METHOD

Abstract

There is provided a method of identifying a resin for isolating or enriching a protein of interest using affinity chromatography. The method comprises the steps of: i) providing the three-dimensional structure of the protein of interest; ii) determining and/or calculating one or more parameters of the protein of interest in its two- and/or three-dimensional form; iii) determining and/or calculating one or more parameters of one or more resin in their two- and/or three-dimensional form; and iv) selecting a resin expected to bind complementarily to the protein of interest based upon one or more of the parameters of the protein of interest.

Claims

1. A method of identifying a resin for isolating or enriching a protein of interest using affinity chromatography, comprising the steps of: i) providing the three-dimensional structure of the protein of interest; ii) determining and/or calculating one or more parameters of the protein of interest in its two- and/or three-dimensional form; iii) determining and/or calculating one or more parameters of one or more resin in their two- and/or three-dimensional form; and iv) selecting a resin expected to bind complementarily to the protein of interest based upon one or more of the parameters of the protein of interest.

2. The method of claim 1, wherein the one or more parameters of the protein of interest comprise one or more of: a) electrostatic potential; b) size; c) amino acid content and/or sequence; d) hydrophobicity/hydrophilicity; e) molecular weight; f) hydrogen bond donors and/or acceptors; g) π-stacking regions; and/or h) cation and/or π regions for cation-π interactions.

3. The method of claim 1, wherein the one or more parameters of one or more resins comprise one or more of: a) electrostatic potential; b) pore size; c) characteristics that will bind to amino acids of the protein of interest; d) hydrophobicity/hydrophilicity; e) average molecular weight; f) hydrogen bond donors and/or acceptors; g) π-stacking regions; and/or h) cation and/or π regions for cation-π interactions.

4. The method of claim 1, wherein the resin is or comprises a polysaccharide-based resin, optionally, a resin based on agarose, alginate, cellulose, chitin, starch, glycogen, callose, laminarin, chrysolaminarin, xylan, arabinoxylan, mannan, fucoidan, pectins and/or galactomannan

5. The method of claim 1, further comprising providing the protein sequence of the protein of interest, optionally, prior to step (i).

6. The method of claim 1, wherein the three-dimensional structure of the protein is: a) retrieved from a database of the three-dimensional structures of proteins, such as the Protein Data Bank (PDB); b) determined using homology modelling; c) determined using NMR techniques; and/or d) determined using X-ray diffraction techniques.

7. The method of claim 1, wherein the resin is selected based upon two or more, three or more, or four or more parameters of the protein.

8. A method of identifying one or more preferred ligands for isolating a protein of interest using affinity chromatography, the method comprising the steps of: i) providing the three-dimensional structure of the protein of interest and creating a model of a receptor-based pharmacophore of the protein of interest using the three-dimensional structure of the protein of interest, and determining and/or calculating one or more parameters of the model of the receptor-based pharmacophore in its two- and/or three-dimensional form; ii) providing a database of molecules; iii) selecting, from the database, molecules that include primary amines and/or carboxylic acid moieties; iv) screening the selected molecules against the model of the receptor-based pharmacophore to find one or more molecules expected to bind complementarily to the protein of interest based upon one or more of the parameters of the model of the receptor-based pharmacophore of the protein of interest; v) selecting, as one or more potential ligands, the one or more molecules expected to bind complimentarily to the protein of interest; vi) calculating the binding affinity of the one or more potential ligands with the protein of interest using a docking algorithm; and vii) selecting, as one or more preferred ligands, one or more potential ligands with the highest binding affinity.

9. The method of claim 8, wherein the binding affinity calculated is a predicted binding affinity.

10. The method of claim 8, wherein the one or more parameters of the model of the receptor-based pharmacophore comprise one or more of: a) electrostatic potential; b) size; c) amino acid content and/or sequence; d) hydrophobicity/hydrophilicity; e) molecular weight; f) hydrogen bond donors and/or acceptors; g) π-stacking regions; and/or h) cation and/or π regions for cation-π interactions.

11. The method of claim 8, further including the steps of: viii) obtaining one or more preferred ligands and determining their ability to bind the protein of interest, optionally using surface plasmon resonance; ix) immobilising positive binding ligands on a bead to further determine binding ability in a binding assay, optionally using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS page); and, optionally, further comprising the step of: x) in silico optimising the binding affinity of one or more positive binding ligands and validating the binding affinity with a further binding assay.

12. (canceled)

13. The method of claim 8, wherein the three-dimensional structure of the protein is: a) retrieved from a database of the three-dimensional structures of proteins, such as the Protein Data Bank (PDB); b) determined using homology modelling; c) determined using NMR techniques; and/or d) determined using X-ray diffraction techniques.

14. The method of claim 8, wherein molecules that include primary amines and/or carboxylic acid moieties are selected from the database.

15. The method of claim 8, wherein the screening step (iv) includes determining and/or calculating one or more parameters of the selected molecules in their two- and/or three-dimensional form; and, optionally, wherein the one or more parameters of the selected molecules comprise one or more of: a) electrostatic potential; b) pore size; c) characteristics that will bind to amino acids of the protein of interest; d) hydrophobicity/hydrophilicity; e) average molecular weight; f) hydrogen bond donors and/or acceptors; g) π-stacking regions; and/or h) cation and/or π regions for cation-π interactions.

16. (canceled)

17. The method of claim 8, wherein in step (v), 20 or more molecules, or 50 or more molecules are selected as potential ligands; and/or wherein in step (vii), 20 or fewer, 10 or fewer, or 5 or fewer molecules are selected as preferred ligands, or one molecule is selected as a preferred ligand.

18. (canceled)

19. The method of claim 1, wherein the method is computer-implemented.

20. A method of isolating or enriching a protein of interest from a protein mixture, wherein the method comprises isolating, purifying or enriching the protein of interest using affinity chromatography with a resin that has been selected or made according to claim 1.

21. The method of claim 17, wherein the protein mixture comprises raw material, industrial side-streams, or waste material; and/or wherein the protein mixture comprises plant material or animal product; and/or wherein the protein of interest is or comprises any one of ovotransferrin, soy protein, casein or whey.

22. (canceled)

23. (canceled)

24. A method of isolating or enriching a protein of interest from a protein mixture, wherein the method comprises isolating, purifying or enriching the protein of interest using affinity chromatography with a resin that has been selected or made according to claim 8.

25. The method of claim 19, wherein the protein mixture comprises raw material, industrial side-streams, or waste material; and/or wherein the protein mixture comprises plant material or animal product; and/or wherein the protein of interest is or comprises any one of ovotransferrin, soy protein, casein or whey.

Description

EXAMPLES

[0120] This methodology enables the selection of a highly diverse set of molecules that are amenable to covalent immobilization ‘on bead’ for use in affinity chromatography.

[0121] The first use of this diversity set is to enable protein enrichment from complex protein matrices (e.g raw material, industrial side-streams, waste material) and to guide further optimisation in a similar fashion to the use of ‘on-bead’ combinatorial libraries for use in protein target binding in the pharmaceutical industry.

[0122] The process can be run in high throughput using miniaturized columns on a 96-well plate to allow probing of protein capture when integrated with high-performance liquid chromatography or LC-MS/MS.

[0123] Once a protein of interest is captured via a specific set of beads, the binding interactions can be investigated computationally to permit protein purification in a second step.

[0124] The specific design of one or a series of small molecule ligand(s) or selection of analogs capable of binding to the surface of the protein of interest is also described. In this way the user may be able to optimise again in a high-throughput fashion, the precise ligand and bead preparation required to produce optimal protein purification conditions.

[0125] As a proof of concept, a specific ligand has been computationally identified that enables the purification of a high-value egg-white protein, Ovotransferrin, which has been shown in numerous studies to harbour a broad range of health benefits (antibacterial, antitumorogenic, antiviral, etc.). Currently, the only described production and purification processes for ovotransferrin require treatment of egg-white with alcohol, addition of heavy metals, treatment with organic solvents or precipitation with high-salt/organic acid concentrations, which renders the remaining egg-white unusable.

[0126] Ovotransferrin is inherently sensitive towards different stress factors, like thermal stress and therefore prone to aggregation and denaturation. Small molecule ligands could also provide a way to stabilise the protein in solution during the production process and to improve the yield and final activity of the protein preparations.

[0127] Importantly, using the method of the invention, we have found a ligand that binds to Ovotransferrin and enables its separation from other egg-white components when immobilised in a bead.

[0128] To achieve highest possible potential of proteins and to explore or exploit the potentially functional and bioactive properties of proteins (e.g. proteins in milk, eggs, soybean etc.), it is important to isolate native proteins from complex matrices by procedures that avoid possible denaturing conditions (such as, high salt conditions, high or low pH conditions, heat or protease treatment/exposure). We outline next two examples of issues observed in the commercial isolation of proteins from soybean and milk respectively where our technology would produce benefits.

[0129] Soybean; Soybeans provide a good source of low-cost protein and have become an important world commodity because they are ubiquitous, have unique chemical composition, good nutritional value, versatile uses, and functional health benefits. Yet, less than about 5% of the soybean protein available is used for food due to the presence of anti-nutritional factors such as trypsin inhibitors, which prevent the uptake of nutrients from the food source. The most common method of reducing the activity of these inhibitors is to heat the soy protein which denatures/destroys not only the trypsin inhibitor proteins, but all other proteins in the matrix also and renders their functionality inactive. An alternative to heating the soy protein to destroy the trypsin inhibitor (TI) protein is to separate the trypsin inhibitor (TI) protein from the remainder of the soy protein. This technique has the advantages of avoiding heat, and can also provide isolated trypsin inhibitor (TI) protein (which itself can be a useful medicinal product).

[0130] Milk; The proteins in milk, which are mainly found as casein proteins or whey proteins, have gained increasing attention over the years. The reason for this increased interest lies in the diversity of milk proteins and because each protein has unique attributes to nutritional, biological, functional and food ingredient applications. The main protein component in milk is casein which is mainly found as micellar casein, formed by macromolecular casein aggregates. Traditionally, treatment of milk generally consists of an initial extraction of casein, such as by precipitation of aggregated micellar casein, e.g. by enzymatic modification using rennet or by acid treatment, providing a precipitate of aggregated casein, a curd, and a liquid whey protein solution. However, this treatment is disadvantageous because the enzymatic modification or the acidic treatment may cause the aggregated casein and/or part of the soluble proteins to be partly degraded and the proteins may lose some of the biological activity. Furthermore, the precipitated casein may entrap some soluble proteins within the aggregate and thereby reducing the yield of soluble proteins or increase impurities in the aggregated casein precipitate.