Topics of Research

 

Autodisplay

and its application for

  1. Assay development and inhibitor testing
  2. Whole cell biocatalysts for synthesis of drugs and building blocks
  3. Directed evolution of enzyme inhibitors and biocatalysts
  4. Biosensor development and diagnostic tools

Autodisplay

The research of Joachim Jose and his group at the Institute of Pharmaceutical and Medicinal Chemistry at the WWU Münster is mainly focused on the development and the application of a new platform technology named Autodisplay. Autodisplay represents an efficient tool for the surface display of recombinant proteins in gram negative bacteria, in particular E. coli.  It is based on the secretion mechanism of the autotransporters, a family of proteins that was discovered by Joachim Jose and co-workers during his time at the Max-Planck-Institute for Biology in Tübingen.

 Autodisplay

Proteins that are transporting themselves to the cell surface are catalogued as autotransporters. They are synthesized within the cell as a polyprotein precursor with a typical signal peptide (SP) at the N terminus that directs the protein across the inner membrane (IM). Subsequently, the C terminus of the precursor folds into the outer membrane (OM) as a porin-like structure, a so called ß-barrel, which facilitates surface translocation of the passenger domain.  By simply replacing the coding region of the natural passenger by the coding region for a recombinant protein in the precursor gene, the recombinant protein can be transported to the cell surface very efficiently and in high yields. This is what became known as Autodisplay.  

A unique feature of Autodisplay in comparison to other surface display tools is that the anchoring domain is not covalently fixed at the cell surface, but is able to move in the plane of the outer membrane. This enables the functional display of multimeric proteins i.e. homodimeric, up to homononameric or heterotetrameric proteins, by simply expressing the monomeric genes as fusions with the autotransporter domains. This is what we called a passenger-driven dimerization or multimerization.

Autodisplay Bovine _ Cyp

Another unique feature of Autodisplay was discovered when bovine adreodoxin was displayed as a recombinant passenger. Autodisplay enables the surface display of functional proteins or enzymes, that contain inorganic prosthetic groups, e.g. iron-sulfur groups, flavins or porphyrins  as  well. Therefore, Autodisplay can be used for the surface display of CYP enzymes or FAD containing oxidoreductases, which is not possible by any other surface display tool known so far.
Finally, a major advantage of Autodisplay is its high expression rate, which yields 105 -106 functional recombinant proteins at the cell surface per single cell of E. coli, without disturbing cell viability of cell integrity.  

Key publications on autodisplay and autotransporters:

  • Jose J, Jähnig F, Meyer TF (1995) Common structural features of IgA1-protease-like outer membrane protein autotransporters. Mol Microbiol, 18: 380-382.
  • Maurer J, Jose J, Meyer TF (1997) Autodisplay: One-component-system for efficient surface display and release of soluble recombinant proteins from Escherichia coli. J Bacteriol, 179:794-804.
  • Jose J (2006) Autodisplay: efficient bacterial surface display of recombinant proteins. Appl Microbiol Biotechnol, 69:607-614.
  • Jose J, Meyer TF (2007) The autodisplay story – from discovery to biotechnical and biomedical applications. Microbiol Mol Biol R, 71:600-619.
  • Tozakidis IE, Sichwart S, Jose J (2015) Going beyond E. coli: Autotransporter based surface display on alternative host organisms. New Biotechnol, DOI:10.1016/j.nbt.2014.12.008

1. Assay development and inhibitor testing

A major application field of Autodisplay is the development of test assays for enzyme inhibitors by displaying the human drug target at the cell surface of E. coli. This provides at first a rapid and simple access to the human target enzyme for which an inhibitor is searched. Whole cells of E. coli displaying the human enzyme can be used for inhibitor testing, as the enzyme is free to access  at the cell surface for any kind of small (or larger) molecule. Purification or any other processing of the target enzyme is not necessary.  Moreover, Autodisplay provides access to expression of human target enzymes that are difficult to express by other expression systems due to the formation of inclusion bodies for example. In most cases we developed  test assay for the human target using instrumental analytics, always with the aim to provide a miniaturized tool that could be as well automated for HT screening.   

 Assay Development

Key publications on assay development and inhibitor testing:

  • Ehmer PB, Jose J, Hartmann RW (2000) Development of a simple and rapid test assay for inhibitors of human 17α-hydroxylase-C17,20-lyase by coexpression of cytochrome P450c17 and NADPH-cytochrome-P450 reductase in Escherichia coli. J Steroid Biochem Mol Biol, 75:57-63.
  • Hartmann RW, Hector M,  Haidar S, Ehmer PB, Reichert W, Jose J (2000) Synthesis and Evaluation of Novel Steroidal Oximes Inhibitors of P450 17 (17α-Hydroxylase/C17-20-Lyase) and 5α-Reductase Type 1 and 2. J Med Chem, 43:4266-4277.
  • Gratz A, Götz, C, Jose J (2010) A CE-based assay for human protein kinase CK2 activity measurement and inhibitor screening. Electrophoresis, 31:634-640.
  • Kaeßler A, Olgen S, Jose J (2011) Autodisplay of catalytically active human hyaluronidase hPH-20 and testing of enzyme inhibitors. Europ J Pharm Sci, 42: 138-147.
  • Götz C, Gratz A, Kucklaender U, Jose J (2012) TF - a novel cell-permeable and selective inhibitor of human protein kinase CK2 induces apoptosis in the prostate cancer cell line LNCaP.Biochim Biophys Acta - General subjects., 7:970-977.
  • Raaf J, Guerra B, Neundorf I, Bopp B, Issinger OG, Jose J, Pietsch M, Niefind K (2013) First structure of protein kinase CK2 catalytic subunit with an effective CK2ß-competitive ligand. ACS Chem Biol, 8:901-907.

2. Whole cell biocatalysts for synthesis of drugs and building blocks

Another major application of Autodisplay is the surface display of enzymes on E. coli in order to obtain whole cell biocatalysts for synthetic purposes. These are not restricted to human enzymes but can comprise biocatalysts of a wide variety of sources including enzymes with prosthetic groups as P450 enzymes (CYPs) or even complex enzyme systems.

 Cyclic Production

In this context it is remarkable, that the outer membrane of E. coli provides sufficient membrane environment to human P450s in order to be enzymatically active. This gives a new access to the synthetic potential of this highly interesting class of enzymes. The list of enzymes displayed on E. coli in an active form contains:  - Esterases - Lipase with its corresponding foldase  - CYP106A2 -  Nitrilases of different origin - Prenyltransferase - Sorbit dehydrogenase - Pyranose Oxidase -  NADH Oxidase - various human Hyaluronidases - Phosphotransferase and others.

Due to the robustness of the cells when using Autodisplay, they can be harvested from a reaction mixture and transferred to a fresh assay, which results in a cyclic exploitation of the whole cell biocatalyst. Synthesis of up to 500 mg product can be facilitated by batch cultures in a laboratory scale, but cells with autodisplayed enzymes can be used for fermentation purposes as well.

Due to the large number of different enzymes displayed on E. coli in an active form by Autodisplay,  a sequential synthesis  approach is possible, in which different whole cell biocatalysts are used as modular synthesis tools. A substrate solution is driving along this module  in a continuous process conduct leading to new compounds that can be tested on their inhibitory potential in an assay as described above, finally yielding new chemical entities and - most hopefully - new lead structures.
 

Sequential Synthesis

Key publications on whole cell biocatalysts for synthesis of drugs and building blocks:

  • Jose J, Bernhardt R, Hannemann F (2002), Cellular surface display of dimeric Adx and whole cell P450-mediated steroid synthesis on E. coli. J Biotechnol, 95: 257-268
  • Schultheiss E, Weiss S, Winterer E, Maas R, Heinzle E, Jose J (2008) Esterase autodisplay: enzyme engineering and whole cell activity determination by microplates with integrated pH-sensors. Appl Environ Microbiol, 74:4782-4791.
  • Detzel C, Maas R, Jose J (2011) Autodisplay of nitrilase from Alcaligenes faecalis in E. coli yields a whole cell biocatalyst for the synthesis of enantiomerically pure R-mandelic acid. ChemCat Chem, 3: 719-725.
  • Kranen E, Steffan N, Maas RM, Li SM, Jose J (2011) Development of a whole cell biocatalyst for the efficient prenylation of indole derivatives by Autodisplay of the aromatic prenyltransferase FgaPT2, ChemCatChem, 3:1200-1207.
  • Schumacher SD, Jose J (2012) Expression of active human P450 3A4 on the cell surface of Escherichia coli by autodisplay. FREE Access in 2014: J Biotechnol, 161:113-120. Elsevier Biotech Article most tweeted in 2012
  • Jose J, Maas RM, Teese MG (2012) Autodisplay of enzymes - molecular basis and perspectives.  J Biotechnol, 161:92-103.
  • Schüürmann J, Quehl P, Festel G, Jose J (2014) Bacterial whole cell biocatalysts by surface display of enzymes: towards industrial application. Appl Microbiol Biotechnol, 98:8031-8046.

3. Directed evolution of enzyme inhibitors and biocatalysts

We succeeded in the surface display of small peptide libraries as well as in the surface display of enzyme libraries. In case of using Autodisplay, such libraries are actually consisting of different cells of E. coli, wherein each cell is bearing a different variant at the cell surface in high numbers. Due to the surface exposure of the variant, the library of E. coli cells can be screened on binding a molecule of interest e.g. a target enzyme. In case the target has been labeled before with a fluorescent dye, fluorescence active cell sorting (FACS) can be used for selecting positive variant. By simple DNA sequencing the primary structure of the variant displayed at the cell surface can be unveiled. Repeated steps of variation and selection will lead to stepwise improved variants design for a specific purpose, thus complying with the strategy of directed evolution. This approach is not restricted to screening by binding as one would assume for enzyme inhibitors, which are known to bind their target enzyme very strongly. Any other adequate selection procedure will do, as long it is suitable for high throughput. Moreover, due to the option of passenger-driven dimerization as described above, combinatorial libraries can be expressed e.g. combinatorial libraries of antibody heavy and light chains, which can subsequently be screened on tumor cell targeting.


Directed Evolution


We successfully applied this approach to identify new inhibitors of human cathepsin G, a target in chronical inflammatory diseases. At current, we are applying this strategy to identify new inhibitors of human protein kinase CK2, a promising target in neoplastic disease. Moreover, we succeeded in displaying combinatorial antibody libraries and were able to demonstrate tumor cell targeting by whole cells of E. coli displaying an antibody variant.

Tumor Cell Targeting

Key publications on directed evolution of enzyme inhibitors and biocatalysts:

  • Jose J, Betscheider D, Zangen D (2005) Bacterial Surface Display Library Screening by target enzyme labelling: Identification of New Human Cathepsin G Inhibitors. Anal Biochem, 346:258-267.
  • Gratz A, Jose J (2008) Protein domain library generation by overlap extension (PDLGO): a tool for enzyme engineering. Anal Biochem, 378:171-176.
  • Thömmes S, Blasshofer F, Jose J (2010) Construction and surface display of antibody libraries in E. coli using autodisplay. Chemie, Ingenieur, Technik (CIT), 9: 1503.
  • Gratz A, Jose J (2011) Focussing mutations to defined domains: protein domain library generation by overlap extension (PDLGO). Methods in Molecular Biology, Volume 279 (eds. Lu, Browse, Wallis), Humana Press, London, 1st Edition, ISBN: 978-1-61779-064-5, pp 153-166.

4. Biosensor development and diagnostic tools

Biosensors utilize the highly selective binding affinity of antibodies for the molecular recognition of a target analyte in a complex mixture such as serum. For the sensitive detection of a target analyte at a very low concentration, the antigen binding sites (Fab region) of antibodies should be directed to the analyte solution (‘orientation control’) and the antibodies should be immobilized with a high density (‘density control’).  This can be solved by Autodisplay, because here, the antibody expressed is to more than 99 % directed towards the outer surface of the outer membrane. Because the outer membrane of E. coli is asymmetric, the outer surface is comprised of LPS, whereas the inner surface contains phospholipids and lipoproteins, this can be exploited for a directed coating of solid matter surfaces of biosensors, including immunoaffinity chips.

 Coating Of Biosensor

This approach can also be used for coating 96 well microplates with an antigen in order to develop new non-sandwich ELISA. But in such case also whole cells of E. coli can be used, that are displaying the antigen of interest, as a simple and rapid source for providing the antigen. As described before, the advantages of Autodisplay such as high expression levels, broad application and robustness of cells displaying the recombinant protein will account for successful application.

Schematic Description Of A Surface Display

Key publications on biosensor development and diagnostic tools:

  • Jose J, Chung JW, Jeon JB, Maas RM, Nam CH, Pyun JC (2009) E. coli with autodisplayed Z-domain of protein A for signal amplification of SPR biosensor. Biosens Bioelectron, 24: 1324-1329.
  • Petermann K, Vordenbäumen S, Braukmann A, Pyun JC, Bleck E, Schneider S, Jose J (2010) Autodisplay of 60 kDa Ro/SS-A and development of a surface display ELISA (SD-ELISA) for SLE patient sera screening. Anal Biochem, 407:72-78.
  • Jose J, Park M, Pyun JC (2010) Highly sensitive immunoassay based on E. coli with autodisplayed Z-domain. Anal Chim Acta, 667:113-118.
  • Park M, Jose J, Pyun JC (2011) SPR biosensor by using E. coli outer membrane layer with autodisplayed Z-domains. Sensor Actuat B-Chem, 154:82-88.
  • Petermann K, Vordenbäumen S, Maas R, Braukmann A, Bleck E, Saenger T, Schneider M, Jose J (2012) Autoantibodies to alphaS1-casein are induced by breast-feeding. Plos One, 7:e32716.
  • Yoo G, Bong JH, Kim S, Jose J, Pyun JC (2014) Microarray based on autodisplayed Ro proteins for medical diagnosis of systemic lupus erythematosus (SLE). Biosens Bioelectron 57:213-218.

Autodisplay Of Bovine