RECOMBINANT PRODUCTION OF GROWTH FACTORS IN ALGAE FOR CELL CULTURE APPLICATIONS

Abstract

The present invention provides a method for producing recombinant growth factors using an algal expression system, which offers advantages over traditional platforms, and the algae-derived growth factors can be used to formulate cell culture media for mammalian cells without the risk of pathogen contamination or endotoxins.

Claims

1. A method of producing growth factors in an algal expression host, comprising: (a) constructing an expression vector comprising a nucleotide sequence encoding a growth factor selected from the group consisting of EGF, aFGF, bFGF and TGF superfamily proteins, operably linked to regulatory elements for expression in algae; (b) introducing said vector into an algal host cell to generate a transgenic algal line; (c) expressing the growth factor from the algal nuclear or chloroplast genome; and (d) purifying the recombinant growth factor from the algal biomass or culture medium.

2. The method of claim 1, wherein the algal host is selected from the group consisting of Chlamydomonas reinhardtii, Nannochloropsis sp., Dunaliella sp. and Haematococcus pluvialis.

3. The method of claim 1 or claim 2, wherein the growth factor is expressed from the chloroplast genome monocistronically or polycistronically.

4. The method of any one of claims 1-3, wherein the nucleotide sequence is a natural sequence or a synthetic codon-optimized sequence.

5. The method of any one of claims 1-4, wherein the vector further comprises a selectable marker gene.

6. The method of any one of claims 1-5, wherein the growth factor is of human, porcine, rat, mouse, feline, canine or equine origin.

7. The method of any one of claims 1-6, wherein artificial intelligence-assisted molecular design is used to enhance thermostability of the growth factor while maintaining its specific activity.

8. A cell culture medium for proliferation and/or differentiation of mammalian cells, comprising a recombinant growth factor produced by the method of any one of claims 1-7.

9. The cell culture medium of claim 8, comprising two or more different growth factors produced by the method of any one of claims 1-7.

10. The cell culture medium of claim 8, further comprising one or more components selected from the group consisting of VEGF, PDGF, FGF-4, FGF-6, TGFs-b, TGF-a, Epo, IGF-I, IGF-II, IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-13, IL-15, IL-18, IL-20, TNF-a, TNF-b, INF-g, G-CSF, GM-CSF, M-CSF, PLGF, NGF, KGF, BMP-4, HGF, leptin, noggin, and thymosin beta 4.

11. The cell culture medium of claim 9, wherein the mammalian cells are stem cells, fibroblasts, keratinocytes or endothelial cells.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] FIG. 1 shows an expression vector for producing human EGF from the chloroplast genome.

[0017] FIG. 2 shows an expression vector for polycistronic production of human aFGF from the chloroplast genome.

[0018] FIG. 3 shows an expression vector for secretory production of bFGF from the nuclear genome.

[0019] FIGS. 4-6 show data demonstrating that algae-produced aFGF promotes growth of human induced pluripotent stem cells comparably to bFGF without affecting pluripotency marker expression.

[0020] FIG. 7 compares the performance equivalence between algal-derived wildtype bFGF and commercial bFGF in stimulating the proliferation of Mesenchymal Stem Cells (MSCs).

[0021] FIG. 8 demonstrates the improved stability of bFGF when applied to Induced Human Pluripotent Stem Cells (iPSCs) and the heat treatment experiment on rat fibroblast cells.

[0022] FIGS. 9A, 9B, and 9C show that the engineered bFGF exhibits significant thermostability, retaining over 50% of its activity even after three days of pre-treatment at 37 C. In contrast, wildtype bFGF loses its entire activity under the same conditions.

[0023] FIG. 10 emphasizes the detrimental impact of endotoxins on the health of animal cells during culture and the lack of endotoxins in the algae-derived growth factors produced by the claimed method of this invention.

[0024] FIG. 11 shows the successful recombinant expression of TGF superfamily proteins from the algal nuclear genome.

DETAILED DESCRIPTION

[0025] The present invention provides methods for producing recombinant growth factors in algae. The general methodology involves: 1) constructing expression vectors encoding growth factors; 2) introducing the vectors into an algal host; 3) expressing the protein from the algal nuclear or chloroplast genome; and 4) purifying the recombinant protein.

[0026] Any protein or peptide that can be encoded by a DNA sequence can be expressed using this system. Suitable algal hosts include, but are not limited to, Chlamydomonas reinhardtii, Nannochloropsis sp., Dunaliella sp. and Haematococcus pluvialis. The gene of interest can be a natural sequence or a synthetic, codon-optimized sequence. Expression can be monocistronically or polycistronically from the chloroplast genome.

[0027] Specific examples are provided for producing PTD-fused human EGF, aFGF, bFGF and TGF superfamily proteins. EGF is expressed monocistronically from the chloroplast genome (FIG. 1). aFGF is expressed polycistronically from the chloroplast genome using repeated coding sequences separated by ribosome binding sites (FIG. 2). bFGF is expressed from the nuclear genome and secreted into the culture medium using a native secretion signal (FIG. 3). Selectable markers such as aadA and aph VIII are included for generating stable transgenic lines.

[0028] Artificial intelligence-assisted molecular design is used to enhance the thermostability of bFGF while maintaining its specific activity. Through molecular dynamics simulations and experimental validation, specific residues and regions contributing to protein flexibility are identified. The resulting bFGF variants exhibit improved thermostability, enabling the development of novel stem cell culture protocols that require less frequent medium changes (FIGS. 5-6).

[0029] The algae-produced growth factors can be further engineered to enhance membrane permeability and thermostability. They can be used individually or in combination in mammalian cell culture media at desired concentrations. Data is presented showing that algae-produced growth factors promote the growth of human induced pluripotent stem cells and mesenchymal stem cells comparably to commercial products (FIGS. 4-5).

[0030] The advantages of the algae-based system include great scalability, lack of pathogen and endotoxin contamination, high yields from polycistronic chloroplast expression, and cost-effectiveness. It provides a novel platform for the large-scale production of recombinant growth factors for cosmetic and cell culture applications.

EXAMPLES

Example 1: Production of EGF in Chlamydomonas reinhardtii Chloroplast

[0031] The coding sequence for human EGF was cloned into the expression vector pEGFCh1 (FIG. 1). The vector contains the aadA gene for spectinomycin resistance. C. reinhardtii cells were transformed with pEGFCh1 by biolistic bombardment and transgenic lines were selected on spectinomycin-containing media. Integration of the transgene into the chloroplast genome was confirmed by PCR. EGF was expressed monocistronically under the control of chloroplast regulatory elements. The recombinant protein was purified from the algal biomass by chromatography methods.

Example 2: Production of aFGF in Chlamydomonas reinhardtii Chloroplast

[0032] The coding sequence for human aFGF was cloned into the expression vector paFGFCh1 (FIG. 2). The vector contains four repeats of the aFGF sequence separated by chloroplast ribosome binding sites for polycistronic expression. The aadA gene was included for selection. Chlamydomonas cells were transformed with paFGFCh1 by electroporation. Transgenic lines were selected and confirmed as in Example 1. aFGF was purified from the algal biomass.

Example 3: Production of Secreted bFGF in Chlamydomonas reinhardtii Nucleus

[0033] The coding sequence for human bFGF and a native secretion signal was cloned into the expression vector pbFGFNuc (FIG. 3). The vector contains the aph VIII gene for paromomycin resistance. C. reinhardtii cells were transformed with pbFGFNuc by glass bead agitation. Transgenic lines were selected on paromomycin-containing media and confirmed by PCR. bFGF was secreted into the culture medium and purified by chromatography.

Example 4: Use of Algae-Produced aFGF in Human Stem Cell Culture

[0034] Algae-produced aFGF from Example 2 was tested for its ability to support the growth of human induced pluripotent stem cells (hiPSCs). hiPSCs were cultured in E8 medium supplemented with either bFGF (control) or aFGF at different concentrations. After 4 days of culture, aFGF and bFGF resulted in similar cell growth (FIG. 4). Over multiple passages, aFGF at 100 ng/ml gave the highest hiPSC yield (FIG. 5). RT-PCR analysis showed that aFGF-cultured hiPSCs expressed the pluripotency markers Oct4, Nanog and Sox2 at levels comparable to bFGF-cultured cells (FIG. 6). This demonstrates that algae-produced aFGF can be used as an alternative to mammalian bFGF in stem cell culture media.

Example 5: Production of TGF Superfamily Proteins from Chlamydomonas Nuclear Genome

[0035] The coding sequences for human TGF-1, BMP-2 and BMP-7 were cloned into algal nuclear expression vectors. Chlamydomonas reinhardtii cells were transformed and transgenic lines were selected.

[0036] FIG. 7 compares the performance equivalence between algal-derived wildtype bFGF with commercial bFGF in stimulating the proliferation of Mesenchymal Stem Cells (MSCs). FIG. 8 demonstrates the improved stability of bFGF when applied to Induced Human Pluripotent Stem Cells (iPSCs), similar to the heat treatment experiment on rat fibroblast cells displayed in FIG. 9A-C. The engineered ebFGF exhibits significant thermostability, retaining over 50% of its activity even after three days of pre-treatment at 37 C. In contrast, wildtype bFGF loses its entire activity under the same conditions. Moreover, FIG. 10 emphasizes the detrimental impact of endotoxins on the health of animal cells during culture. The algae-derived growth factors produced from algae using the claimed method of this invention lack endotoxins and support distinct cell morphology compared to bacterial-derived counterpart. The colorimetric western blot result of FIG. 11 shows the representative complex growth factors produced by the method of this invention in algae. Primary antibody against HA epitope was used.

[0037] The above examples illustrate the production and application of algae-derived growth factors. The products are free of contaminating pathogens and endotoxins, making them safe and reliable for cosmetic and cell culture uses. The expression vectors and methods are applicable to producing other growth factors and cytokines of interest from algae.

[0038] All publications, patents, and patent applications cited herein are incorporated by reference in their entirety as if each individual publication, patent or patent application were specifically and individually indicated to be incorporated by reference. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention.

[0039] It should be understood that the foregoing relates only to the exemplary embodiments of the present invention and that numerous changes may be made therein without departing from the scope of the invention as defined by the following claims.