Platelet-Derived Growth Factor B Mutant, Preparation Method Therefor and Use Thereof

• US 20170253642A1
• Filed: 07/16/2015
• Published: 09/07/2017
• Est. Priority Date: 05/16/2014
• Status: Active Grant

• Current Assignee: Institute of Biotechnology Academy of Military Medical Sciences P.L.A. China.
• Original Assignee: Institute of Biotechnology Academy of Military Medical Sciences P.L.A. China.
• Inventors: CHEN, Wei, ZHANG, Xiaopeng, YU, Changming, FU, Ling, DAI, Mengmeng, ZHANG, Jun, XU, Junjie, HOU, Lihua, LI, Jianmin
• Granted Patent: US 10,450,358 B2
• Time in Patent Office: Days
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 38/18 Growth factors; Growth regulators
• CPC Class Codes: A61P 1/02 Stomatological preparations, e.g. drugs for caries, aphtae, periodontitis
• CPC Class Codes: A61P 17/02 for treating wounds, ulcers, burns, scars, keloids, or the like
• CPC Class Codes: A61P 19/00 Drugs for skeletal disorders
• CPC Class Codes: A61P 19/02 for joint disorders, e.g. arthritis, arthrosis
• CPC Class Codes: A61P 19/08 for bone diseases, e.g. rachitism, Paget's disease
• CPC Class Codes: B01D 15/203 Equilibration or regeneration
• CPC Class Codes: B01D 15/327 with hydrophobic interaction
• CPC Class Codes: B01D 15/34 Size selective separation, e.g. size exclusion chromatography, gel filtration, permeation
• CPC Class Codes: B01D 15/361 Ion-exchange
• CPC Class Codes: B01D 15/426 Specific type of solvent
• CPC Class Codes: C07K 1/16 by chromatography
• CPC Class Codes: C07K 1/18 Ion-exchange chromatography
• CPC Class Codes: C07K 1/20 Partition-, reverse-phase or hydrophobic interaction chromatography
• CPC Class Codes: C07K 14/49 Platelet-derived growth factor [PDGF]
• CPC Class Codes: C07K 16/22 against growth factors ; against growth regulators
• CPC Class Codes: C07K 19/00 Hybrid peptides , i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• CPC Class Codes: C12N 15/63 Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
• CPC Class Codes: C12N 5/10 Cells modified by introduction of foreign genetic material

Abstract

Provided are a platelet-derived growth factor B derivative, the encoding nucleic acid molecule thereof, and a vector and host cell having the nucleic acid molecule. Also provided are a preparation method for the mutant, and the use of the mutant in preparing medications for promoting cell division, cell proliferation, wound healing, skin regeneration, bone and tooth defect regeneration, and joint repair.

Claims

1. A platelet-derived growth factor B mutant having mutations at amino acid positions 101 and 109 of wild-type platelet-derived growth factor B and having platelet-derived growth factor B activity.

2. The platelet-derived growth factor B mutant of claim 1 having a mutation at amino acid position 6 of the wild-type platelet-derived growth factor B and having platelet-derived growth factor B activity.

3. The platelet-derived growth factor B mutant of claim 1 having a mutation at amino acid position 32, 33, or both 32 and 33 of the wild-type platelet-derived growth factor B and having platelet-derived growth factor B activity.

4. The platelet-derived growth factor B mutant of claim 1 having an N-terminal deletion of 5 amino acids compared with the wild-type platelet-derived growth factor B and having platelet-derived growth factor B activity.

5. The platelet-derived growth factor B mutant of claim 1 having mutations to alanine at amino acid positions 6, 101 and 109 of the wild-type platelet-derived growth factor B.

6. The platelet-derived growth factor B mutant claim 1 having mutations to alanine at amino acid positions 101 and 109 of the wild-type platelet-derived growth factor B.

7. The platelet-derived growth factor B mutant of claim 1 having mutation(s) to proline, valine or isoleucine at amino acid positions 32, 33 or both positions 32 and 33 of the wild-type platelet-derived growth factor B.

8. The platelet-derived growth factor B mutant of claim 1, wherein said platelet-derived growth factor B is a mammalian derived platelet-derived growth factor B.

9. The platelet-derived growth factor B mutant of claim 1, wherein the amino acid sequence thereof is the sequence set forth in SEQ ID NO:

3 or SEQ ID NO;

5.

10. The platelet-derived growth factor B mutant of claim 1 having a further substitution, deletion or addition of one or more amino acids in its amino acid sequence as compared to the wild-type platelet-derived growth factor B and having platelet-derived growth factor B activity.

11. A platelet-derived growth factor homodimer or heterodimer formed by two platelet-derived growth factor B mutants of claim 1 via intra- and/or inter-chain disulfide bonding, or formed by one platelet-derived growth factor B mutant of claim 1 and one platelet-derived growth factor A via intra- and/or inter-chain disulfide bonding.

12. A nucleic acid molecule encoding the platelet-derived growth factor B mutant of claim 1.

13. The nucleic acid molecule of claim 12 having a nucleotide sequence selected from the group consisting of sequences set forth in SEQ ID NO:

4 and SEQ ID NOs;

6-9.

14. A vector comprising the nucleic acid molecule of claim 12.

15. A host cell comprising the vector of claim 14.

16. The host cell of claim 15, wherein the host cell is a eukaryotic cell.

17. The host cell of claim 16, wherein the host cell is Pichia pastoris.

18. A method for preparation of the platelet-derived growth factor B mutant of claim 1, comprising the steps of culturing a host cell that comprises a nucleic acid vector that encodes the platelet-derived growth factor B mutant and expressing the mutant.

20. The purification method of claim 19, wherein the platelet-derived growth factor B mutant is the platelet-derived growth factor B mutant of claim 1.

24. An antibody capable of specifically binding to the platelet-derived growth factor B mutant of claim 1.

27. A method for promoting cell division, proliferation, promoting wound healing, skin regeneration, bone and tooth defect regeneration, and joint repair, comprising the step of administering to a subject in need thereof an effective amount of the platelet-derived growth factor B mutant of claim 1.

19. A method for purifying platelet-derived growth factor B or a mutant thereof comprising the steps of successively subjecting a culture supernatant or a cell lysate containing the platelet-derived growth factor B or a mutant thereof to hydrophobic interaction chromatography, ion exchange chromatography and gel filtration chromatography.

21. The purification method of claim 19, characterized in one or more of the following 1)-3):

1) the chromatographic medium used for hydrophobic interaction chromatography is Phenyl Sepharose 6 Fast Flow;

2) the chromatographic medium used for ion exchange chromatography is Source 30S;

3) the chromatographic media used for gel filtration chromatography is Hiload Superdex 75 prep grade.

22. The purification method of claim 19, wherein,

said hydrophobic interaction chromatography comprises the steps of:

(1) adjusting the conductivity of the culture supernatant or cell lysate containing the platelet-derived growth factor B or a mutant thereof with a conditioning buffer, and the final system with said conditioning buffer added is 10-50 mM phosphate buffer, 0.8-1M (NH₄)₂SO₄, pH 6.8-7.5;

(2) equilibrating the column with an equilibration buffer, and the formulation of the equilibration buffer is 10-50 mM phosphate buffer, 0.8-1M (NH₄)₂SO₄, pH 6.8-7.5;

(3) after loading the sample onto the column, washing the column with the equilibration buffer;

(4) eluting with an elution buffer and collecting the protein of interest, and the formulation of the elution buffer is 10-50 mM phosphate buffer, 30%-50% ethylene glycol, pH 6.8-7.5;

said ion exchange chromatography comprises the steps of;

(1) diluting the elution peak of hydrophobic interaction chromatography with an equilibration buffer to a conductivity of 6 mS/cm or less, and the formulation of the equilibration buffer is 10-50 mM phosphate buffer, pH 6.8-7.5;

(2) equilibrating the column with the equilibration buffer;

(3) after loading the sample onto the column, washing the column with the equilibration buffer;

(4) eluting with elution buffer gradient and collecting the protein of interest, and the formulation of the elution buffer is 10-50 mM phosphate buffer, 0.8-1.2 mM NaCl, pH 6.8-7.5;

said gel filtration chromatography comprises the steps of;

(1) equilibrating the column with a phosphate buffer, and the formulation of the phosphate buffer is 10-50 mM phosphate buffer, 0.1-0.5M NaCl, pH 6.8-7.5;

(2) loading the elution peak of the ion exchange chromatography, and the volume of each loading is not more than 0.3 to 4% of the column volume;

(3) continuing to wash the column with the phosphate buffer in step (1), collecting the protein of interest, and obtaining the purified platelet-derived growth factor B or the mutant thereof.

23. (canceled)

25. A method for expressing platelet-derived growth factor in a cell in a homogenous form comprising the steps of modifying the amino acid sequence of wild-type platelet-derived growth factor that is to be expressed in the cell, wherein the modification comprising one or more of the following a) to c):

a) mutations at amino acid positions 101 and 109;

b) a mutation at amino acid position 6;

c) mutation(s) at amino acid positions 32 and/or 33;

d) N-terminal deletion of 5 amino acids; and

expressing the modified platelet-derived growth factor.

26. The method of claim 25, characterized in one or more of the following i)-iii):

i) mutating the amino acids at positions 101 and 109 to alanine;

ii) mutating the amino acid at position 6 to alanine;

iii) mutating the amino acid(s) at positions 32 and/or 33 to proline, valine, or isoleucine.