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

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.

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.

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.

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 (NH.sub.4).sub.2SO.sub.4, 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 (NH.sub.4).sub.2SO.sub.4, 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)

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

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.

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.

Description

DESCRIPTION OF THE DRAWINGS

[0102] FIG. 1. The schematic diagram of the structure of PDGF-B protein. A. PDGF-B precursor protein was removed of N-terminal signal peptide, propeptide and C-terminal propeptide sequence by proteolysis to become a mature protein. .box-tangle-solidup. represents protease hydrolysis sites; B, two PDGF-B monomers form a PDGF-BB homodimer via interchain disulfide bonds, and each PDGF-B monomer contains eight cysteines, forming three pairs of intrachain disulfide bonds (C16-C60, C49-C97, C53-C99) and two pairs of interchain disulfide bonds (C43-C52, C52-C43). SP: signal peptide; PRO: the pro-sequence preceding the growth factor domain.

[0103] FIG. 2. SDS-PAGE electrophoresis analysis of PDGF-BB expressed and secreted by Pichia pastoris. Non-reducing (left) and reducing (right) SDS-PAGE analysis were performed on three different batches of fermented and purified recombinant PDGF-BB.sup.Thr6. Under the non-reducing condition, the protein is a single band. Upon DTT treatment, the PDGF-B monomers exhibit various forms with heterogeneous molecular weights.

[0104] FIG. 3. The co-effect of the proteolysis and glycosylation contributes to the formation of several monomers of PDGF-B.sup.Thr6. (A) PDGF-B.sup.Thr6 protein was separated by SDS-PAGE under reducing condition for N-terminal amino acid sequence analysis. The first five amino acid residues at N-terminus in the first, second and third bands are all TIATP, and the first five amino acid residues at N-terminus in the fourth and fifth bands are TNANF, suggesting that protease cleavage occurs at Arg32-Thr33. (B) PDGF-B.sup.Thr6 protein was subjected to WB (middle) and glycoprotein staining (right) analysis under reducing conditions. WB detection bands correspond to bands 3 and 5 of Coomassie Brilliant Blue staining (left), and the glycoprotein staining bands correspond to bands 1, 2 and 4 of Coomassie Brilliant Blue staining. (C) PDGF-B.sup.Thr6 protein was treated with PNGase F under reducing condition and stained for glycoprotein. There was no change in terms of band type, molecule weight (left) and glycoprotein staining results before and after treatment with PNGase F. IFN-ω serves as positive control for glycosidase cleavage and glycoprotein staining.

[0105] FIG. 4. Prediction results of O-linked glycosylation sites of PDGF-B.sup.Thr6 protein sequence. Thr6, Thr101 and Thr109 are potential O-linked glycosylation modification sites.

[0106] FIG. 5. Purity the purified protein PDGF-M2 upon HPLC detection.

[0107] FIG. 6. Analysis of post-translational modification sites of PDGF-B expressed in Pichia pastoris. (A) The schematic diagram of site mutation of PDGF-M1 and PDGF-M2 mutants. (B) WB detection showed PDGF-M1 and PDGF-M2 monomers as single bands, and control PDGF-BThr6 as two bands. (C) The PDGF-M1 and PDGF-M2 monomers were detected by SDS-PAGE, Coomassie Brilliant Blue staining result showed PDGF-M2 as a single protein band and PDGF-M1 as two protein bands (as shown by the arrows in the figure). (D) Glycoprotein staining can hardly detect PDGF-M1 and PDGF-M2 protein monomers.

[0108] FIG. 7. The biological activity of PDGF-M2 is higher than that of PDGF-BB.sup.Thr6, and there is statistically significant difference between them. The experiment was repeated three times, with EC50 expressed as mean±standard deviation, P=0.039.

[0109] FIG. 8. The effect of mutation of Arg32 on the expression. (A) The SDS-PAGE results of the expression products of 7 clones of each of codon-optimized strains PDGF-IM-P, PDGF-IM-V and PDGF-IM-I. The protein expression amount of PDGF-IM-P is significantly higher than the other two strains. (B) The codon-optimized strains PDGF-IM-P, PDGF-IM-V and PDGF-IM-I were screened for multiple insert copies by G418 resistance, respectively. Six clones were selected under the G418 concentration of 2.0 mg/ml or 4.0 mg/ml for expression in a tube. SDS-PAGE analysis showed that the expression amount of PDGF-IM-P high-copy screening strain is significantly higher than that of the other two strains.

[0110] FIG. 9. LC/MS plots of PDGF-B wild-type and PDGF-M2 mutants.

DETAILED DESCRIPTION OF THE INVENTION

[0111] The embodiments of the present invention will be described in detail below in combination with the examples, however, those skilled in the art will appreciate that the following examples are merely intended to illustrate the invention and should not be construed as limiting the scope of the invention. Those without the specific conditions specified in the examples should be carried out under normal conditions or the conditions recommended by the manufacturer. The reagents or instruments without manufacturers specified are all commercially available conventional products.

[0112] In previous studies, we have successfully employed Pichia pastoris expression system to express rhPDGF-BB.sup.Thr6 with five amino acids deleted at N-terminus with a expression level of up to 100 mg/L (see CN Patent No.: ZL200410068993.2). PDGF-B.sup.Thr6 is selected as research subject in order to ensure homogeneity of expressed protein without biological activity impaired. However, further studies demonstrate that, rhPDGF-B.sup.Thr6 monomer expressed by Pichia pastoris still exhibits various forms with heterogeneous molecular weights ranging from 10 to 15 kDa (FIG. 2).

[0113] The following examples are carried out by engineering based on rhPDGF-B.sup.Thr6, and all the descriptions of the sites or positions are based on the wild-type PDGF-B (109 amino acids).

[0114] Genbank number of the amino acid sequence of the wild-type PDGF-B is NM-002608.2.

[0115] The amino acid sequence of rhPDGF-BB.sup.Thr6 is:

TABLE-US-00001 (SEQ ID NO: 1) TIAEPAMIAECKTRTEVFEISRRLIDRTNANFLVWPPCVEVQRCSGCCNN RNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCETVAAA RPVT.

[0116] The nucleic acid sequence of rhPDGF-BB.sup.Thr6 is:

TABLE-US-00002 (SEQ ID NO: 2) 5′-ACCATTGCTGAGCCGGCCATGATCGCCGAGTGCAAGACGCGCACCG AGGTGTTCGAGATCTCCCGGCGCCTCATAGACCGCACCAACGCCAACTT CCTGGTGTGGCCGCCCTGTGTGGAGGTGCAGCGCTGCTCCGGCTGCTGC AACAACCGCAACGTGCAGTGCCGCCCCACCCAGGTGCAGCTGCGACCTG TCCAGGTGAGAAAGATCGAGATTGTGCGGAAGAAGCCAATCTTTAAGAA GGCCACGGTGACGCTGGAAGACCACCTGGCATGCAAGTGTGAGACAGTG GCAGCTGCACGGCCTGTGACC-3′.

[0117] Materials and Methods

[0118] Construction of Recombinant Expression Clones

[0119] DNA sequences encoding various PDGF mutants were synthesized by Shanghai Sangon Inc. The gene fragments were cloned into the expression vector pMEX9K (see patent ZL02117906.9) via restriction sites XhoI and EcoRI and confirmed by sequencing. The recombinant plasmids were extracted, linearized by SalI digestion, and then transformed into Pichia pastoris expression strain GS115 competent cells by electroporation. The yeast transformants were screened by histidine-deficient MD plates and the positive recombinant yeast strains were identified by PCR.

[0120] Induction Expression of Recombinant Proteins

[0121] The single clones of the recombinant yeast strain were inoculated into a flask of 25 mL BMGY medium (BMGY medium is prepared as follows: 10 g yeast extract powder and 20 g tryptone were weighed, dissolved in 700 ml water, autoclaved at 121° C. for 20 min; cooled to room temperature, added 100 ml 1 M potassium phosphate buffer, 100 ml 10×YNB and 100 ml 10×GY, and stored at 4° C. Wherein: 10×YNB (13.4% yeast nitrogen source base), 10×GY (10% glycerol), 1 M potassium phosphate buffer (132 ml 1M K.sub.2HPO.sub.4 and 868 ml 1M KH.sub.2PO.sub.4 were measured, adjusted to pH 6.0±0.1 with phosphoric acid or KOH, autoclaved at 121° C. for 30 min and stored at room temperature.) Yeast Extract (LP0021) is a product of OXOID Inc. and Peptone (211677) is a product of B&D Inc.), cultured and propagated at 28-30° C. with 220-250 rpm to OD.sub.600=2-6 (about 16-18 hours). 25 ml yeast culture was inoculated into a flask containing 1 L BMGY, and continued to culture and propagate at 28-30° C. with 220-250 rpm to OD.sub.600=2-6. Yeasts were collected by centrifugation at room temperature with 1500-3000 g for 5 min. The supernatant was removed and the yeasts were resuspended with 1 L BMMY medium to initiate expression induction. The induction temperature was 28° C. and the rotational speed was 220 rpm. Methanol was added every 24 hours until the final concentration is 0.5%, and the induction time was 72 hours. After the induction was complete, the supernatant containing the recombinant protein was collected by centrifugation with 7000 rpm at room temperature.

[0122] Purification of Recombinant Proteins

[0123] The expression supernatant of Pichia pastoris was adjusted into an appropriate buffer by centrifugation and filtration, and subsequently subjected to hydrophobic interaction chromatography (Phenyl Sepharose 6 Fast Flow), ion exchange chromatography (Source 30S) and gel filtration chromatography (Hiload Superdex 75 prep grade) to obtain the protein of interest with a purity>95% (FIG. 5). The chromatographic media are all products from GE Amersham Bioscience Inc.

[0124] Hydrophobic chromatography was carried out as follows. (1) Yeast expression supernatant was adjusted for conductivity with ½ volume of conditioning buffer (60 mM PB, 3M (NH.sub.4).sub.2SO.sub.4, pH 7.2). (2) As described in the instruction, the column was equilibrated with an equilibration buffer (20 mM PB, 1M (NH.sub.4).sub.2SO.sub.4, pH 7.2). (3) The sample was loaded to the column, thereafter the column was washed with the equilibration buffer until the baseline is flat. (4) The column was eluted with an elution buffer (20 mM PB, 50% ethylene glycol, pH 7.2) to collect the protein of interest.

[0125] Ion exchange chromatography was carried out as follows. (1) The Phenyl HS elution peak was diluted with an equilibration buffer (20 mM PB, pH 7.2) to a conductivity of 6 mS/cm or less. (2) According to the method in the instruction, the column was equilibrated with the equilibration buffer. (3) The sample was loaded to the column, thereafter the column was washed with the equilibration buffer until the baseline is flat. (4) The column was eluted with a gradient of elution buffer (20 mM PB, 1M NaCl, pH 7.2) to collect the protein of interest.

[0126] Gel filtration chromatography was carried out as follows. (1) The column was equilibrated with PBS buffer (20 mM PB, 0.15M NaCl, pH 7.2). (2) The Source 30S elution peak was loaded with a loop, and the volume of each loading was not more than 3% of the column volume. (3) The column was washed with PBS buffer to collect the protein of interest.

[0127] SDS-PAGE Detection of Recombinant Proteins

[0128] 30 μl purified protein with a suitable concentration was added to 10 μl 4×SDS-PAGE buffer (with and without 20 mM DTT) respectively, denatured at 100° C. for 5 min and centrifuged. Then 30 μl of the supernatant was taken for SDS-PAGE electrophoresis analysis (separation gel is 15%). Following the electrophoresis, the gel was stained with Coomassie Brilliant Blue R250.

[0129] Western Blot (WB) Detection of Recombinant Proteins

[0130] The sample was prepared in the same way as in SDS-PAGE. 3 μl sample was taken for SDS-PAGE. Following the electrophoresis, the proteins were transferred to a nitrocellulose membrane with 300 mA constant current for 1 h and blocked with 5% skim milk/TBST at room temperature for 1 h. The primary antibody PDGF-B (F-3) (Santa Cruz Biotechnology, SC-365805) was 1:1000 diluted, coated at room temperature for 1 h, and washed with TBST for several times. HRP-labeled secondary antibody (Cell Signaling Technology, #7076) was 1:10000 diluted, incubated at room temperature for 1 h, and washed with TBST. The substrate was added and imaged with an LAS400 mini gel imaging system (GE).

[0131] Sequencing of N-Terminal Amino Acid Sequence

[0132] Sample preparation and SDS-PAGE processes were the same as the above. Following the completion of the electrophoresis, the proteins were transferred to a PVDF membrane with CAPS electroblotting buffer at 300 mA constant current for 1 h, and stained with 0.1% Coomassie Brilliant Blue R250, immediately after that, fully decolored with 50% methanol until the protein bands were visible. The protein bands to be determined were cut off and sent to Chromatography Laboratory of Biomedical Analysis Center, Military Medical Academy for determination.

[0133] Detection of Protein Glycosylation

[0134] 5 μl of the sample and the positive control IFN-co were added into 3 μl of 10× glycoprotein denaturation buffer (containing NEB PNGase F enzyme) and 15 μl of water, and heated at 100° C. to denature for 10 min. After cooling, 3 μl NP-40, 3 μl G7 buffer (containing NEB PNGase F enzyme) and 2 μl peptide N-glycosidase F (PNGase F) (a product of New England Biotech Inc. (NEB)) were added and digested at 37° C. for 3 h. Following the completion of the digestion, the sample was heated at 100° C. to inactivate the enzyme and then subjected to SDS-PAGE electrophoresis. Following the completion of the electrophoresis, staining was performed using a glycoprotein staining kit (Themo Scientific, #24562). Firstly, the gel was added into 100 ml 50% methanol and fixed for 30 min; the gel was washed several times with 3% acetic acid, transferred to 25 ml Oxidizing Solution and shaken gently for 15 min; the gel was washed several times with 3% acetic acid, transferred to 25 ml Glycoprotein Staining Reagent and shaken gently for 15 min; thereafter, the gel was transferred to 25 ml Reducing Solution and shaken gently for 5 min; then, the gel was washed with 3% acetic acid and rinsed with deionized water.

[0135] Detection of PDGF-B Biological Activity

[0136] BALB/C 3T3 cells (purchased from Beijing Xiehe Cell Resource Center) were cultured in DMEM complete medium (Life Technology) containing 10% FBS under a condition of 37° C. and 5% carbon dioxide. After digestion and collection, the cells were prepared as cell suspension containing 5.0×10.sup.4 cells per ml with a complete broth, inoculated into a 96-well cell culture plate (100 μl per well), and followed by culturing under a condition of 37° C. and 5% carbon dioxide. 24 hours later, the medium was exchange into a maintenance medium (DMEM containing 0.4% FBS), followed by culturing under a condition of 37° C. and 5% carbon dioxide. Upon 24 hours of culturing, the culture medium was discarded and added a pre-gradient diluted PDGF-BB solution (100 μl per well). The cells were cultured for another 64 to 72 hours under the action of proteins, and were assayed for cell proliferation with WST-1 method as follows: 10 μl WST-1 solution (Roche, 11644807001) was added into each well, cultured under a condition of 37° C. and 5% carbon dioxide for 3 hours, and then measured for the absorbance at a wavelength of 450 nm using a microplate reader (reference wavelength: 630 nm). The experimental data were processed by a four-parameter regressive calculation method. The EC.sub.50 values of the two proteins were calculated respectively. The experiment was repeated three times. The difference statistical analysis between the two, groups was performed by t-test.

Example 1. The Co-Effect of the Proteolysis and Glycosylation Contributes to the Formation of Diverse Monomers of PDGF-BB.SUP.Thr6

[0137] The inventors firstly suspected that proteolysis is the cause of the formation of diverse PDGF-B monomers. By reducing SDS-PAGE electrophoresis, different monomers of PDGF-B were separated and five bands were detected via Coomassie Brilliant Blue staining (FIG. 3A). The five protein bands were subjected to sequencing for N-terminal amino acid sequence, and the results showed that the first five amino acid residues at N-terminus in the first, second and third bands were all TIAEP, corresponding to the correct N-terminal sequence of PDGF-B.sup.Thr6, while the first five amino acid residues at N-terminus in the fourth and fifth bands were TNANF. The alignment of protein sequences determined that the fourth and fifth protein fragments were the truncated proteins generated by proteolytic cleavage at Arg32-Thr33 (FIG. 3A).

[0138] However, this cannot explain the reason for the formation of at least 5 kinds of PDGF monomers. The difference in molecular weights between bands 1, 2, 3 and bands 4, 5 might be due to C-terminal cleavage. To answer this question, the inventors performed WB assay using a specific monoclonal antibody (F-3) against PDGF-B C-terminus (Santa Cruz Biotechnology, SC-365805). The result showed that only two of the five bands were detected. But interestingly, the two bands bound to the antibody appeared to correspond to the third and fifth protein fragments (FIG. 3B). If the first, second, and fourth protein fragments cannot be detected by the antibodies due to the C-terminal cleavage, their molecular weights should be smaller. However, this is clearly not consistent with the result of electrophoresis assay. This means that there are other reasons to be found.

[0139] In order to analyze whether PDGF-B was glycosylated, PDGF was digested with peptide N-glycosidase F (PNGase F), and SDS-PAGE and glycoprotein staining were performed simultaneously. PNGaseF is an amidase which can act on almost all N-glycan chains in a glycopeptide/glycoprotein, cleaves between the innermost GlcNAc and asparagine residues of the sugar chain moiety, and converts asparagine into aspartic acid (10), and is the most widely used enzyme in the identification of N-glycoprotein in the glycoprotein proteomics research. The recombinant IFN-w protein expressed in Pichia pastoris acts as a positive control of N-glycosylated protein. Coomassie Brilliant Blue staining result showed that there was no change in the relative molecular weight of PDGF-B protein before and after the cleavage, indicating that PDGF-B protein did not undergo N-glycosylation (FIG. 3C, left). However, the glycoprotein staining result indicated that PDGF-B is indeed a glycoprotein (FIG. 3C, right). This means that PDGF-B secreted by Pichia pastoris was O-glycosylated. Meanwhile, further analysis showed that only three protein fragments were detected in the sugar staining, which should correspond to bands 1, 2 and 4 in the SDS-PAGE result respectively (FIG. 3B). This is also consistent with the result of the above WB assay: the first, second and fourth protein fragments were glycosylated at C-terminus thereof, thus affecting the binding of PDGF-B.sup.Thr6 to the antibody.

[0140] Combining the above experimental results, the inventors deduced that the several forms of PDGF-B.sup.Thr6 monomers resulted from the co-effect of the proteolysis and differentially post-translational glycosylation occurring between amino acids Arg32-Thr33 at positions 27/28 (Table 1).

TABLE-US-00003 TABLE 1 Analysis on PDGF-B.sup.Thr6 modifications Band modification type 1 complete PDGF, O-glycosylation 2 complete PDGF, O-glycosylation 3 complete PDGF 4 Arg32-Thr33 truncated, O-glycosylated 5 Arg32-Thr33 truncated Note: The complete PDGF in the table refers to PDGF-B.sup.Thr6, i.e., a PDGF-B with 5 amino acids deleted at N-terminus.

Example 2. Construction of PDGF-MI and PDGF-M2 Modifiers and Detection of Protein Properties

[0141] Moreover, the inventors would like to confirm the above-mentioned deduction, and expected the expression of PDGF-B in Pichia pastoris to be homogenous. Firstly, the inventors would like to determine the possible O-glycosylation sites. The prediction of the glycosylation sites of PDGF-B.sup.Thr6 protein sequence was performed using online website CBS (www.CBS.dtu.dk) (11). The result showed that Thrs at positions 6, 101 and 109 are the possible O-glycosylation modification sites (FIG. 4). Compared with N-glycosylation, there is no definite motif for O-glycosylation sites, so the prediction thereof is also relatively difficult. However, the predicted potential glycosylation sites at positions 6, 101 and 109 are consistent with our results: there should be glycosylation modifications (Thr101, Thr109) at C-terminus of the PDGF-B.sup.Thr6 protein, since they hindered the binding to the antibody; there should be glycosylation modification site(s) before Thr33, which could explain the fact that there was only one (Band 4) glycosylation-modified variant for the digested PDGF-B, but there were two bands (Bands 1, 2) for glycosylated PDGF-B monomer without digestion (FIG. 3B; Table 1). In order to confirm the predicted results, we constructed two mutants PDGF-M1 and PDGF-M2 of PDGF-B.sup.Thr6. Two glycosylation sites at C-terminus were mutated in PDGF-M1, and all three potential glycosylation sites were mutated in PDGF-M2 (See FIG. 6A for the pattern of mutations). Meanwhile, in order to remove the protease cleavage site Arg32-Thr33, we mutated Arg32 to Pro, considering the evolutionary selection of amino acids. We noted that the mature PDGF-B protein has 60% amino acid sequence homology with PDGF-A, and both have a high similarity in terms of structure and function, whereas the amino acid in the corresponding position of PDGF-A protein is Pro.

[0142] The amino acid sequence of PDGF-M1 is:

TABLE-US-00004 (SEQ ID NO: 3) TIAEPAMIAECKTRTEVFEISRRLIDPTNANFLVWPPCVEVQRCSGCCN NRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCEAVA AARPVA;

[0143] The nucleotide sequence of PDGF-M1 is:

TABLE-US-00005 (SEQ ID NO: 4) ACCATTGCTGAGCCGGCCATGATCGCCGAGTGCAAGACGCGCACCGAGG TGTTCGAGATCTCCCGGCGCCTCATAGACCCCACCAACGCCAACTTCCT GGTGTGGCCGCCCTGTGTGGAGGTGCAGCGCTGCTCCGGCTGCTGCAAC AACCGCAACGTGCAGTGCCGCCCCACCCAGGTGCAGCTGCGACCTGTCC AGGTGAGAAAGATCGAGATTGTGCGGAAGAAGCCAATCTTTAAGAAGGC CACGGTGACGCTGGAAGACCACCTGGCATGCAAGTGTGAGGCAGTGGCA GCTGCACGGCCTGTGGCC.

[0144] The amino acid sequence of PDGF-M2 is:

TABLE-US-00006 (SEQ ID NO: 5) AIAEPAMIAECKTRTEVFEISRRLIDPTNANFLVWPPCVEVQRCSGCCN NRNVQCRPTQVQLRPVQVRKIEIVRKKPIFKKATVTLEDHLACKCEAVA AARPVA;

[0145] The nucleotide sequence of PDGF-M2 is:

TABLE-US-00007 (SEQ ID NO: 6) GCCATTGCTGAGCCGGCCATGATCGCCGAGTGCAAGACGCGCACCGAGG TGTTCGAGATCTCCCGGCGCCTCATAGACCCCACCAACGCCAACTTCCT GGTGTGGCCGCCCTGTGTGGAGGTGCAGCGCTGCTCCGGCTGCTGCAAC AACCGCAACGTGCAGTGCCGCCCCACCCAGGTGCAGCTGCGACCTGTCC AGGTGAGAAAGATCGAGATTGTGCGGAAGAAGCCAATCTTTAAGAAGGC CACGGTGACGCTGGAAGACCACCTGGCATGCAAGTGTGAGGCAGTGGCA GCTGCACGGCCTGTGGCC.

[0146] DNA sequences encoding PDGF-M1 and PDGF-M2 were inserted into expression vector pMEX9K, and integrated into the Pichia pastoris strain GS115. The expression was induced by methanol and proteins were purified via chromatography. The purified PDGF-B proteins were subjected to SDS-PAGE, glycoprotein staining and Western blotting detection, in order to determine the properties of the engineered protein. WB result showed that only single band could be detected after the two mutants were reduced, and the relative molecular mass is about 12 kDa, consistent with the expected one (FIG. 6B). SDS-PAGE result showed that PDGF-M2 is a single band, but PDGF-M1 still has a minor band above the major band (FIG. 6C). It is presumed that this band results from the glycosylation of Thr6. Glycoprotein staining result showed that the glycosylation levels of the two mutants were very low compared to those before mutation and hardly to be detected with glycoprotein staining (FIG. 6D). The above results indicated that mutation of R at position 32 to P removed the potential Kex2 protease cleavage site and prevented the formation of Thr33 truncated PDGF-B monomer, while the mutations of three glycosylation sites Thr6, 101 and 109 also abolishes the post-translational glycosylation modifications of the protein at different degrees, thereby rendering the expression of PDGF-B protein in Pichia pastoris homogenous.

Example 3. Detection of Cell Proliferation Enhancing Activity of PDGF-M2

[0147] In order to analyze whether the mutations of glycosylation sites Thr6-Ala, Thr101-Ala and Thr109-Ala and the mutation of Arg32-Pro KEX cleavage site could affect the biological activity of PDGF-BB, the inventors determine the proliferation activity of PDGF-B.sup.Thr6 and PDGF-M2 on Balb/c 3T3 cells using WST-1 method. The results showed that EC50 of PDGF-B.sup.Thr6 is 5.434±0.6475 ng/ml, while EC50 of PDGF-M2 is 3.492±0.4078 ng/ml. The t-test showed that the protein activity after engineering is higher than before engineering, with P value of 0.0117 (FIG. 7).

Example 4. Effect of PDGF-M2 Arg32 Mutation on Expression Level

[0148] In order to enhance the expression level of PDGF-M2, the inventors carried out codon-optimization on PDGF-M2 (PDGF-IM-P) according to the codon preference of Pichia pastoris during protein expression using online tool JAVA Condon Adaptation Tool. The optimized encoding DNA sequence is as follows:

[0149] The DNA sequence encoding PDGF-IM-P:

TABLE-US-00008 (SEQ ID NO: 7) 5′GCTATCGCTGAACCAGCTATGATCGCTGAATGTAAGACTAGAAC TGAAGTTTTCGAAATCTCTAGAAGATTGATCGACCCAACTAACGCTAAC TTCTTGGTTTGGCCACCATGTGTTGAAGTTCAAAGATGTTCTGGTTGTT GTAACAACAGAAACGTTCAATGTAGACCAACTCAAGTTCAATTGAGACC AGTTCAAGTTAGAAAGATCGAAATCGTTAGAAAGAAGCCAATCTTCAAG AAGGCTACTGTTACTTTGGAAGACCACTTGGCTTGTAAGTGTGAAGCTG TTGCTGCTGCTAGACCAGTTGCT-3′.

[0150] The protein sequence thereof is same as PDGF-M2.

[0151] On the basis of codon optimization, Arg32 was mutated to Val (PDGF-IM-V, Val codon used is GTT) and Ile (PDGF-IM-I, Ile codon used is ATC), the expression level was compared with that of PDGF-M2 in order to analyze the effect of mutation of Arg32 on protein expression.

[0152] The nucleotide sequence of PDGF-IM-V is:

TABLE-US-00009 (SEQ ID NO: 8) GCTATCGCTGAACCAGCTATGATCGCTGAATGTAAGACTAGAACTGAAG TTTTCGAAATCTCTAGAAGATTGATCGACGTTACTAACGCTAACTTCTT GGTTTGGCCACCATGTGTTGAAGTTCAAAGATGTTCTGGTTGTTGTAAC AACAGAAACGTTCAATGTAGACCAACTCAAGTTCAATTGAGACCAGTTC AAGTTAGAAAGATCGAAATCGTTAGAAAGAAGCCAATCTTCAAGAAGGC TACTGTTACTTTGGAAGACCACTTGGCTTGTAAGTGTGAAGCTGTTGCT GCTGCTAGACCAGTTGCT.

[0153] The nucleotide sequence of PDGF-IM-I is:

TABLE-US-00010 (SEQ ID NO: 9) GCTATCGCTGAACCAGCTATGATCGCTGAATGTAAGACTAGAACTGAAG TTTTCGAAATCTCTAGAAGATTGATCGACATCACTAACGCTAACTTCTT GGTTTGGCCACCATGTGTTGAAGTTCAAAGATGTTCTGGTTGTTGTAAC AACAGAAACGTTCAATGTAGACCAACTCAAGTTCAATTGAGACCAGTTC AAGTTAGAAAGATCGAAATCGTTAGAAAGAAGCCAATCTTCAAGAAGGC TACTGTTACTTTGGAAGACCACTTGGCTTGTAAGTGTGAAGCTGTTGCT GCTGCTAGACCAGTTGCT.

[0154] The DNA sequences encoding PDGF-IM-P, PDGF-IM-V and PDGF-IM-I were ligated to the sequences such as restriction site(s), terminator(s), cloned into expression vector pMEX9K, and integrated into expression strain GS115. Following histidine-deficient MD plate screening, nine clones were randomly selected, and subjected to expression induced by methanol in a tube. The SDS-PAGE electrophoresis analysis of the culture supernatant demonstrated that the expression amount of PDGF-IM-P protein was significantly higher than other two strains (FIG. 8A).

[0155] The screening of GS115/PDGF-IM-P, GS115/PDGF-IM-V, and GS115/PDGF-IM-P clones with multiple inserts was carried out with G418. The expression of clones grown on plates with 2.0 mg/ml and 4.0 mg/ml G418 was analyzed, respectively. The result demonstrated that the average expression level of PDGF-IM-P was higher than the other two strains (FIG. 8B). This indicates that the mutation of Arg32 site would affect the secretion and expression level of PDGF in Pichia pastoris, and the mutation of this site to Pro is relatively favorable for expression.

Example 5 LC/MS Detection of the Glycosylation of PDGF-B and PDGF-M2 Mutants

[0156] Method

[0157] The recombinant PDGF-B wild-type and PDGF-M2 mutants were reduced with DTT (2.5 mM) at 37° C. for 30 min, and diluted with buffer A (an aqueous solution containing 0.1% formic acid), followed by liquid chromatography and mass spectrometry (LC/MS) analysis. The proteins were separated on Easy-spray column (15 cm×75 m ID, 3-μm C18 particles) using EASY-nLC system (Thermo Fisher Scientific), eluted with a linear gradient of buffer B (containing a solution of 0.1% formic acid in methanol; 0-90%, 20 min) at a flow rate of 300 nl/min. High resolution spectra were obtained using Q Exactive Mass Spectrometer (Thermo Fisher Scientific) under a condition of a resolution of 60,000, m/z 350-1600 and de-convoluted using Xtract software (Thermo Scientific).

[0158] Results

[0159] Analysis of the PDGF-B wild-type and PDGF-M2 mutant by high resolution LC/MS demonstrated that for wild-type PDGF, different isoforms containing up to 6 carbohydrate residues were detected, wherein the content of the isoform containing three carbohydrate residues was the highest. Meanwhile, glycosylation could hardly be detected for the PDGF-M2 (M2) mutant (as shown in FIG. 9).

[0160] Although the specific embodiments of the invention have been described in detail, those skilled in the art will appreciate that in accordance with all the teachings which have been disclosed, various modifications and substitutions may be made to those details and these changes are all within the scope of the present invention. The scope of the invention is given by the appended claims and any equivalents thereof.

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