Engineered Platelet-Derived Growth Factor Variants

Abstract

The present invention relates to improved platelet-derived growth factor (PDGF) polypeptide variants, particularly polypeptides comprising the sequence of any one of SEQ ID NOs: 2 to 4 or a sequence having at least 80% similarity thereto, wherein the PDGF polypeptide exhibits increased stability, biological activity and / or solubility compared to the wild-type PDGF polypeptide of SEQ ID NO: 1. The present invention further provides nucleic acid sequences encoding the PDGF polypeptides of the present invention, and also provides a method for growing animal cells, comprising culturing the animal cells in a culture medium containing the PDGF polypeptide of the present invention.

Description

[Technical Field]

[0001] The present invention relates to improved growth factor polypeptide variants, particularly variants of platelet-derived growth factor (PDGF), and their uses, including use in cell culture media. [Background technology]

[0002] Growth factors are naturally occurring cell signaling molecules that play many essential roles, including regulating cell proliferation and development and cell differentiation.

[0003] One of the most common mechanisms for regulating cell signaling in living organisms is the degradation of signaling molecules. Therefore, most wild-type growth factors have high turnover rates and are not "built" to persist, making them less suitable for industrial situations where a constant level of stimulation for cell proliferation should be maintained.

[0004] There are a number of drawbacks associated with this rapid degradation of wild-type growth factors, including wasteful and inefficient use of components and the introduction of variability in the context of cell culture.

[0005] Growth media used in cell culture typically contain a combination of growth factors. To overcome the exponential depletion of these naturally occurring cell growth signaling stimuli, high levels of growth factors are used to keep the stimulation above a minimum threshold, and the medium must be replaced frequently (e.g., every two days) even if there are still sufficient nutrients.

[0006] In the growing field of cultivated meat, cell culture is a fundamental aspect of the process. One of the limiting steps in the production of cultivated meat is the high cost of the cell growth medium. Climate and other environmental concerns continue to drive the demand for cultivated meat, and therefore also the need for improved growth media to replace animal serum-based media.

[0007] To address these concerns, a series of engineered polypeptides are provided that have improved properties, such as increased stability (including thermostability). These polypeptides can be used as replacements for naturally occurring wild-type growth factors in growth media. The improved properties also make the polypeptides useful in a number of other areas.

[0008] Platelet-derived growth factors (PDGFs) are a family of cell signaling proteins whose members are involved in diverse cellular processes. PDGF polypeptides are commonly used in growth media, and there is a continuing need for PDGF polypeptides with improved properties, such as increased stability, biological activity, and / or solubility. PDGFB is one member of this family and binds to a specific PDGF receptor. PDGFB can exist as a homodimer (also known as PDGFbb) or a heterodimer with PDGFA (known as PDGFab). PDGFB can stimulate the proliferation and differentiation of various cell types and assist in the process of angiogenesis.

[0009] The present invention fulfills this need by providing novel polypeptides with improved properties. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] National Center for Biotechnology Information (NCBI): ncbi.nlm.nih.gov [Non-patent document 2] Gregorio et al., “A User's Guide to Cell-Free Protein Synthesis,” Methods Protoc., 2019 [Non-patent document 3] https: / / www.ebi.ac.uk / Tools / msa / clustalo / Summary of the Invention [Means for solving the problem]

[0011] Provided herein is a platelet-derived growth factor (PDGF) polypeptide that contains at least one modification selected from the group consisting of an amino acid substitution, an amino acid deletion, an amino acid insertion, and combinations thereof, compared to the sequence set forth in SEQ ID NO:1, and that exhibits increased stability (including thermal stability), biological activity, and / or solubility compared to the PDGFB polypeptide of SEQ ID NO:1.

[0012] Also provided herein is a PDGF polypeptide comprising the sequence of any one of SEQ ID NOs: 2-4 or a sequence having at least 80% similarity thereto, which exhibits increased stability (including thermal stability), biological activity and / or solubility compared to the wild-type PDGFB polypeptide of SEQ ID NO: 1.

[0013] Also provided herein are nucleic acid sequences encoding the PDGF polypeptides of the present invention.

[0014] Also provided are cells comprising a polypeptide of the invention or a nucleic acid sequence of the invention.

[0015] Also provided is a method for growing animal cells comprising culturing the animal cells in a culture medium containing a PDGF polypeptide of the present invention. [Brief explanation of the drawings]

[0016] [Figure 1] 1 shows an alignment of the amino acid sequences of the polypeptides of the present invention (SEQ ID NOS: 2 to 4). [Figure 2] 1 shows an alignment of the amino acid sequences of the polypeptides of the present invention (SEQ ID NOs: 2 to 4) and the wild-type PDGFB sequence (SEQ ID NO: 1). [Figure 3A-3C]1 depicts a schematic of the plasmid inserts encoding the polypeptides of SEQ ID NOs: 2-4. [Figure 4A-4B] 1 illustrates the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis demonstrating the expression of a polypeptide of the present invention. [Figures 5A-5C] 1 shows a comparison of the receptor kinase activity (biological activity) of a PDGF polypeptide of the present invention compared to wild-type PDGFB, with the grey line representing the polypeptide of the present invention and the black line representing wild-type PDGFB. DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides novel PDGF polypeptide sequences, including PDGFB polypeptide sequences, that have improved properties compared to known PDGF polypeptides, e.g., increased stability, including increased thermal stability (increased melting temperature (Tm)). The present invention also provides nucleic acids encoding the PDGF polypeptides of the present invention.

[0018] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature and laboratory procedures in cell culture, molecular genetics, organic chemistry, nucleic acid chemistry, and hybridization used herein are those well known and commonly used in the art. Standard techniques are used for nucleic acid and peptide synthesis. The techniques and procedures are generally performed according to conventional methods in the art. The nomenclature used herein and the synthetic biology laboratory procedures described below are those well known and commonly used in the art.

[0019] Polypeptides of the Invention In some embodiments, the present invention provides a polypeptide comprising at least one modification selected from the group consisting of an amino acid substitution, an amino acid deletion, an amino acid insertion, and a combination thereof, compared to the sequence set forth in SEQ ID NO:1, wherein the polypeptide exhibits increased stability compared to the PDGFB polypeptide of SEQ ID NO:1.

[0020] In some embodiments, the present invention provides a polypeptide comprising at least one modification selected from the group consisting of an amino acid substitution, an amino acid deletion, an amino acid insertion, and a combination thereof, compared to the sequence set forth in SEQ ID NO:1, wherein the polypeptide is biologically active and exhibits altered stability and / or solubility compared to the PDGFB polypeptide of SEQ ID NO:1.

[0021] In some embodiments, the present invention provides a polypeptide comprising at least one modification selected from the group consisting of an amino acid substitution, an amino acid deletion, an amino acid insertion, and a combination thereof, compared to the sequence set forth in SEQ ID NO:1, wherein the polypeptide is biologically active and exhibits a level of stability and / or solubility equivalent to that exhibited by the PDGFB polypeptide of SEQ ID NO:1.

[0022] In some embodiments, the polypeptide of the invention is a PDGF polypeptide.

[0023] In some embodiments, the polypeptide of the invention is a PDGFB polypeptide.

[0024] In some embodiments, the polypeptides of the invention are dimers, including homodimers.

[0025] In some embodiments, the polypeptides of the invention bind to at least one platelet-derived growth factor receptor (PDGFR).

[0026] In some embodiments, the polypeptides of the invention bind to at least PDGFRB, which is also known as PDGFRbeta.

[0027] In some embodiments, the polypeptide of the present invention is a recombinant polypeptide, a natural polypeptide or a synthetic polypeptide, preferably a recombinant polypeptide.

[0028] Substitutions may be conservative amino acid substitutions, i.e., substitution of one amino acid residue for another that shares the chemical and physical properties of the amino acid side chain (e.g., charge, size, hydrophobicity / hydrophilicity). Conservative substitutions are intended to include substitutions within the following groups of amino acid residues: gly, ala; val, ile, leu; asp, glu; asn, gln; ser, thr; lys, arg; and phe, tyr.

[0029] The terms "wild-type PDGFB," "WT PDGFB," "naturally occurring PDGFB," or "native PDGFB" refer to PDGFB that occurs in nature, without any modifications.

[0030] The human wild-type PDGFB protein comprises the sequence of SEQ ID NO: 1 and is shown below:

[0031] [ka]

[0032] PDGFB has multiple functions, including stimulating the proliferation and differentiation of various cell types and can assist in the angiogenic process.

[0033] PDGFB binds to and stimulates the platelet-derived growth factor receptor (PDGFR), and this biological activity is required for effective function of the PDGFB protein, both for naturally occurring and engineered forms of PDGFB.

[0034] In some embodiments, polypeptides of the invention exhibit increased biological activity compared to the PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention exhibit increased stability (e.g., increased thermostability) compared to the PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention exhibit increased solubility, e.g., in Escherichia coli (E. coli) or yeast, compared to the PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention exhibit both increased stability (e.g., increased thermostability) and increased biological activity compared to the PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention exhibit increased stability (e.g., increased thermostability), increased biological activity, and increased solubility compared to the PDGFB polypeptide of SEQ ID NO: 1.

[0035] A preferred approach for measuring stability, including thermostability, is by assessing biological activity over time, for example, by using the ONE-Glo luciferase assay (Promega). The ONE-Glo luciferase assay detects and quantifies expression of a luciferase reporter gene in response to signaling triggered by PDGF binding to PDGFR. This assay is described in detail in Example 6 and also in Example 3.

[0036] The melting temperature of a polypeptide can also be used to quantify thermal stability. Wild-type PDGFB polypeptide has a melting temperature (Tm) of about 50°C. Thermal stability can be measured by differential scanning fluorimetry, in which the change in fluorescence of a dye, such as Spyro Orange, that preferentially binds unfolded protein over folded protein is measured by PCR, e.g., real-time PCR, to determine the melting temperature (°C).

[0037] In some embodiments, polypeptides of the invention exhibit melting temperatures equivalent to the melting temperature of the PDGFB polypeptide of SEQ ID NO: 1. In this context, equivalent can mean within 20°C, within 10°C, or within 5°C.

[0038] In some embodiments, polypeptides of the invention exhibit a melting temperature that is higher than the melting temperature of the PDGFB polypeptide of SEQ ID NO: 1. The increase in melting temperature can be an increase of about 1°C, 2°C, 3°C, 4°C, 5°C, 10°C, 15°C, 20°C, 25°C, or 30°C.

[0039] The term "biological activity" refers to the ability of a polypeptide to bind to a relevant receptor. Biological activity, as used herein, can refer to the ability of a polypeptide of the present invention to bind to a platelet-derived growth factor receptor (PDGFR), such as PDGFRB. Biological activity can be determined by the ONE-Glo luciferase assay (Promega). This assay is described in detail in Example 3. EC50 (half maximal effective concentration) quantifies biological activity.

[0040] In some embodiments, a polypeptide of the invention has a lower EC50 than the PDGFB polypeptide of SEQ ID NO: 1, when the EC50 of each polypeptide is measured under the same conditions.

[0041] It is understood that for purposes of comparing the EC50 of a polypeptide of the invention with the EC50 of the PDGFB polypeptide of SEQ ID NO: 1, both EC50s are measured under the same conditions in the same experiment. Thus, in one embodiment, improved biological activity of a polypeptide of the invention means a lower EC50 than the PDGFB polypeptide of SEQ ID NO: 1 when measured under the same conditions.

[0042] In some embodiments, polypeptides of the invention have an EC50 that is at least two-fold lower than the EC50 of the wild-type PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have an EC50 that is at least five-fold lower than the EC50 of the wild-type PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have an EC50 that is at least ten-fold lower than the EC50 of the wild-type PDGFB polypeptide of SEQ ID NO: 1.

[0043] In some embodiments, polypeptides of the invention have an EC50 at least 0.05 ng / ml below the EC50 of the wild-type PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have an EC50 at least 0.075 ng / ml below the EC50 of the wild-type PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have an EC50 at least 0.1 ng / ml below the EC50 of the wild-type PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have an EC50 at least 0.125 ng / ml below the EC50 of the wild-type PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have an EC50 at least 0.15 ng / ml below the EC50 of the wild-type PDGFB polypeptide of SEQ ID NO: 1.

[0044] In some embodiments, the polypeptides of the present invention can bind to platelet-derived growth factor receptor (PDGFR). In some embodiments, the polypeptides of the present invention can bind to PDGFRA and / or PDGFRB. In some embodiments, the polypeptides of the present invention can bind to PDGFRA and PDGFRB.

[0045] "Solubility," as used herein, refers to the ability of a polypeptide to fold into a functional protein. Solubility can be an indicator of the expression level of a functional protein by a particular expression platform. Solubility can be determined by using SDS-PAGE and Western blot. This assay is described in detail in Example 4. The solubility percentage quantifies the solubility.

[0046] The wild-type PDGFB polypeptide of SEQ ID NO: 1 is not soluble. Accordingly, it is an object of the present invention to provide soluble variants. In one embodiment, the polypeptides of the present invention are soluble.

[0047] Insoluble proteins require denaturing purification, which involves unfolding and then refolding the protein during purification. The refolding step is inefficient and sometimes does not work. In contrast, soluble proteins (e.g., polypeptides of the present invention) can be purified by native purification, which does not require unfolding the protein. This is one advantage of the present invention.

[0048] In some embodiments, the polypeptides of the present invention have a solubility of at least 30%. In some embodiments, the polypeptides of the present invention have a solubility of at least 40%. In some embodiments, the polypeptides of the present invention have a solubility of at least 50%. In some embodiments, the polypeptides of the present invention have a solubility of at least 60%. In some embodiments, the polypeptides of the present invention have a solubility of at least 70%. In some embodiments, the polypeptides of the present invention have a solubility of at least 80%. In some embodiments, the polypeptides of the present invention have a solubility of at least 90%. In some embodiments, the polypeptides of the present invention have a solubility of 100%.

[0049] In some embodiments, the polypeptides of the invention are more stable than and have a higher or substantially the same level of biological activity as wild-type PDGFB (e.g., the polypeptide of SEQ ID NO: 1). In some embodiments, the polypeptides of the invention are more stable than and exhibit substantially the same solubility as wild-type PDGFB (e.g., the polypeptide of SEQ ID NO: 1).

[0050] In some embodiments, the polypeptides of the present invention are soluble and bioactive. In some embodiments, the polypeptides of the present invention are soluble, bioactive, and thermostable at 39°C to 49°C. In some embodiments, the polypeptides are soluble, bioactive, and thermostable at 40°C.

[0051] In some embodiments, polypeptides of the invention are soluble and exhibit substantially the same or greater stability as the PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention are biologically active and exhibit increased stability compared to the PDGFB polypeptide of SEQ ID NO: 1. In some embodiments, polypeptides of the invention are soluble, biologically active, and exhibit increased stability compared to the PDGFB polypeptide of SEQ ID NO: 1.

[0052] In some embodiments, the polypeptide of the invention comprises the sequence of any one of SEQ ID NOs: 2-4.

[0053] In some embodiments, the polypeptide of the present invention consists of any one of SEQ ID NOs: 2-4.

[0054] In some embodiments, the polypeptides of the present invention comprise a sequence having at least 80% similarity to any one of SEQ ID NOs: 2-4. In some embodiments, the polypeptides of the present invention comprise a sequence having at least 85% similarity to any one of SEQ ID NOs: 2-4. In some embodiments, the polypeptides of the present invention comprise a sequence having at least 90% similarity to any one of SEQ ID NOs: 2-4. In some embodiments, the polypeptides of the present invention comprise a sequence having at least 92% similarity to any one of SEQ ID NOs: 2-4. In some embodiments, the polypeptides of the present invention comprise a sequence having at least 95% similarity to any one of SEQ ID NOs: 2-4. The polypeptides of the embodiments can be PDGFB polypeptides. The polypeptides of this embodiment can bind to at least one platelet-derived growth factor receptor (PDGFR).

[0055] In some embodiments, the polypeptide of the present invention comprises a sequence having at least 96% similarity to any one of SEQ ID NOs: 2-4. In some embodiments, the polypeptide of the present invention comprises a sequence having at least 97% similarity to any one of SEQ ID NOs: 2-4. In some embodiments, the polypeptide of the present invention comprises a sequence having at least 98% similarity to any one of SEQ ID NOs: 2-4. In some embodiments, the polypeptide of the present invention comprises a sequence having at least 99% similarity to any one of SEQ ID NOs: 2-4. The polypeptide of the embodiment can be a PDGFB polypeptide. The polypeptide of this embodiment can bind to at least one platelet-derived growth factor receptor (PDGFR).

[0056] In a preferred embodiment, a polypeptide of the invention comprises a sequence having at least 90% similarity to SEQ ID NO:2 or SEQ ID NO:4.

[0057] In a preferred embodiment, a polypeptide of the invention comprises a sequence having at least 92% similarity to SEQ ID NO:3.

[0058] In a preferred embodiment, the polypeptide of the present invention comprises a sequence having at least 95% similarity to any one of SEQ ID NOs: 2-4.

[0059] The percent similarity (or "percent similarity") between two sequences can be calculated by multiplying the number of matches in the pair by 100 and dividing by the length of the aligned region, including gaps. Identity scoring counts only perfect matches and does not take into account the degree of amino acid similarity to each other. Gaps at the ends of the sequences are not included, and internal gaps are included in the length. Alignments can be generated using programs known in the art. For purposes herein, alignment of nucleotide sequences can be performed using the blastn program set to default parameters, and alignment of amino acid sequences can be performed using the blastp program set to default parameters (see National Center for Biotechnology Information (NCBI): ncbi.nlm.nih.gov).

[0060] In some embodiments, a polypeptide of the present invention comprises the sequence of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, a polypeptide of the present invention comprises a sequence that is at least 80% similar to the sequence of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, a polypeptide of the present invention comprises a sequence that is at least 85% similar to the sequence of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, a polypeptide of the present invention comprises a sequence that is at least 90% similar to the sequence of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, a polypeptide of the present invention comprises a sequence that is at least 92% similar to the sequence of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, a polypeptide of the present invention comprises a sequence that is at least 95% similar to the sequence of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, a polypeptide of the present invention consists of the sequence of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, a polypeptide of the invention comprises a sequence that is at least 96% similar to the sequence of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, a polypeptide of the invention comprises a sequence that is at least 97% similar to the sequence of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, a polypeptide of the invention comprises a sequence that is at least 98% similar to the sequence of SEQ ID NO:2 or SEQ ID NO:4. In some embodiments, a polypeptide of the invention comprises a sequence that is at least 99% similar to the sequence of SEQ ID NO:2 or SEQ ID NO:4.

[0061] In some embodiments, a polypeptide of the present invention comprises the sequence of SEQ ID NO:2. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 80% similarity to the sequence of SEQ ID NO:2. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 85% similarity to the sequence of SEQ ID NO:2. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 90% similarity to the sequence of SEQ ID NO:2. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 92% similarity to the sequence of SEQ ID NO:2. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 95% similarity to the sequence of SEQ ID NO:2. In this embodiment, a polypeptide of the present invention may be more stable than wild-type PDGFB (e.g., the polypeptide of SEQ ID NO:1). In some embodiments, a polypeptide of the present invention consists of the sequence of SEQ ID NO:2.

[0062] In some embodiments, a polypeptide of the invention comprises a sequence having at least 96% similarity to the sequence of SEQ ID NO: 2. In some embodiments, a polypeptide of the invention comprises a sequence having at least 97% similarity to the sequence of SEQ ID NO: 2. In some embodiments, a polypeptide of the invention comprises a sequence having at least 98% similarity to the sequence of SEQ ID NO: 2. In some embodiments, a polypeptide of the invention comprises a sequence having at least 99% similarity to the sequence of SEQ ID NO: 2. In this embodiment, a polypeptide of the invention may be more stable than wild-type PDGFB (e.g., the polypeptide of SEQ ID NO: 1).

[0063] In a preferred embodiment, the polypeptide of the invention comprises a sequence having at least 92% similarity to the sequence of SEQ ID NO:2.

[0064] In a preferred embodiment, the polypeptide of the invention comprises a sequence having at least 95% similarity to the sequence of SEQ ID NO:2.

[0065] In some embodiments, a polypeptide of the present invention comprises the sequence of SEQ ID NO:3. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 80% similarity to the sequence of SEQ ID NO:3. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 85% similarity to the sequence of SEQ ID NO:3. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 90% similarity to the sequence of SEQ ID NO:3. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 92% similarity to the sequence of SEQ ID NO:3. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 95% similarity to the sequence of SEQ ID NO:3. In this embodiment, a polypeptide of the present invention may be more stable than wild-type PDGFB (e.g., the polypeptide of SEQ ID NO:1). In some embodiments, a polypeptide of the present invention consists of the sequence of SEQ ID NO:3.

[0066] In some embodiments, a polypeptide of the invention comprises a sequence having at least 96% similarity to the sequence of SEQ ID NO: 3. In some embodiments, a polypeptide of the invention comprises a sequence having at least 97% similarity to the sequence of SEQ ID NO: 3. In some embodiments, a polypeptide of the invention comprises a sequence having at least 98% similarity to the sequence of SEQ ID NO: 3. In some embodiments, a polypeptide of the invention comprises a sequence having at least 99% similarity to the sequence of SEQ ID NO: 3. In this embodiment, a polypeptide of the invention may be more stable than wild-type PDGFB (e.g., the polypeptide of SEQ ID NO: 1).

[0067] In a preferred embodiment, the polypeptide of the invention comprises a sequence having at least 92% similarity to the sequence of SEQ ID NO:3.

[0068] In a preferred embodiment, the polypeptide of the invention comprises a sequence having at least 95% similarity to the sequence of SEQ ID NO:3.

[0069] In some embodiments, a polypeptide of the present invention comprises the sequence of SEQ ID NO:4. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 80% similarity to the sequence of SEQ ID NO:4. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 85% similarity to the sequence of SEQ ID NO:4. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 90% similarity to the sequence of SEQ ID NO:4. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 92% similarity to the sequence of SEQ ID NO:4. In some embodiments, a polypeptide of the present invention comprises a sequence having at least 95% similarity to the sequence of SEQ ID NO:4. In this embodiment, a polypeptide of the present invention may be more stable than wild-type PDGFB (e.g., the polypeptide of SEQ ID NO:1). In some embodiments, a polypeptide of the present invention consists of the sequence of SEQ ID NO:4.

[0070] In some embodiments, a polypeptide of the invention comprises a sequence having at least 96% similarity to the sequence of SEQ ID NO: 4. In some embodiments, a polypeptide of the invention comprises a sequence having at least 97% similarity to the sequence of SEQ ID NO: 4. In some embodiments, a polypeptide of the invention comprises a sequence having at least 98% similarity to the sequence of SEQ ID NO: 4. In some embodiments, a polypeptide of the invention comprises a sequence having at least 99% similarity to the sequence of SEQ ID NO: 4. In this embodiment, a polypeptide of the invention may be more stable than wild-type PDGFB (e.g., the polypeptide of SEQ ID NO: 1).

[0071] In a preferred embodiment, the polypeptide of the invention comprises a sequence having at least 92% similarity to the sequence of SEQ ID NO:4.

[0072] In a preferred embodiment, the polypeptide of the invention comprises a sequence having at least 95% similarity to the sequence of SEQ ID NO:4.

[0073] The present invention further relates to fragments, analogs, and derivatives of the polypeptides of the invention, wherein the "fragment," "derivative," and "analog" retain essentially the same biological function or activity as the polypeptide set forth in any one of SEQ ID NOS: 2-4. As such, the fragment, analog, or derivative may have a melting temperature of at least 40°C, an EC50 that is equal to or lower than the EC50 of wild-type PDGFB (e.g., the polypeptide of SEQ ID NO: 1) when the EC50 of the fragment, derivative, or analog and the EC50 of the wild-type polypeptide are determined under the same conditions, and / or is soluble.

[0074] The polypeptides and polynucleotides of the present invention are preferably provided in an isolated form, and preferably are purified to homogeneity. "Isolated" means that the polypeptide or polynucleotide is separated from its natural environment.

[0075] In some embodiments, a polypeptide of the invention comprises a sequence that is the consensus sequence of the alignment shown in FIG.

[0076] In some embodiments, a polypeptide of the invention comprises a sequence that is the consensus sequence of the alignment shown in FIG.

[0077] In some embodiments, the consensus sequence comprises amino acid residues common to all of SEQ ID NOs: 2 to 4. In some embodiments, the consensus sequence comprises amino acid residues common to all of SEQ ID NOs: 2 to 4 that differ from SEQ ID NO: 1.

[0078] In some embodiments, a polypeptide of the invention comprises a sequence that is the consensus sequence of an alignment of one of Figures 1 or 2, and for a residue that is not common among all of SEQ ID NOs: 2-4, the polypeptide of the invention comprises one of the residues of one of SEQ ID NOs: 2-4 at that position.

[0079] In some embodiments of the invention, the minimum length of a polypeptide of the invention is 80% of the length of the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments of the invention, the minimum length of a polypeptide of the invention is 85% of the length of the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments of the invention, the minimum length of a polypeptide of the invention is 90% of the length of the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments of the invention, the minimum length of a polypeptide of the invention is 95% of the length of the wild-type PDGFB sequence of SEQ ID NO: 1.

[0080] In some embodiments of the invention, the maximum length of a polypeptide of the invention is twice the length of the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments of the invention, the maximum length of a polypeptide of the invention is 80% greater than the length of the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments of the invention, the maximum length of a polypeptide of the invention is 70% greater than the length of the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments of the invention, the maximum length of a polypeptide of the invention is 60% greater than the length of the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments of the invention, the maximum length of a polypeptide of the invention is 50% greater than the length of the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments of the invention, the maximum length of a polypeptide of the invention is 40% greater than the length of the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments of the invention, the maximum length of a polypeptide of the invention is 30% greater than the length of the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments of the invention, the maximum length of a polypeptide of the invention is 20% greater than the length of the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments of the invention, the maximum length of a polypeptide of the invention is 10% greater than the length of the wild-type PDGFB sequence of SEQ ID NO: 1.

[0081] In some embodiments, the polypeptides of the invention are the same length as the wild-type PDGFB sequence of SEQ ID NO:1.

[0082] In some embodiments, polypeptides of the invention have lengths from 50% shorter to 50% longer than the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have lengths from 40% shorter to 40% longer than the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have lengths from 30% shorter to 30% longer than the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have lengths from 20% shorter to 20% longer than the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have lengths from 10% shorter to 10% longer than the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have lengths from 5% shorter to 5% longer than the wild-type PDGFB sequence of SEQ ID NO: 1. In some embodiments, polypeptides of the invention have lengths from 2% shorter to 2% longer than the wild-type PDGFB sequence of SEQ ID NO: 1.

[0083] Nucleic acid sequences of the present invention In some embodiments, the present invention provides a nucleic acid sequence encoding any of the polypeptides disclosed herein.

[0084] In some embodiments, the present invention provides a nucleic acid sequence encoding a polypeptide of any one of SEQ ID NOs:2-4.

[0085] In some embodiments, the present invention provides a nucleic acid sequence having at least 80% identity to the nucleic acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the present invention provides a nucleic acid sequence having at least 85% identity to the nucleic acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the present invention provides a nucleic acid sequence having at least 90% identity to the nucleic acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the present invention provides a nucleic acid sequence having at least 92% identity to the nucleic acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the present invention provides a nucleic acid sequence having at least 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the nucleic acid sequences of the present invention encode a PDGF polypeptide having increased stability compared to the PDGFB polypeptide of SEQ ID NO: 1.

[0086] In some embodiments, the present invention provides a nucleic acid sequence having at least 96% identity to the nucleic acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the present invention provides a nucleic acid sequence having at least 97% identity to the nucleic acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the present invention provides a nucleic acid sequence having at least 98% identity to the nucleic acid sequence of any one of SEQ ID NOs: 5-7. In some embodiments, the present invention provides a nucleic acid sequence having at least 99% identity to the nucleic acid sequence of any one of SEQ ID NOs: 5-7.

[0087] In a preferred embodiment, the present invention provides a nucleic acid sequence having at least 92% identity to the nucleic acid sequence of any one of SEQ ID NOs: 5 to 7, which encodes a PDGF polypeptide having increased stability compared to the PDGFB polypeptide of SEQ ID NO: 1.

[0088] In a preferred embodiment, the present invention provides a nucleic acid sequence having at least 95% identity to the nucleic acid sequence of any one of SEQ ID NOs: 5 to 7, which encodes a PDGF polypeptide having increased stability compared to the PDGFB polypeptide of SEQ ID NO: 1.

[0089] The nucleic acid molecules of the present invention can be mRNA, DNA, cDNA or genomic DNA.

[0090] In some embodiments, the present invention provides a nucleic acid sequence that hybridizes to the complementary strand of the nucleic acid sequence of any one of SEQ ID NOs: 5-7.

[0091] In some embodiments, the present invention provides a nucleic acid sequence that is the reverse complement of a nucleic acid sequence of the present invention.

[0092] In some embodiments, the present invention provides a vector comprising a polynucleotide of the present invention.

[0093] In some embodiments, the invention provides cells comprising the polypeptides or nucleic acid sequences described herein. The cells may be genetically engineered to express the vectors of the invention.

[0094] In some embodiments, the cell is a bacterial cell, a yeast cell, a plant cell, an insect cell, or a mammalian cell. In some embodiments, the bacterial cell is an E. coli cell or a Corynebacterium glutamicum cell. In some embodiments, the cell is an E. coli cell. In some embodiments, the cell is a yeast cell.

[0095] In some embodiments, the present invention provides the use of a polypeptide described herein in a cell culture medium. In some embodiments, the cell culture medium is an animal cell culture medium. The cell culture medium may be serum-free.

[0096] A cell culture medium is a medium used for the survival, proliferation, and / or storage of cells. In some embodiments, the cell culture medium of the present invention is used for the culture of fibroblasts, myoblasts, adipocytes, mesenchymal stem cells, or iPSCs.

[0097] The cell culture medium of the present invention may additionally comprise one or more additional growth factors, serum or serum replacement, one or more hormones, one or more antibiotics, one or more trace elements and / or one or more antioxidants.

[0098] In some embodiments, the present invention provides a method for growing cells, the method comprising culturing the cells in a culture medium containing a polypeptide described herein. In some embodiments, the cells are animal cells. In some embodiments, the cell culture medium is an animal cell culture medium.

[0099] In some embodiments, the present invention provides methods for preparing the polypeptides described herein. In some embodiments, the methods include recombinant production and synthesis of the polypeptides described herein. In some embodiments, the methods are cell-free or cell-based methods.

[0100] In some embodiments, the method comprises culturing a cell described herein under conditions that allow for expression of a polypeptide described herein, and optionally recovering the expressed polypeptide.

[0101] In some embodiments, the method comprises cell-free protein synthesis.

[0102] In some embodiments, the method comprises the steps of: i) culturing a nucleic acid sequence of the invention in a lysate of a cell having ribosomal activity; and ii) synthesizing a protein encoded by the nucleic acid using a cell-free protein synthesis reaction.

[0103] Cell-free protein synthesis is described, for example, in Gregorio et al., "A User's Guide to Cell-Free Protein Synthesis," Methods Protoc., 2019, the contents of which are incorporated by reference.

[0104] It will be understood that the specific embodiments described herein are shown by way of illustration and not by way of limitation of the invention. The principal features of this invention can be employed in various embodiments without departing from the scope of the invention. Those skilled in the art will recognize, or be able to ascertain using no more than routine research, numerous equivalents to the specific procedures described herein. Such equivalents are considered to be within the scope of this invention and are covered by the claims. All publications mentioned in this specification are indicative of the level of skill of those skilled in the art to which this invention pertains.

[0105] All publications are herein incorporated by reference to the same extent as if each individual publication was specifically and individually indicated to be incorporated by reference.

[0106] The use of the word "a" or "an" when used in conjunction with the term "comprising" in the claims and / or this specification can mean "one," but is also consistent with the meanings of "one or more," "at least one," and "one or more than one." The use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer to alternatives only or the alternatives are mutually exclusive, in which case the present disclosure supports a definition that refers to alternatives only and "and / or."

[0107] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error for the subsequent characteristic.

[0108] As used in this specification and claims, the words "comprising" (and any form of comprising, e.g., "comprise" and "comprises"), "having" (and any form of having, e.g., "have" and "has", "including" (and any form of including, e.g., "includes" and "include")), or "containing" (and any form of containing, e.g., "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps.

[0109] The term "or combinations thereof," as used herein, refers to all permutations and combinations of the listed items preceding the term. For example, "A, B, C, or combinations thereof" is intended to include at least one of A, B, C, AB, AC, BC, or ABC, and, where order is important in the particular context, also BA, CA, CB, CBA, BCA, ACB, BAC, or CAB. Continuing with this example, combinations containing repeats of one or more items or terms are explicitly included, such as BB, AAA, MB, BBC, AAABCCCC, CBBAAA, and CABABB. Those of skill in the art will understand that the number of items or terms in any combination is typically not limited unless otherwise clear from the context.

[0110] Any part of this disclosure may be read in combination with any other part of this disclosure, unless the context makes clear otherwise.

[0111] All of the polypeptides, nucleic acids, and media disclosed and claimed in this application can be made and executed without undue experimentation in light of the present disclosure. While the present invention has been described in terms of preferred embodiments, it will be apparent to those skilled in the art that variations can be applied to the polypeptides, nucleic acids, media, and / or methods, and in the method steps or sequence of steps, described herein without departing from the concept, spirit, and scope of the invention. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope, and concept of the invention as defined by the appended claims.

[0112] The invention is described in more detail in the following non-limiting examples. [Example]

[0113] The following examples are useful in demonstrating the present invention.

[0114] Example 1 Polypeptide Variants and Alignments The following three variants are disclosed herein:

[0115] Variant 2 (SEQ ID NO: 2):

[0116] [ka]

[0117] Variant 3 (SEQ ID NO: 3):

[0118] [ka]

[0119] Variant 7 (SEQ ID NO: 4):

[0120] [ka]

[0121] The nucleic acid sequences corresponding to SEQ ID NOs: 2-4 (variants 2, 3 and 7) are provided as SEQ ID NOs: 5-7 and are shown below:

[0122] Variant 2 (SEQ ID NO: 5):

[0123] [ka]

[0124] Variant 3 (SEQ ID NO: 6):

[0125] [ka]

[0126] Variant 7 (SEQ ID NO: 7):

[0127] [ka]

[0128] Alignments were generated using the Clustal Omega Multiple Sequence Alignment tool from EMBL-EBI, version O (1.2.4), obtained from https: / / www.ebi.ac.uk / Tools / msa / clustalo / .

[0129] An alignment of the amino acid sequences of variants 2, 3, and 7 (SEQ ID NOs: 2 to 4) is shown in FIG.

[0130] An alignment of the amino acid sequences of variants 2, 3, and 7 (SEQ ID NOs: 2 to 4) and the wild-type PDGFB sequence (SEQ ID NO: 1) is shown in FIG.

[0131] A number of differences can be seen that all three variants share but differ from wild-type PDGFB, such as the I30V substitution.

[0132] Example 2 Polypeptide Expression By converting the primary amino acid sequence to DNA sequence via codon optimization, we generated plasmid inserts for each of the three variants of SEQ ID NOs: 2 to 4. These plasmids are shown in Figures 3A to 3C.

[0133] The insert containing the engineered variant was digested with BsaI and ligated into the pET-28a(+) backbone containing the modification, pGT94. E. coli strain BL21(DE3) was used for transformation with 5 μl of the reaction mix. Electroporation was performed using the protocol provided for the MicroPulser Electroporator (Bio-Rad). Single colonies were picked and sequences were verified via Sanger sequencing (Azenta).

[0134] An overnight culture was prepared using 10 ml of LB supplemented with 50 μg / ml kanamycin. The starter culture was diluted 1:100 into 50 ml of autoinducible expression medium (Auto Induction Media, Formedium) for 24 hours at 250 rpm and 30°C. The culture was pelleted at 10,000 x g for 10 minutes at 4°C and stored at -20°C.

[0135] Lysis was performed in buffer A (50 mM Tris, 300 mM NaCl, pH 8) supplemented with 5% glycerol, 1 mM DTT, and protease inhibitor cocktail (Roche). Sonication (Branson Ultrasonics) was performed at 45%, 10 s on / off. Crude lysates were pelleted at 10,000 × g for 30 min at 4°C.

[0136] The insoluble fraction of the crude lysate was resolubilized in Buffer D1 (100 mM NaH2PO4, 10 mM Tris, 8 M urea, 10 mM imidazole, 1 mM DTT, 0.05% Tween-20, pH 8) at 4°C. The crude lysate was clarified at 1000 x g for 5 min at 4°C. Ni-charged magnetic beads (Genescript) were pre-equilibrated with Buffer D1 and incubated with the clarified lysate at 4°C prior to affinity chromatography. The beads were washed twice with 7 column volumes using Buffer D2 (100 mM NaH2PO4, 150 mM NaCl, 8 M urea, 20 mM imidazole, pH 8), followed by five washes with 10 column volumes using Buffer D3 (50 mM NaH2PO4, 500 mM NaCl, 20 mM imidazole, pH 8). The protein was eluted by adding 2 column volumes of buffer E1 (50 mM NaHPO4, 500 mM NaCl, 250 mM imidazole, pH 8) and incubating therein for 5 min, followed by elution with 1.5 column volumes of E1. 10 μl of the eluate was analyzed by SDS-PAGE (40 mA, 60 min, Bis-Tris / MES). The process was performed on ice.

[0137] The results of SDS-PAGE are shown in Figure 4A.

[0138] Approximately 20 μg of protein was loaded onto a Bis-Tris gel (Genscript) and then blotted onto a nitrocellulose membrane (100 V, 1 hour, 4°C) using a Trans-Blot module (Bio-Rad). The membrane was blocked with TBS supplemented with 5% skim milk powder. Variants were detected using a 1:1000 dilution of mouse anti-His primary antibody (Biolegend) followed by a 1:2500 dilution of horseradish peroxidase (HRP)-conjugated goat anti-mouse secondary antibody (Biolegend). Bands were identified using ECL Western Blotting Substrate (Promega) according to the manufacturer's instructions.

[0139] The results of the Western blot are shown in Figure 4B.

[0140] As can be seen from Figures 4A and 4B, all variants are successfully expressed.

[0141] Example 3 Biological activity of polypeptides Bioactivity was assessed using the ONE-Glo luciferase assay (Promega). HEK293T cells were seeded at 5,000 cells / well in clear, flat-bottom 96-well plates and transfected with pGL4.33 (Promega) containing a serum response element (SRE) at a 2:1 ratio using Viafect transfection reagent (Promega) according to the manufacturer's instructions. Cells were starved overnight in DMEM-F12 supplemented with 0.5% FBS and 1% penicillin-streptomycin. Purified protein variant eluates were serially diluted in DMEM-F12 supplemented with 1% penicillin-streptomycin and treated for 6 hours with DMEM-F12 supplemented with 20% FBS + 20 ng / ml PMA as a positive control and DMEM-F12 as a negative control. One-Glo buffer and substrate were reconstituted and added to the samples according to the manufacturer's instructions. All experiments were performed in triplicate.

[0142] For accurate quantification, 10 μl of the diluted sample was diluted into 90 μl DMEM F12 and analyzed using a His-Tag ELISA Detection kit (Genscript) according to the manufacturer's instructions. The luminescence of the samples was measured. Relative luminescence units (RLU) were normalized using a negative control. Dose-response curves were plotted using Graphpad-Prism software, and EC50 values ​​were calculated.

[0143] The results are shown in Figures 5A to 5C.

[0144] As can be seen from the graph, all of the variants shown exhibit biological activity. The biological activity is comparable to that of wild-type PDGFB. Variant 3 exhibits higher affinity for the receptor (i.e., enhanced biological activity) compared to wild-type PDGFB.

[0145] Example 4 Polypeptide solubility - SDS-PAGE and Western blot

[0146] To assess solubility, 100 μl of crude and clarified lysates were collected before and after pelleting during the lysis process. Total protein content was quantified using a Bradford assay (Bio-Rad) using bovine serum albumin as a standard. 30 μg of protein was loaded per well and analyzed by Western blot. The ratio of soluble to total protein was calculated by quantifying the intensity of the bands in both fractions using ImageJ.

[0147] The ratio was calculated using the following formula:

[0148]

number

[0149] The results are shown in Table 1.

[0150] [Table 1]

[0151] A reading of 0 in Table 1 indicates a level above the detection limit.

[0152] Table 1 shows that all variants are soluble. All variants exhibit improved solubility compared to wild-type PDGFB, with variant 3 having the greatest solubility.

[0153] Example 5 Polypeptide thermal stability A trait predictor trained on a public dataset of PDGFB variants was used to predict the thermal stability of the polypeptide.

[0154] The results are shown in Table 2.

[0155] [Table 2]

[0156] All three variants also exhibit increased thermostability compared to wild-type PDGFB, with the greatest increases in thermostability seen in variants 3 and 7.

[0157] These data, taken together with the solubility and bioactivity data, indicate that the polypeptides having the sequences of SEQ ID NOs: 2-4 have improved properties.

[0158] Example 6 Stability - determined by measuring biological activity over time The biological activity of polypeptide variants can be assessed over time to determine stability, including thermal stability. This assay confirms that the variants retain an appropriate level of biological activity. Purified protein variants were added to DMEM-F12 supplemented with 1% penicillin and streptomycin. Protein variants were incubated at 37°C for 10 days, with samples collected every 24 hours. The biological activity of each sample was measured using the ONE-Glo luciferase assay (Promega) as previously described. The biological activity of each protein sample was compared to the wild-type protein incubated under identical conditions by generating an EC50 derived from a sigmoidal curve.

[0159] array

[0160] [Table 3]

Claims

1. A platelet-derived growth factor (PDGF) polypeptide comprising at least one modification selected from the group consisting of an amino acid substitution, an amino acid deletion, an amino acid insertion, and a combination thereof, compared to the sequence set forth in SEQ ID NO: 1, wherein the PDGF polypeptide exhibits increased stability, solubility, and / or biological activity compared to the PDGFB polypeptide of SEQ ID NO:

1.

2. 2. The PDGF polypeptide of claim 1, comprising the sequence of any one of SEQ ID NOs: 2-4, or a sequence having at least 80%, 85%, 90%, 92%, or 95% sequence similarity to any one of SEQ ID NOs: 2-4, and having a melting temperature of at least 40°C.

3. 3. The PDGF polypeptide of claim 1, comprising the sequence of SEQ ID NO: 2 or SEQ ID NO: 4 or a sequence having at least 80%, 85%, 90%, 92% or 95% sequence similarity to SEQ ID NO: 2 or SEQ ID NO: 4, and having a melting temperature of at least 40°C.

4. 3. The PDGF polypeptide of claim 1 or 2, comprising the sequence of SEQ ID NO: 2 or a sequence having at least 80%, 85%, 90%, 92% or 95% sequence similarity to SEQ ID NO: 2, and having a melting temperature of at least 40°C.

5. 3. The PDGF polypeptide of claim 1, comprising the sequence of SEQ ID NO: 3 or a sequence having at least 80%, 85%, 90%, 92% or 95% sequence similarity to SEQ ID NO: 3, and having a melting temperature of at least 40°C.

6. 3. The PDGF polypeptide of claim 1 or 2, comprising the sequence of SEQ ID NO: 4 or a sequence having at least 80%, 85%, 90%, 92% or 95% sequence similarity to SEQ ID NO: 4, and having a melting temperature of at least 40°C.

7. 3. The PDGF polypeptide of claim 1 or 2, comprising the sequence of SEQ ID NO: 2 or a sequence having at least 80%, 85%, 90%, 92% or 95% sequence similarity to SEQ ID NO: 2, and having more biological activity than the PDGFB polypeptide of SEQ ID NO:

1.

8. 3. The PDGF polypeptide of claim 1, comprising the sequence of SEQ ID NO: 3 or a sequence having at least 80%, 85%, 90%, 92% or 95% sequence similarity to SEQ ID NO: 3, and having more biological activity than the PDGFB polypeptide of SEQ ID NO:

1.

9. 3. The PDGF polypeptide of claim 1 or 2, comprising the sequence of SEQ ID NO: 4 or a sequence having at least 80%, 85%, 90%, 92% or 95% sequence similarity to SEQ ID NO: 4, and having more biological activity than the PDGFB polypeptide of SEQ ID NO:

1.

10. 10. The PDGF polypeptide of any one of claims 1 to 9, which is soluble and capable of binding to a platelet-derived growth factor receptor (PDGFR), optionally PDGFRB.

11. (a) a nucleic acid sequence encoding a PDGF polypeptide according to any one of claims 1 to 10; (b) a nucleic acid sequence having at least 80% similarity to the nucleic acid sequence of any one of SEQ ID NOs: 5 to 7 and encoding a PDGF polypeptide according to any one of claims 1 to 10; (c) a nucleic acid sequence that hybridizes to the complement of any one of the nucleic acid sequences of SEQ ID NOs: 5-7 and encodes a PDGF polypeptide of any one of claims 1 to 10; or (d) a nucleic acid sequence that is the reverse complement of a nucleic acid sequence defined in (a), (b), or (c). A nucleic acid comprising a sequence selected from the group consisting of:

12. 12. A cell comprising a PDGF polypeptide or nucleic acid sequence according to any one of claims 1 to 11.

13. 13. The cell of claim 12, which is a bacterial cell, a yeast cell, a plant cell, an insect cell or a mammalian cell.

14. 14. A method for preparing a PDGF polypeptide according to any one of claims 1 to 10, comprising culturing a cell according to claim 12 or 13 under conditions that allow expression of the polypeptide, and optionally recovering the expressed polypeptide.

15. 11. A method for preparing a PDGF polypeptide according to any one of claims 1 to 10, comprising the steps of: i) culturing a nucleic acid sequence according to claim 11 in a cell lysate having ribosomal activity; and ii) synthesizing a protein encoded by the nucleic acid using a cell-free protein synthesis reaction.

16. 11. A method for growing animal cells, comprising culturing said animal cells in a culture medium containing a PDGF polypeptide according to any one of claims 1 to 10.

17. 11. Use of a PDGF polypeptide according to any one of claims 1 to 10 in an animal cell culture medium.

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