Community Research and Development Information Service - CORDIS

ERC

PeptidePadlock Report Summary

Project ID: 615945
Funded under: FP7-IDEAS-ERC
Country: United Kingdom

Mid-Term Report Summary - PEPTIDEPADLOCK (Peptide padlocks evolved towards infinite affinity for antibody nanoassembly and ultrasensitive cell capture)

SpyTag is a peptide that was engineered to form a spontaneous isopeptide bond when it encounters its protein partner SpyCatcher. This peptide padlock, derived from the CnaB2 domain from Streptococcus pyogenes FbaB, is now applied widely in biological research and bionanotechnology. Key features are that both partners are genetically encoded, can be fused at any location in a partner of interest, and react under a wide range of conditions. However, SpyTag’s performance can be improved in many different parameters for molecular assembly in the test-tube, in cell-culture and in different organisms.
We have successfully generated large numbers of variant SpyTag and variant SpyCatcher and shown that they enable enhanced labelling and interaction at the outer membrane of Escherichia coli. Enhanced properties of SpyTag and SpyCatcher variants have been fully characterized in terms of second order rate-constants and reaction dependence upon variation in pH, temperature, buffer salts and detergent levels. We have also performed Differential Scanning Calorimetry for characterization of the thermal stability of variants. We have been working to ensure that variants are selected such that increases in reaction rate do not come at the cost of decrease in thermostability or decrease in expression yield. We have also created an orthogonal peptide-protein interaction: SnoopTag forms a spontaneous covalent bond with SnoopCatcher. These partners were generated by splitting of the RrgA domain from Streptococcus pneumoniae, testing a wide range of separation sites and performing structure-based and computational design to optimize each partner for expression efficiency and reaction speed. SnoopTag and SnoopCatcher show no interaction with SpyTag or SpyCatcher.
Step-wise extension from a solid-phase allowed the production of programmed protein teams, including with branched architectures. We performed extensive optimization of the solid-phase attachment, using the well-known maltose binding protein/amylose interaction. However, we tested and combined a wide range of point mutants of maltose binding protein to enhance the amylose binding affinity, to reduce losses after each round. However, we maintained efficient elution of the assembled protein teams from the resin in response to the addition of maltose. In contrast to other anchoring and elution methods, using such gentle elution with no extra protein required (e.g. a protease), greatly enhanced our ability to transition directly into cellular testing of the eluted protein teams. This assembly approach was initially validated with single domain protein units, in particular affibodies, nanobodies and fluorescent proteins. Since then, we have applied this approach to the assembly of precise antibody chains after expression of tagged antibody fragments in mammalian cells. This solid-phase assembly with dual reactive tags was an important advance because any approach using SpyTag/SpyCatcher alone generates a series of variants with different lengths and branchings. Assembly of the protein teams has been carefully characterized by gel electrophoresis, size-exclusion chromatography and mass spectrometry.
We are now applying this new generation of protein teams to enhance magnetic capture of cancer cells. Our initial validation has used cell-line models with well-established levels of surface HER2. This is also a good model system because of our ability to tune precisely the antibody fragment’s affinity for HER2 and because of the insight we have made into how modulating membrane fluidity changes magnetic capture efficiency. Optimization of the sensitivity of magnetic cell capture by tailored assembly of antibody teams will then enable us to improve circulating tumour cell capture, towards enhanced diagnosis and increased insight into cancer biology.

Contact

Gill Wells, (Head of European Team)
Tel.: +44 1865 289800
Fax: +44 1865 289801
E-mail
Record Number: 195448 / Last updated on: 2017-03-13
Follow us on: RSS Facebook Twitter YouTube Managed by the EU Publications Office Top