Expressed Lanthanide Binding Tags as Visualization Tools

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Schematic showing the expression and proteolytic processing of LBT-Ubiquitin conjugates.Graph showing the competition analysis of Tb-loaded HIS-LBT-Ub in the presence of competing metal cations (5 mM Na+, Ca2+, Mg2+ and 5 μM Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Fe3+). The competing ion was added to the LBT-conjugate (5 μM) with 10 μM Tb3+ in 10 mM HEPES buffer pH 7 (λex=285 nm).Graph comparing the standardized emission maxima at 544 nm of 10 μM HIS-LBT-Ub protein with 10 μM Tb3+ and various additives.The diffraction pattern of a HIS-LBT-Ub crystal. Note that the edge of the plate is at 2 Å.Schematic showing the preparation of semisynthetic LBT-RNAse conjugates.
Categories
Inventors
Professor Barbara Imperiali
Department of Chemistry, MIT
External Link (web.mit.edu)
Katherine Franz
Department of Chemistry, MIT
External Link (chem.duke.edu)
Managed By
Michelle Hunt
MIT Technology Licensing Officer
Patent Protection

Lanthanide Binding Tags

US Patent 7,101,667
Publications
Lanthanide-Binding Tags as Luminescent Probes for Studying Protein Interactions
Journal of the American Chemical Society, May 12, 2006, p. 7346-7352
Engineering Encodable Lanthanide-Binding Tags into Loop Regions of Proteins
Journal of the American Chemical Society, Dec 23, 2010, p. 808-819
Lanthanide-Binding Tags as Versatile Protein Coexpression Probes
ChemBioChem, April 4, 2003, p. 265-271
Encoded Loop-Lanthanide-Binding Tags for Long-Range Distance Measurements in Proteins by NMR and EPR Spectroscopy.
Journal of Biomolecular NMR, Nov 2015, p. 275-282
An Encodable Lanthanide Binding Tag with Reduced Size and Flexibility for Measuring Residual Dipolar Couplings and Pseudocontact Shifts in Large Proteins
Journal of Biomolecular NMR, Jan 2016, p. 75-85

Applications

Together with luminescence enhanced photography, lanthanide binding tags conjugated to proteins of interest can be used as fluorescent probes in 1D or 2D gel electrophoresis. They may also be used to directly analyze protein-protein interactions in place of traditional GFP-based protein-protein interaction assays or static and time-dependent variation in protein-protein distances in Forster Resonance Energy Transfer (FRET) experiments. In addition, lanthanide binding tags may be useful as heavy-atom derivatives in macromolecular X-ray crystallography.

Problem Addressed

Traditional luminescent protein probes, such as fluorescent tags, have a relatively short lifetime of emission and are susceptible to photobleaching, leading to lowered signal quality due to the interference of strong background signals and the overtime loss of probe signal. To address these issues, lanthanide ions (Ln3+), which have a long emission lifetime, have been incorporated in indirect probing methods such as dissociation-enhanced lanthanide fluoroimmunoassay (DELFIA). However, these methods involve many time-consuming steps. On a separate issue, macromolecular crystallography traditionally relies on heavy-atom derivatization using the MIR method and constraints of the primary peptide sequence to resolve the phase problem. These methods are often fraught with difficulty because heavy atom soaking often alters the crystal cell structure. New tagging methods that increase fluorescent emission lifetime, as well as decrease heavy atom interference with target protein folding and crystallization, would greatly improve localization and imaging techniques.

Technology

This invention involves the incorporation of short oligopeptide motifs designed to complex trivalent Ln3+ ions into native amino acid sequences. Thus far, these motifs have been successfully fused with the protein RNAse at both the C- and N-terminals along with a polyhistidine tag for purification of the target protein. The complexes these motifs form with Ln3+ ions show physical properties including fluorescence and anomalous X-ray scattering. They exhibit high binding affinity for Ln3+ within nM and pM concentrations and low binding affinity for trace essential transition metal ions. They have low susceptibility to endogenous proteases. Their short sequence means that they are less likely to interfere with native protein function than traditional fluorescent constructs. Finally, they have a long emission lifetime that allows for the elimination of background signals from intrinsic fluorescent material, which means they can be used for highly selective detection and quantitation of the expression of tagged proteins. In terms of their potential in x-ray crystallography, they have been shown to absorb edges for anomalous scattering within the tunable range of synchrotron light sources (7-12 keV). The high binding affinity between the motifs and Ln3+ ions prevents Ln3+ ions from interfering with target protein folding and crystallization, and the low thermal motion of the Ln3+ ion within the peptide-binding site allows for reliable experimental results.

Advantages

  • Maximum signal intensity for optimal sensitivitiy
  • Long-lived fluorescent species that can be observed in time-resolved experiments
  • Selective binding to lanthanide and low affinity for trace essential transition metal ions
  • Low susceptibility to endogenous proteases
  • Good stability