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Biotin Protein Ligase as Protein: DNA Connecting System

Background

Biotin protein ligase (BirA) is an essential protein and the sole enzyme capable of biotin transfer onto the biotin carboxyl carrier protein subunit of acetyl-coA carboxylase. Site‑specific biotinylation of proteins has a wide range of applications in biochemistry and cell biology. Despite this, DNA binding properties of Escherichia coli (Ec) BirA have not yet been exploited.

James Cook University present Ec BirA as a new protein:DNA linking system and its proof-of-concept application in quantitative immuno‑PCR (qIPCR). This technology can also be used to create bispecific antibodies.

The self-assembly of GFP-fused biotin protein ligase with bioO repressor DNA is triggered in the presence of ATP and biotin. The resulting high affinity Protein:DNA complex is applied to detect anti-GFP IgG immobilized on a surface. The release of bioO DNA from the surface is triggered by removing ATP and biotin, and detected by qPCR. The nature of the Ec holoBirA-GFP:bioO offers an avenue to self-assemble two different antitarget proteins. The system could be universally applied to assemble two different protein domains (e.g. mAbs) or even catalytic domains of enzyme to reduce entropy and increase catalytic rates.

Specifications for Quantitative ImmunoPCR

  • Sample types: Biological fluids, environmental samples, etc.
  • Assay format: 96-well plate, real-time qPCR
  • Sensitivity: pM currently – may be lower (to be tested)
  • Range: 5 log
  • Assay time: 3hr
  • Limitations:- High background if sufficient care not taken during setup
  • Platform: Real-time thermal cycler

Fig. 1: Principle of Ec BirA:bioO self-assembly and DNA release mechanism. A) Two Ec holoBirA assemble with bioO into a stable protein:DNA complex following formation of bio-5’-AMP. B) Ec BirA-GFP:bioO self-assembles in biotin/ATP buffer and detects anti-GFP IgG. DNA release is triggered by removing biotin and ATP. Free bioO is quantified by qPCR. C) Mixed Ec BirA complex displaying two different fusion protein domains.

  • Platform technology with multiple applications
  • In-built DNA release mechanism
  • Integrated approaches to measure protein & DNA
  • Benefits in qIPCR:
    • Easy to use
    • Low concentration of reagent (5 nM)
    • Low volume of sample (<20 μL)
    • Stable, self-assembling complex
    • Dual detection possible (two Ag or Ab)
  • Benefits in creating bispecific antibodies:
    • Faster than other reported chemical linkers
    • Much faster & lower cost than genetic methods to create bispecific mAbs
    • useful to create prototypes of bispecific mAbs to screen
  • Various assays:
    • Proximity ligation
    • qIPCR
    • Protein arrays
    • DNA arrays
  • Clinical diagnostics
  • Drug development
  • Biomarker development
  • Biologics development (bispecific mAbs)
  • Industrial biotechnology

The university is interested in licensing the technology to companies developing molecular assays and companies conducting R&D on bispecific antibodies.

Seeking:

  • Development partner
  • Commercial partner
  • Licensing

Provisional patent filed.

Contact Details

Photo of Pradeep Sadasivan Pillai

Pradeep Sadasivan Pillai

Associate, Development and Commercialisation

pradeep.sadasivanpillai@jcu.edu.au
Phone: (07) 4781 6458