PolyBa'cs Ltd 2009
- 13. Applications The following slides present data from various published studies representing information in the public domain. PolyBatics has additional data from unpublished studies available for review under terms of confidentiality. Copyright PolyBa'cs Ltd 2010
- 14. Separations Science Different ligands can be displayed on the beads alone, or in combination with other functional domains (e.g. reporter molecules). Currently we have created a high binding capacity replacement for agarose immobilized Protein A for use in discovery, development and the production of antibodies. The Z-domain from protein A was displayed on the surface of the polyester beads and compared to commercial Protein A agarose beads used in IgG purification. 1 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 1: BioLabs Ladder 2: Pure IgG (1mg/ml) 3: PB WT Unbound 4: PB WT Elu'on 5: Pure IgG (1mg/ml) 6: GE Unbound 7: GE Elu'on 8: Pure IgG (1mg/ml) 9: PBZ Unbound 10: PBZ Elu'on 5µl of 1µg/ml hIgG was loaded onto a 10% SDS-‐PAGE 5µl of 5µg/ml hIgG was loaded onto a 10% SDS-‐PAGE gel gel Copyright PolyBa'cs Ltd 2009
- 15. Eluted human IgG in drained bead mass 100 75 % 50 25 0 PB 1 2 PB: PolyBatics ZZ polyester beads 1, 2: Commercial protein A agarose beads
- 16. Separations Science (cont’d) Display of single chain antibody fusions demonstrated a 75-fold increase in binding affinity as a result of the properly oriented natively folded protein when compared to the soluble counterpart. In this example a single chain variable fragment of the anti-ß-galactosidase antibody was C-terminally tagged to the synthase. M A B C M kDa 212.0 158.2 LacZ 116.4 97.2 66.4 BSA 55.6 42.7 Determina@on of the binding capacity of an@-‐β-‐galactosidase scFv-‐displaying beads. 34.6 Beads were incubated with increasing concentra'ons of β-‐glactosidase, and the conversion of ONPG was measured to detect β-‐galactosidase ac'vity. Control beads M, Molecular weight standard were only incubated with the highest β-‐galactosidase concentra'on. An absorp'on maximum was observed for ≥2.66µg of β-‐galactosidase. A, Protein mixture B, Proteins purified with wildtype beads C, Proteins purified with an'-‐LacZ an'body displaying beads Grage, Copyright PolyBa'cs Bioconj. Chemistry, 19(1):254-‐62. K. and Rehm, B.H.A. (2008) Ltd 2009
- 17. Separations Science (cont’d) Schema@c of the bead containing the gold binding protein and the IgG binding zz-‐domain. 100nm TEM of polyester beads with colloidal gold bound to the surface A protein profile showing polymer beads displaying both gold and silica plus IgG binding func@onali@es Schema@c overview of hybrid genes used in this study. The bracket combining the last two constructs illustrates that both genes are located on the same plasmid. Binding is indicated by +, nonbinding by -‐; -‐* indicates a slightly increased unspecific silica binding compared to the control. Jahns, A.K., Haverkamp, R.G., Rehm, B009 Copyright PolyBa'cs Ltd 2 .H.A.,(2008) Bioconj. Chem. 19:2072-‐2080
- 18. Vaccines Small size combined with the ability to display multiple domains on the surface of the beads has allowed us to engineer virus- like particles. Animal studies using antigens from Mycobacterium tuberculosis displayed on the beads elicit both humoral and cell mediated response to the virus like particles. An'body responses in mice immunized with wild-‐type beads or Ag85A-‐ESAT-‐6 beads. Levels of an'bodies of the IgG1 isotype ( ) or the IgG2 isotype ( ) were measured by ELISA. Each data point represents results from 7 mice ± SD. Results are expressed in reciprocal an'body 'tres, represen'ng the dilu'on required to obtain half of the maximal amount of OD signal. * Significantly greater than the non-‐vaccinated control group (p<0.01). ** Significantly greater than all other vaccine groups (p<0.01). IFN-‐γ responses in mice immunized 3 'mes with (A) wild-‐type beads, (B) Ag85A-‐ESAT-‐6 beads, (C) SDS-‐PAGE analysis of proteins aaached to the polyester beads. Lane A, Ag85A-‐ESAT-‐6 beads with Emulsigen or (D) unvaccinated. Release of IFN-‐γ was measured by ELISA. molecular weight markers; Lane B, beads isolated from E. coli harboring plasmid * Significantly greater than the non-‐vaccinated control group (p<0.01). ** Significantly greater than pHAS-‐Ag85A-‐ESAT-‐6; Lane C, beads isolated from E. coli harboring plasmid pHAS. all other vaccine groups (p<0.01). The presence of the Ag85A-‐ESAT-‐6-‐PhaC fusion protein (indicated with the arrow) was confirmed by tryp'c pep'de fingerprin'ng analysis using MALDI-‐TOF-‐MS. Parlane NA, Wedlock DN, Buddle B2009 BH, (2009) Appl Environ Microbiol 75:7739-‐7744 Copyright PolyBa'cs Ltd M, Rehm
- 19. Spleen M.bovis CFU (log10) 4 3 5 6 PBS Vaccines Ag85A.ESAT6 BNP * (E.coli) Copyright PolyBa'cs Ltd 2009 Wild type BNP (E.coli) Ag85A.ESAT6 BNP (Lactococcus) Wild type BNP (Lactococcus) Lung M. bovis CFU (log10) 4 3 5 6 7 recAg85A.ESAT6 PBS ** BCG Ag85A.ESAT6 BNP * (E.coli) Wild type BNP (E.coli) Ag85A.ESAT6 BNP * (Lactococcus) Wild type BNP (Lactococcus) recAg85A.ESAT6 * BCG
- 20. Diagnostics Display of antigens on the beads in conjunction with reporter molecules or signal tags provides for multiplexed diagnostic assays. Here we present FACS and ELISA examples. MOG-‐phaP granules were incubated with serial dilu'ons of pooled an'sera from five MOG or OVA immunized mice. Granules were extensively washed and then incubated with bio'nylated an'-‐mouse IgG, followed by PE-‐conjugated streptavidin. The data was depicted in a "normalized" fashion (% of Max). One representa've experiment, of at least two experiments performed, is depicted. ELISA demonstra'ng specific binding of IgG to ZZ domain-‐displaying beads isolated from L. lac3s; ZZ beads L. lac3s indicates beads isolated from L. lac3s NZ9000(pNZ-‐ZZCAB) that produce PhaP-‐IL2 and PhaP-‐MOG granules were incubated with enterokinase for the ZZ-‐PhaC; control beads L. lac3s indicates granules isolated from L. lac3s NZ9000(pNZ-‐CAB) that indicated period of 'me, washed and then tested for the amount of na've produce PhaC only; and ZZ beads E. coli indicates granules isolated from E. coli producing ZZ-‐ proteins remaining at the surface of the granules. In (A), the an'-‐IL2 PC61 and PhaC. Isolated PHB granules were bound to ELISA plates, and HRP-‐conjugated rabbit an'-‐mouse an'-‐MOG 8-‐18C5 monoclonal an'bodies were used for this purpose as IgG was used to detect the func'onal display of the ZZ domain. described for Figure 1. Brockelbank, J.A., Peters, V., Rehm, B.H.A. (2006) Appl. Environ. Microbiol. 72 (11): 7394 Baeckstroem, T.B., Brockelbank, J.A., Rehm, B.H.A. (2007) BMC Biotechnology 7:3 Mifune, J., Grage, K., Rehm, B.H.A., (2009) Appl. Environ. Microbiol. 75(14):4668-‐4675 Copyright PolyBa'cs Ltd 2009
- 21. Diagnostics Copyright PolyBa'cs Ltd 2009
- 22. Biocatalysis Engineering the beads provides for the stable formation and display of enzymes on the surface of the beads. Creation of a synthase LacZ fusion resulted in reaction kinetics of a Km of 630 µM and a Vmax of 17.6 nmol/min for orthonitrophenyl-D- galactopyranoside as a substrate.1 Display of α-amylase on the surface of the PHB beads has also been evalauted. The immobilized BLA indicated a Michalis-Menten-type kinetics, with a Km of 5 µM, suggesting a high binding affinity compared with the Km values of 9.6 µM 44 µM for free BLA and BLA chemically cross-linked to cellulose beads, respectively. Qualita've analysis of BLA (α-‐amylase) ac'vity using starch agar Single bead multi-enzyme schemes (e.g. amylase and Lugol’s iodine staining. Sectors are as follows: A, α-‐amylase + lipase + protease)are currently under (Sigma 3403); B, BLA-‐PhaC beads; C, control PhaC beads. Protein amounts for the numbered posi'ons are as follows: 1, 56 µg; 2, investigation. 180 µg; 3 and 5, 21 µg; and 4 and 6, 70 µg Peters, V., Rehm, B.H.A., Appl Enviro. Mirco (2006) 72:1777-‐1783 Rasiah, I. A., Rehm, B.H. A. Appl. Enviro. Micro. (2009) 75:2012-‐2016 Copyright PolyBa'cs Ltd 2009
- 23. Functionalisation of biopolyester beads Polyester synthase scFv GFP Antigen TB Enzyme Drugs, dyes, Drugs, nutrients biotinylated molecule LacZ dyes, nutrients Drugs, Streptavidin dyes, Drugs, nutrients Inorganic dyes, nutrients compound antibody IL2 Cytokine doxorubicin Protein A (ZZ domain) Copyright PolyBa'cs Ltd 2010
- 24. Materials Processing Backscaaering electron density image showing size distribu'on of lyophilized PHB beads. Copyright PolyBa'cs Ltd 2009
- 25. Acknowledgements Prof. Bernd Rehm Dr. Maahew Plassmeyer Dr. Anika Jahns Dr. Panimalar Vijayan Yoga Maspolim Siddarth Runwal Amos Chua Thank you! Natalie Parlane Dr. Andy Herbert Prof. Conan Fee Dr. Bryce Buddle Dr. Uwe Goaschalk Dr. Wiltrud Treffenfeldt Copyright PolyBa'cs Ltd 2009