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EP-4735887-A1 - PROTEIN BINDING ASSAYS

EP4735887A1EP 4735887 A1EP4735887 A1EP 4735887A1EP-4735887-A1

Abstract

Provided herein are methods, and compositions for the analysis of protein binding. The methods are applicable to synthesis of proteins on a microfluidic device and assays using the expressed proteins.

Inventors

  • BARNES, COLIN
  • MCINROY, Gordon Ross

Assignees

  • Nuclera Ltd

Dates

Publication Date
20260506
Application Date
20240627

Claims (20)

  1. 1. A method for identifying protein binding using a fluorescent assay in a droplet on a digital microfluidic device having an array of electrodes comprising: a. taking a droplet containing a protein (X) immobilised on a solid support; b. exposing the protein (X) to potential binding partners (Y) wherein the binding partners carry a detectable tag; c. removing unbound binding partners; d. adding a detector species which makes the binding partners fluorescent; and e. determining the level of fluorescent signal within the droplets, thereby measuring the level of binding partner (Y) bound to the protein (X).
  2. 2. The method according to claim 1, wherein the proteins (X) are expressed on the device and captured onto solid supports.
  3. 3. The method according to claim 1 or claim 2, wherein the binding partners (Y) are expressed on the device.
  4. 4. The method according to any one of claims 1 to 3, wherein the solid supports are magnetic beads.
  5. 5. The method according to any one of claims 1 to 4, wherein the detectable tag and detector species are components of a fluorescent protein.
  6. 6. The method according to claim 5, wherein the detectable tag contains ccGFPn.
  7. 7. The method according to any one of claims 1 to 6, wherein the detector species is ccGFPi-io.
  8. 8. The method according to any one of claims 1 to 7, wherein the binding partner (Y) is an amino acid sequence.
  9. 9. The method according to claim 8, wherein the binding partner (Y) contains a single chain binding sequence such as a VHH or nanobody.
  10. 10. The method according to claim 9, wherein the proteins (X) and binding partner (Y) are co-expressed in droplets on the digital microfluidic device having an array of electrodes.
  11. 11. The method according to claim 10, wherein the expressed protein and binding partner are co-expressed in the same droplet.
  12. 12. The method according to claim 10, wherein the expressed protein and binding partner are expressed in separate droplets on the device.
  13. 13. The method according to claim 12, wherein a population of (n) expressed proteins are expressed in separate droplets, a population of (m) potential binding partners are expressed in separate droplets and the droplets of expressed protein is split into at least (m) number of droplets and binding partners are each split into at least (n) number droplets and the droplets are combined to perform (n)x(m) number of binding assays simultaneously on the device.
  14. 14. A method according to any one of claims 2 to 13, wherein the protein expression occurs in human lysate system, a rabbit reticulocyte lysate (RRL) system, a Chinese Hamster Ovary (CHO) lysate system, a wheat germ cell-free system, a E. coli whole cell lysate system or in a system of purified recombinant elements (PURE) or a mixture thereof.
  15. 15. The method according to any one of claims 1 to 14, wherein the droplets are in an oil layer and the oil layer contains surfactant.
  16. 16. The method according to any one of claims 1 to 15, comprising the steps of: a. expressing target proteins (X) in droplets on a digital microfluidic device having an array of electrodes; b. binding the expressed proteins to magnetic beads; c. immobilising and washing the beads to remove unbound proteins; d. exposing the beads to potential binding partners (Y) having a detectable tag; e. washing the beads to remove unbound binding partners; f. exposing the beads to a detector species; g. optionally washing the beads to remove detector species; and h. measuring fluorescence from the assembled detector, thereby measuring the presence of the binding partners on the beads.
  17. 17. The method according to any one preceding claim, wherein the proteins are immobilised via a binding interaction.
  18. 18. The method according to claim 17, wherein the binding interaction uses a purification tag selected from: Alfa-tag (SRLEEELRRRLTE) Avi-tag (GLNDIFEAQKIEWHE) C-tag (EPEA) Calmodulin-tag (KRRWKKNFIAVSAANRFKKISSSGAL) Dogtag (DIPATYEFTDGKHYITNEPIPPK) E-tag (GAPVPYPDPLEPR) FLAG (DYKDDDDK) G4T (EELLSKNYHLENEVARLKK) HA (YPYDVPDYA) His (HHHHHH) Isopeptag (TDKDMTITFTNKKDAE) lanthanide binding tag (LBT) (FIDTNNDGWIEGDELLLEEG) Myc (EQKLISEEDL) NE-Tag (TKENPRSNQ.EESYDDNES) Poly Glutamate-tag (EEEEEEE) Poly Arginine-tag (RRRRRRR) RholD4-tag (TETSQ.VAPA) SBP-tag (MDEKTTGWRGGHVVEGLAGELEQ.LRARLEHHPQ.GQ.REP) Sdytag (DPIVMIDNDKPIT) SH3 (STVPVAPPRRRRG) SNAC (GSHHW) Snooptag (KLGDIEFIKVNK) Softag 1 (SLAELLNAGLGGS) Softag 3 (TQ.DPSRVG) Spot-tag (PDRVRAVSHWSS) Spytag (AHIVMVDAYKPTK) S-tag (KETAAAKFERQHMDS) Strep-tag (AWAHPQ.PGG) (AWRHPQ.FGG) Strep-tag II (WSHPQFEK) T7tag (MASMTGGQ.Q.MG) TC-tag (EVHTNQ.DPLD) Ty-tag (CCPGCC) VSV-tag (YTDIEMNRLGK) Xpress-tag (DLYDDDDK).
  19. 19. The method according to any one of claims 1 to 18, comprising the steps of: a. expressing target proteins (X) in droplets on a digital microfluidic device having an array of electrodes, wherein the expressed proteins have a tag selected from Strep-tag or Strep-tag II; b. binding the expressed proteins to magnetic beads having streptavidin or strep- tactin; c. immobilising and washing the beads to remove unbound proteins; d. exposing the beads to potential binding partners (Y) having a detectable ccGFPn tag; e. washing the beads to remove unbound binding partners; f. exposing the beads to a detector species comprising ccGFPi-io; g. optionally washing the beads to remove detector species; and h. measuring fluorescence from the assembled detector ccGFPi-ii, thereby measuring the presence of the binding partners on the beads.
  20. 20. The method according to any one preceding claim, wherein the assay contains a ligand which inhibits or displaces the binding of X and Y in order to determine the binding of the ligand by disrupting signal generated by the binding of X and Y.

Description

PROTEIN BINDING ASSAYS FIELD OF THE INVENTION Provided herein are methods and compositions for the on-device expression and binding assays on the expressed proteins. The methods are applicable to performing protein binding assays on a microfluidic device. BACKGROUND TO THE INVENTION The invention relates to methods for expression of proteins on a microfluidic device, particularly cell-free protein synthesis. Cell-free protein synthesis (CFPS) regimes are attractive alternatives to cell-based expression systems as they can be treated as reagents rather than organisms, making them amenable to in-vitro experimentation techniques. Additionally, cell-free systems are less sensitive to toxic protein synthesis; are open systems that can be modulated via addition of elements due to the lack of a cell membrane; are adaptable to high-throughput experiments; and can be used to good effect in small volumes. However, many of the cellular expression regulatory control paradigms still apply (e.g. incorrect ribosome binding motifs can lead to poor binding and poor transcription; incorrect codon usage can lead to inefficient translation etc). Efficient protein synthesis relies on having the correct nucleic acid expression construct in the correct conditions. Protein synthesis and purification can be improved by attaching additional amino acids to the protein of interest, for example sequences improving solubility or tags for purification. In order to efficiently screen the optimal cell-free conditions for expression of a particular protein sequences it is desirable to provide a population of nucleic acid expression constructs. Furthermore, in order to identify the best DNA construct to generate a protein of interest it is desirable to provide a population of nucleic acid expression constructs. The invention herein describes methods for the screening of nucleic acid constructs suitable for cell- free protein expression, and the use thereof. When performing cell-free protein synthesis at microfluidic scale in a microfluidic device, such as a digital microfluidic device, it is useful to perform assays on the proteins that are synthesized from said cell-free protein synthesis reaction. However, it is difficult to perform real-time detection of proteins in a cell-free protein synthesis reaction environment. The reaction contains many other proteins and biomolecules at high concentration, making non-specific protein detection via standard protein staining methods difficult (e.g., Coomassie Brilliant Blue G-250, SYPRO™ Ruby, Silver staining). Immunostaining or affinity-based purification followed by non- specific proteins staining are equally unhelpful as significant washing on a solid support must be performed to prevent background interference. As washing is known to be difficult in microfluidic and digital microfluidic devices, background interference may become debilitating. Currently existing luminescent complementation approaches cannot achieve prolonged realtime detection in cell-free protein synthesis reactions, which often extend beyond 6 hours. This limit is due to a combination of reasons including O2 consumption by the luminescencegenerating enzyme that competes with cell-free protein synthesis O2 requirements and temporary or permanent exhaustion of luminescent substrate over 3 - 24 hours of recombinant protein expression detection. Proteins of interest may be expressed as a fusion to a fluorescent protein, such as green fluorescent protein (GFP). However, GFP is a 26.9 kDa protein, which is the typical size for most fluorescent proteins. Tags of this size increase the total size of the protein of interest, especially if the protein of interest must be tagged with other large fusion proteins such as maltose-binding protein (MBP), which is 42.5 kDa. Given the average size of a human protein is ~52 kDa and the average size of an E. coli protein is ~35 kDa (Kim, Y. E. et al. Annu. Rev. Biochem. 2013. 82:323- 355), the addition of a comparably sized fluorescent protein tag can significantly change the biological function and biophysical characteristics of a protein. Many pieces of prior art disclose the use of sub-component tags for monitoring expression in a cellular system. For example US 7,666,606 discloses protein-protein interaction detection systems using microdomains. Schinn et al. Biotechnol. Bioeng 114 10 October 2017 2412-2417. (https://onlinelibrarv.wilev.com/doi/10.1002/bit.263Q5) discloses Rapid in-vitro screening for the location-dependent effects of unnatural amino acids on protein expression and activity - Schinn - 2017 - Biotechnology and Bioengineering - Wiley Online Library. WO2022/038353 describes a method for measuring protein expression levels using split fluorescent protein systems. The level of expressed protein is measured in the presence of an excess of detector species, thereby measuring a single interaction which assembles a fluorescent protein. US20050221343 provides a protein labelling an