Genetically engineered yeast cell, recombinant protein produced by genetically engineered yeast cell and application thereof

Abstract

The present invention provides a method for increasing cell growth and protein expression in in vitro cell culture using recombinant growth factors produced by genetically engineered yeast strains. A recombinant growth factor is introduced into a culture medium of a cell to be cultured, thereby increasing the growth rate or protein expression rate of the cultured cell. In one embodiment, recombinant growth factors are used that (i) genetically the same or similar species as the cultured cell and / or (ii) genetically the same genus as the cultured cell. In another embodiment, recombinant growth factors having engineered amino acid sequences are used to improve stability, biological activity, or other characteristics that promote cell growth and / or protein expression of the cultured cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. non-provisional patent application serial number 17 / 831,351, filed on June 2, 2022, which is a continuation-in-part application PCT / IB2020 / 061200, filed on November 27, 2020, which claims priority to U.S. provisional patent application serial number 62 / 942,568, filed on December 2, 2019. This application also claims priority to U.S. provisional patent application serial number 63 / 423,488, filed on November 7, 2022. All of the above applications are hereby incorporated by reference in their entirety. Technical Field

[0003] Embodiments discussed herein generally relate to genetically engineered strains of yeast and improved methods for producing recombinant proteins, cell products or cultured meat. Embodiments discussed herein also generally relate to improved methods for cell growth and protein expression using growth factors. Background Art

[0004] Animal meat is high in protein, providing all the amino acids needed to build proteins used to support body functions. Edible meat has traditionally been obtained from farm-raised animals or fish. However, agriculture and aquaculture used to produce animal meat require a lot of energy and resources and have a high carbon footprint. Meat produced through agriculture or aquaculture can pose a public health risk because the production method can expose meat to diseases, contaminants and toxins. Many issues such as population growth, increasing demand for meat, environmental concerns, limited land and water resources, loss of biodiversity, and negative perceptions associated with animal slaughter have prompted scientists to develop technologies to produce meat through alternative methods.

[0005] In vitro meat production is the process of using cell culture technology to cultivate animal muscle tissue or organ tissue in the laboratory to make meat and meat products. In addition to foods such as meat, in vitro cell culture can also be used to produce the active ingredients of various products or the products themselves (including but not limited to dietary supplements, hair care, skin care, wound care, cosmetics, supplements, drugs and other medicinal applications). In vitro meat and meat products as used herein include animal protein products and non-meat products including soluble forms and solid forms. Although still in the early stages of development, in vitro meat and meat products can provide many advantages over traditional meat products, such as health and environmental advantages, and benefits to animal welfare. This is the next generation of emerging technology, which is part of the broader field of cell agriculture or manufacturing agricultural products from cell cultures.

[0006] The cells used to produce in vitro meat can be cells obtained from animal biopsies (e.g., muscle cells, somatic cells, stem cells, etc.), which can then be separated from the animal and grown in a culture medium in a bioreactor or other type of sterile controlled environment. The cells can be grown into a semi-solid or solid that simulates an animal organ by attaching to an edible three-dimensional scaffold placed in a bioreactor. The starting cells can be primary cells directly obtained from animal tissues, or continuous cell lines. If grown under the right conditions in an appropriate culture medium, primary cells will grow and proliferate, but can only grow and proliferate a limited number of times related to the telomere length at the end of the cell DNA. On the other hand, continuous cell lines can be cultured in vitro for a long time. Cell biology research has established procedures for converting primary cells into immortal continuous cell lines. Primary cells can be converted into continuous cell lines by preventing telomere shortening using viral oncogenes, chemical treatments, or overexpression of telomerase reverse transcriptase.

[0007] The culture medium may contain components necessary for cell proliferation, such as amino acids, salts, vitamins, growth factors, and buffer systems for controlling pH. Current methods generally add exogenous animal products, such as fetal bovine serum (FBS) or recombinant mammalian growth factors or cytokines, to the culture medium before use to provide important macromolecules, growth factors, and immune molecules that maintain cell proliferation and increase cell yield. However, this approach has several disadvantages. First, growing cells in a culture medium without animal components is an important factor to consider for scientists engaged in in vitro meat production research. FBS comes from unborn calves and is therefore inconsistent with the goal of being animal product-free.

[0008] Second, typically more than 95% of the media cost is attributed to protein components. Recombinant human growth factors (e.g., insulin growth factor 1 (IGF-1)), human serum albumin (HSA), and fetal bovine serum (FBS) are usually of research or pharmaceutical grade and are often added to the basal media in excess, which increases the media cost and is unnecessary in most industrial applications.

[0009] Third, while these supplements are effective in promoting the growth and differentiation of human cells, they have low bioactivity against cross-species cells (i.e., cells from distant species, such as fish and birds). This results in long culture cycles and low cell quality. To compensate for the low bioactivity of non-human cells, excessive levels of human protein factors or FBS are added to the growth medium, which leads to high costs. Due to limited commercial sources of non-mammalian growth factors, in vitro cell culture of non-mammalian cells (e.g., fish) is worse than that of mammalian cells. The use of mammalian growth factors to promote the growth of non-mammalian cells is ineffective because mammalian growth factors may not bind to or act poorly on cell receptors on non-mammalian cells; products manufactured in this way may also raise consumer concerns about safety and potential allergic reactions.

[0010] Fourth, most industry practices use natural growth factors to culture cells, which can be inefficient because natural growth factors may be unstable or have low inherent biological activity in their natural form. Therefore, large doses or frequent feeding of growth factors are required to compensate for these problems.

[0011] The above-mentioned deficiencies not only lead to high in vitro meat production costs, but also increase the health risks of consumers of the final product. In order to address such deficiencies and provide an improved method for increasing cell growth and protein expression in in vitro cell culture in a more economical and efficient but simpler manner, the present invention provides a novel yeast strain that can produce recombinant natural or modified growth factors to enhance cell growth. In some embodiments, the recombinant growth factor is introduced into the culture medium of the cells to be cultured, thereby increasing the growth rate or protein expression rate of the cultured cells.

[0012] In some embodiments, the recombinant growth factor to be supplemented into the cultured cells has a similar species origin as the cultured cell type and / or its amino acid sequence is modified to have better biological activity and stability than the native sequence.

[0013] By providing growth factors of similar species origin to the cultured cell type and / or modified growth factors with improved biological activity and stability characteristics, the present invention significantly reduces the amount of growth factors that need to be supplemented and the risks to consumers. The present invention does not require a complex setup, and the products produced using the present invention can be produced on a large scale and are easy to use in most applications without the need for additional cumbersome purification procedures. In addition, the yeast strains of the present invention can be used to produce a variety of mammalian and non-mammalian growth factors, so their application range is wide. In general, the present invention provides novel yeast strains and improved recombinant protein production methods that promote cell growth in in vitro culture and can produce in vitro meat products and other types of cell products at a lower cost and in a simplified manner. The resulting products are suitable for a variety of applications, including cell agriculture, cosmetics, pharmaceuticals, and biotechnology. Summary of the invention

[0014] Embodiments of the present disclosure provide genetically engineered strains of yeast and improved methods for increasing cell growth and protein expression in in vitro cell culture.

[0015] The present invention aims to provide novel genetically engineered yeast strains and an alternative method to produce recombinant proteins, such as recombinant growth factors for in vitro cell culture. In some embodiments, the recombinant growth factors are introduced into the culture medium of the cells to be cultured to increase the growth rate or protein expression rate of the cultured cells.

[0016] It is an object of the present invention to provide an alternative method for culturing cells using species-specific or genus-specific growth factors. This method not only reduces the cost of the culture medium by reducing the amount of growth factor used, but also shortens the culture time and improves the quality of the cells by enhancing the cell response. The use of species-specific or genus-specific growth factors can help to enhance the cell response (e.g., growth and differentiation) and break the maximum cell response encountered when using non-species-specific or non-genus-specific growth factors. Another object of the present invention is to provide an alternative method for culturing cells using growth factors that have modified amino acids for improved stability and / or biological activity compared to the use of natural growth factors.

[0017] The object of the present invention is to provide a method for producing a recombinant growth factor that can be used for both non-mammalian and mammalian cell culture; for example, the resulting recombinant growth factor is derived from fish and can be used to culture fish cells or cells of related species. Another object of the present invention is to provide a method for producing a recombinant natural growth factor or a modified growth factor whose amino acid sequence has been modified to improve stability, biological activity, or other properties that promote cell growth and / or protein expression in cultured cells. Since growth factors of different species and amino acid sequences can vary greatly in their efficacy in stimulating cell growth, cell culture users can use the present invention to generate different growth factor constructs and evaluate their efficacy, and select the construct that the relevant cell type responds most to a specific application. In addition, the cell products produced using the present invention have a purity that is generally suitable for most types of applications and can be easily used without complex purification procedures. In some embodiments where a higher purity is required for specific applications such as pharmaceutical or medical applications, the resulting in vitro cell product can be further purified using conventional purification techniques. In summary, the application of the present invention not only provides flexibility for its application, but also significantly reduces the production cost of large-scale culture.

[0018] According to one embodiment of the present disclosure, a genetically modified yeast cell encoding a recombinant growth factor is provided. The recombinant growth factor may be a natural amino acid sequence or a modified amino acid sequence, and the modified amino acid sequence has improved stability and / or biological activity compared to the natural form.

[0019] According to one embodiment of the present disclosure, a method for increasing the growth or protein expression rate of multiple cells in in vitro cell culture is provided. The method comprises the following steps: culturing multiple cells in a nutrient medium under growth conditions suitable for initiating cell division, and introducing at least one recombinant growth factor into the nutrient medium. In some embodiments, the recombinant growth factor (a) is optimized to increase the growth or protein expression rate of multiple cells, or (b) (i) is a species that is genetically identical or similar to the multiple cells and / or (ii) is genetically identical to the multiple cells.

[0020] According to one embodiment of the present disclosure, a method for producing meat by in vitro cell culture includes isolating tissue from an animal or plant source and preparing a cell suspension. The method also includes introducing a growth factor, wherein the growth factor (a) is optimized to increase the growth or protein expression rate of multiple cells or (b) (i) is the same or similar species as the cell gene and / or (ii) is the same genus as the cell gene. In addition, the method also includes culturing cells on a food-grade scaffold in a culture medium, and the cells grow into a solid or semi-solid structure that simulates an animal organ.

[0021] In further embodiments, growth factors that support cell growth may be produced by other microorganisms such as bacteria or fungi that contain and express a growth factor transgene.

[0022] In view of the complex and expensive process involved in purifying expressed growth factors from microorganisms, embodiments of the present invention develop a method for culturing cells using growth factors directly from yeast culture without complicated purification procedures. In some embodiments, the recombinant growth factor for introduction into in vitro culture is produced by genetically modified yeast cells containing recombinant growth factor genes and obtained from cell culture medium in which the genetically modified yeast cells are cultured. In one embodiment, the recombinant growth factor is purified from the yeast cell culture medium before introduction into in vitro culture. In another embodiment, the recombinant growth factor is introduced into the in vitro culture by introducing the yeast cell culture medium containing the growth factor into the in vitro culture.

[0023] Aspects of the present invention can be genetically engineered to yeast clones containing the IGF-1 gene of a particular species (e.g., fish IFGF-1 gene). In one aspect, recombinant yeast can be grown in yeast culture medium to allow protein expression of the IGF-1 gene. In one aspect, IGF-1 can be secreted into the yeast culture medium. In another embodiment, the yeast culture medium containing IGF-1 can be centrifuged at about 1500g for 5 minutes to separate the yeast from the culture medium. The supernatant can be collected and centrifuged again at about 7200g for about 15 minutes at about 4°C. The clarified supernatant can then be filtered with a filter of 2 μm or less pore size. The filtered yeast culture medium containing IGF-1 can be diluted up to 10,000 times and then added to a cell culture medium to culture fish cells or other types of cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] The present disclosure may be better understood by referring to the detailed description in conjunction with the accompanying drawings, in which the components are not necessarily drawn to scale, emphasis instead being placed upon illustrating the principles of the present disclosure.

[0025] Figure 1 A flow chart showing a method for producing meat by in vitro cell culture according to one embodiment of the present disclosure.

[0026] Figure 2 The graphs show the changes in cell number after MCF-7 cells were treated with different concentrations (1 pg / ml to 100 ng / ml) of recombinant human IGF-1 (Oryzogen). The cells harvested on day 10 were directly counted.

[0027] Figure 3Graph showing relative fluorescence of MCF-7 cells after treatment with 15 nM recombinant human IGF-1 (from 3 different suppliers), recombinant mouse IGF-1 and recombinant fish (tuna, sea bream) IGF-1, respectively. Cells were harvested on day 7 and subjected to the CyQUANT cell proliferation assay.

[0028] Figure 4 Relative fluorescence of swim bladder cells after treatment with 10 nM of three different recombinant fish IGF-1 clones (fish IGF-1 (A), IGF-1 (B) and IGF-1 (C)) is shown. Cells were harvested on day 3 and subjected to the CyQUANT cell proliferation assay.

[0029] Figure 5 Shown is a graph showing the relative fluorescence of swim bladder cells after treatment with 1% of three different recombinant fish IGF-1 in yeast medium. Cells were harvested on day 3 and subjected to the CyQUANT cell proliferation assay.

[0030] Figure 6 Shown is a Western blot analysis of recombinant native and engineered grouper IGF-1 in yeast culture using an anti-6X His antibody.

[0031] Figure 7 Shown is the characterization of recombinant native and engineered grouper IGF-1 purified from yeast culture using immobilized metal affinity chromatography. Panel (a) shows analysis by total protein staining and panel (b) shows analysis by Western blotting using an anti-6X His antibody.

[0032] Figure 8 Two graphs are shown, illustrating the results of resazurin assay of (a) MCF-7 cells and (b) FHM cells treated with 0 nM, 0.1 nM, 1 nM and 10 nM of purified grouper IGF-1, respectively.

[0033] Fig. 9 Two graphs are shown illustrating the results of the resazurin assay for (a) MCF-7 cells and (b) FHM cells treated with 1:100 and 1:1000 dilutions of yeast culture containing grouper IGF-1, respectively. DETAILED DESCRIPTION

[0034] Referring now to the drawings, and in particular to Figure 1, an in vitro meat production method 10 is shown. As used herein, "in vitro meat production" refers to a cell-based meat production process or a cell-based agricultural process in which tissues from animals and / or plants are cultured in a laboratory using cell culture technology to make meat and meat products. In block 12, tissues from animals or plants are separated. In one embodiment, the tissue is from a bony fish of the class Osteichthyes, which includes saltwater fish such as grouper, sea bass, or yellow croaker. In other embodiments, other types of animal tissues (e.g., bovine tissue) may be separated. In some embodiments, block 12 may involve collecting organ tissues, such as fish bladders, from fish and making a cell suspension. Although the following description primarily describes tissues from fish sources, it should be understood that these concepts can be applied to tissues from other types of animal sources and / or plant sources to provide other types of in vitro meat and / or animal protein products, as well as vegetarian meat and / or protein products.

[0035] Many of the isolated cells are adult cells, and various methods established in medical research can be used to continuously proliferate them (block 14). For example, specific genes, such as Yamanaka factors, can be used to reprogram adult cells into stem cells, such as induced pluripotent stem cells (iPSCs). Alternatively, isolated adult cells can be converted into continuous cell lines by overexpression of telomerase reverse transcriptase. In other embodiments, other types of cells, such as adult stem cells and embryonic stem cells, can be isolated. In this regard, it should be understood that the methods of the present disclosure include cell lines of all sources.

[0036] In the next block 16, cells are grown as solid or semi-solid structures simulating animal organs (such as fish organs) by attaching / attaching to food-grade biocompatible scaffolds in sterile chambers or containers (such as bioreactors). The sterile chamber or container can be temperature-controlled and can have inlets and outlets for introducing and removing substances, such as chemicals, nutrients and cells. The food-grade biocompatible scaffold becomes part of the final edible product and is made of plant-based or fungus-based materials (such as but not limited to agarose, alginate, chitosan, mycelium and konjac glucomannan). Alginate is a biopolymer naturally derived from brown algae and has biocompatibility. In addition, plant-based chitosan from fungi has antibacterial properties. In some embodiments, block 16 is performed in the sterile container without antibiotics or antimicrobial compounds. Block 18 involves supplying culture medium to the bioreactor to support cell survival and growth. The culture medium may be a buffer solution containing, for example, but not limited to, the following components: inorganic salts (e.g., calcium chloride (CaCl2), potassium chloride (KCl), sodium chloride (NaCl), sodium bicarbonate (NaHCO3), sodium dihydrogen phosphate (NaH2PO4), magnesium sulfate (MgSO4), etc.), amino acids, vitamins (e.g., thiamine, riboflavin, folic acid, etc.), and other components such as glucose, β-mercaptoethanol, ethylenediaminetetraacetic acid (EDTA), and sodium pyruvate. Non-limiting examples of growth medium include, but are not limited to, Leibovitz's L-15 medium, Eagle's minimum essential medium (MEM), medium 199, Dulbecco's modified Eagle medium (DMEM), Ham's F12 nutrient mixture, Ham's F10 nutrient mixture, MacCoy's 5A medium, Glasgow modified Eagle medium (GMEM), Iscove's modified Dulbecco's medium, and RPMI 1640.

[0037] According to block 20, food-grade growth factors and cytokines are introduced into the culture medium in the bioreactor to support cell growth and proliferation. The growth factors and cytokines may include, but are not limited to, insulin growth factor 1 (IGF-1), insulin, interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), interleukin 11 (IL-11), fibroblast growth factor (FGF), epidermal growth factor (EGF), and transferrin. In some embodiments, the present invention uses growth factors of (i) the same or similar species as the isolated / cultured cells and / or (ii) the same genus as the isolated / cultured cells (i.e., cells grown in block 16). It has been found that the use of growth factors of (i) the same or similar species as the isolated / cultured cells and / or (ii) the same genus as the isolated cells has higher biological activity on the isolated cells than the use of growth factors and serum of species distantly related to the isolated cells. Due to the enhanced compatibility, it is not necessary to provide large doses of "suboptimal" growth factors when culturing these cells using growth factors from (i) genetically identical or similar species and / or (ii) genetically identical genus. The higher bioactivity can also help reduce the amount of growth factor required in the culture medium, shorten the culture period and improve cell quality. The cost of the culture medium can be reduced because lower levels of growth factors in the culture medium are required to stimulate cell growth and differentiation.

[0038] Additionally, the use of suboptimal growth factors can limit the magnitude of the maximal cellular response (growth, differentiation). In some cases, certain responses cannot be achieved no matter how much suboptimal growth factor is provided. Species-specific and / or genus-specific growth factors can help overcome these limitations.

[0039] Species-specific or genus-specific growth factors can effectively act on the receptors of isolated cells. The use of species-specific or genus-specific growth factors is more effective than traditional growth media, which are usually supplemented with high levels of human protein growth factors and / or FBS, regardless of the species of the isolated cells. Species-specific or genus-specific amino acid sequence changes and post-translational modifications of growth factors and cell receptors may explain this phenomenon.

[0040] As will be discussed in more detail in Example 1 below, in order to determine which species of specific growth factors exert the highest biological activity on the target cells to be cultured, the target cells are first inoculated in complete medium (i.e., basal medium + FBS). After reaching the target confluence (about 20%-70%), the target cells are treated with growth factors of different species at a certain concentration range (e.g., 1pM-1μM). The target cells are kept in an incubator until the time point required for the study parameters (e.g., cell growth, differentiation markers, cell products) is reached. For example, when there is a difference in cell confluence between experimental groups (about 2-10 days), cell growth can be measured by trypan blue exclusion, CyQUANT assay, or any other appropriate cell proliferation / death assay. Based on the EC50 value (half-maximal effective concentration) of the growth factor, the biological activity of growth factors of various species is compared. For cost-effectiveness, the growth factor with the lowest EC50 value should be used to culture the target cells. However, if the goal is to achieve the shortest culture time or the highest cell quality, it is best to select the growth factor that triggers the highest maximum cell response. The optimal dose of a growth factor is defined as the lowest concentration required to elicit a maximal cellular response.

[0041] In some embodiments, block 20 may involve co-culturing the bioengineered cells with the isolated / cultured cells in the absence of fetal bovine serum (FBS). The bioengineered cells are engineered to secrete the above-mentioned growth factors and cytokines, and supply these biomolecules to the isolated cells according to the needs of growth and proliferation. "Bioengineered" cells as used herein are not equivalent to genetically modified cells. Bioengineered cells have specific genes that overexpress one or more specific proteins. Bioengineered cells can be fish cells, or other types of animal cells, such as cattle cells. Bioengineered cells and isolated cells can be genetically similar or identical species. In addition, bioengineered cells and isolated cells can belong to the same genus. As a non-limiting example, bioengineered fish cells can be co-cultured with isolated fish cells, or bioengineered cattle cells can be co-cultured with isolated cattle cells. In some specific examples, if chicken cells are used as isolated cells, the bioengineered cells can be chicken cells or bird cells. In another specific example, if yellow croaker cells are used as isolated cells, the bioengineered cells can be yellow croaker cells or other fish cells. Bioengineered cells are not present in the final meat product. The co-cultivation method disclosed herein eliminates the need for animal-derived fetal bovine serum (FBS) in the culture medium. In addition, the co-cultivation method continuously supplies food-grade specific growth factors and cytokines to the isolated cells growing in situ, and simplifies the production process and reduces the cost of the production process, wherein the growth factors (i) are of the same or similar species as the isolated cells and / or (ii) belong to the same genus as the isolated cells. However, in other embodiments, FBS or other serum or other products containing recombinant growth factors can be used to supply growth factors, cytokines and other nutrients to support cell growth during block 16.

[0042] In some embodiments, block 20 comprises a recombinant growth factor of a genetically identical or similar species to the isolated cells. In yet other embodiments, a recombinant growth factor of the same genus as the isolated cells is used. In some embodiments, the recombinant growth factor is introduced into a growth medium in a purified form or a crude purified form, such as a yeast culture (or a supernatant of a yeast culture) obtained from a recombinant yeast cell culture expressing the recombinant growth factor. Compared with the effects of distant growth factors and serum on isolated cells, such recombinant growth factors are used to exert higher biological activity on isolated cells. Such recombinant growth factors can be produced using bacteria, yeast, insects, mammals or any other appropriate protein expression system. Protein purification is carried out by, but is not limited to, affinity chromatography, ion exchange chromatography, size exclusion chromatography or a combination of these strategies.

[0043] It is expected that the recombinant growth factor produced using the present invention can be used to support or promote the growth of various cells and cells from various sources. In some embodiments, the recombinant growth factor is a recombinant fish growth factor, which can be used to support the growth of fish cells. For example, a recombinant growth factor of a species Epinephelus akaara that is genetically similar to fish muscle cells or swim bladder cells of Epinephelus akaara is used. The recombinant growth factor used to cultivate Epinephelus akaara is IGF-1, insulin and / or transferrin of Epinephelus akaara. The concentration range of the IGF-1 is 10ng / ml to 100ng / ml. The concentration range of the insulin is 1μg / ml-10μg / ml. The concentration range of the transferrin is 0.5μg / ml-5μg / ml.

[0044] In some embodiments, the recombinant fish growth factor is used to support or promote the growth of non-fish cells (eg, human cells or other mammalian cells). In yet another embodiment, the recombinant fish growth factor is used to support or promote the growth of non-mammalian cells other than fish cells.

[0045] In some embodiments, the recombinant growth factor is a recombinant bovine growth factor, which can be used to support the growth of bovine cells, other mammalian cells, or non-mammalian cells.

[0046] In some embodiments, the recombinant growth factor is a recombinant avian growth factor, which can be used to support the growth of avian cells, other non-mammalian cells, or mammalian cells.

[0047] Alternatively, according to block 22, protein expression in cells can increase the biomass yield in the resulting meat product. As used herein, "biomass yield" refers to the amount of digestible material (e.g., protein) in the resulting meat product, which can be used to generate energy after consumption. Similarly, as an optional feature, block 22 can involve increasing protein expression by changing the microRNA level in the cell, wherein the cell is operated before cultivation. MicroRNA is an endogenous, short, non-coding single-stranded RNA sequence that participates in regulating post-transcriptional gene expression. Block 22 can optionally involve increasing the amount of up-regulated microRNA that increases protein expression by promoting messenger RNA (mRNA) translation, and / or reducing the amount of down-regulated microRNA that reduces protein expression by inhibiting mRNA translation. MicroRNA levels can be increased or reduced by introducing microRNA, microRNA mimics or microRNA inhibitors into cells. MicroRNA mimics have the same function as microRNA, but can be more stable and effective in regulating protein expression. In some embodiments, electroporation can be used to introduce a free vector carrying instructions for expressing a specific microRNA into a cell. Alternatively or in combination therewith, an adeno-associated virus can be used as a vector to carry free instructions for expressing a specific microRNA. Reducing the amount of a target down-regulated microRNA can be achieved by introducing an inhibitor of the target microRNA into the cell via transfection. It should be noted that the method of increasing protein expression / biomass yield according to the present disclosure is performed without altering the cell genome.

[0048] Example 1 - Identification of species-specific or genus-specific growth factors

[0049] This example describes a method for identifying species-specific or genus-specific growth factors. It includes two main steps, namely, a cell growth stimulation step and a cell growth measurement step.

[0050] Step 1: Stimulate cell growth

[0051] MCF-7 human epithelial cell line was cultured in a humidified incubator (34°C; 5% CO2; 95% air) using DMEM / F12 complete medium, 10% FBS. Cells were split at a ratio of 1:4 to 1:8 for daily maintenance.

[0052] When cells reached approximately 80% confluence, cells were detached using trypsin / EDTA. To study the effects of growth factors on cell growth, cells were plated at 3 x 10 4 Cells / cm 2 Seed the cells at a density of 1:1 in complete medium in a 24-well plate (if measuring cell growth by cell counting) or a 96-well plate (if measuring cell growth by the CyQUANT Cell Proliferation Assay Kit). Return the cells to the incubator.

[0053] After 24 hours, the medium was removed. The cells were pre-adapted to serum-free conditions by adding serum-free medium DMEM / F12, 0.1% human serum albumin. The cells were placed in an incubator and cultured in this medium for at least 16 hours.

[0054] Prepare a 10x effective concentration of growth factor (e.g., IGF-1) in serum-free medium for each species / genus to be tested for growth stimulation (e.g., recombinant human IGF-1, human IGF-1-LR3, mouse IGF-1, sea bream IGF-1, and tuna IGF-1). Add the 10x growth factor to the wells and treat the cells with 1x growth factor (e.g., add 50 μl of 10x growth factor to a well containing 450 μl of serum-free medium) (e.g., 1 pM-1 μM). Return the cells to the incubator.

[0055] Observe cells under a microscope daily for signs of cell growth. When there is a clear difference in cell confluence between experimental groups (usually detected between days 2 and 10), quantify cell growth by cell counting, CyQUANT cell proliferation assay, or any other cell proliferation / death assay.

[0056] Step 2: Measuring cell growth steps

[0057] There are two common methods for measuring cell growth, the Trypan blue exclusion method and the CyQUANT Cell Proliferation Assay Kit.

[0058] Trypan blue exclusion

[0059] Cells should have been treated in 24-well plates. Aspirate the media and detach the cells using trypsin-EDTA.

[0060] Add 1 volume of complete medium to the wells to stop trypsin activity. Pipette 3-5 times in the wells to ensure that all cells have been detached.

[0061] Collect the cell suspension into a 1.5 ml tube and centrifuge at 400 x g for 5 minutes to pellet the cells.

[0062] Remove the supernatant without disturbing the cell pellet and resuspend the cell pellet in 200 μl DMEM / F12 basal medium.

[0063] Mix 10 μl of cell suspension with 10 μl of 0.4% Trypan blue solution

[0064] After 2 minutes, add 10 μl of the cell / Trypan blue mixture to each chamber of the Countess II FL disposable slide or a hemocytometer. If using the Countess II FL system, insert the slide into the slide holder of the Countess II FL automated cell counter and determine the cell concentration and % viability. If using a hemocytometer, count the cells with a microscope.

[0065] The number of cells in each experimental group was calculated, and the number of live cells in each well was the live cell concentration (cells / ml).

[0066] CyQUANT Cell Proliferation Assay Kit

[0067] The CyQUANT Cell Proliferation Assay Kit quantifies cell growth by measuring the nucleic acid content of a sample. Cells should be plated in 96-well plates, preferably in triplicate per experimental group.

[0068] Use a multichannel pipette to aspirate as much medium as possible. Avoid scratching the bottom of the well with the pipette tip.

[0069] Freeze the plates in a -80°C freezer. Plates can be stored at -80°C for up to 4 weeks.

[0070] Thaw plates and assay kit reagents at room temperature.

[0071] Mix the kit reagents in each well according to Table 1.

[0072] Table 1

[0073] Element Volume per well (μl) Cell lysis buffer 10μl Autoclaved MilliQ water 189.5μl CyQUANT GR stock solution 0.5μl Total = 200 μl

[0074] When the plate is completely thawed, add 200 μl of CyQUANT GR dye / lysis buffer mixture to each sample well and three empty wells (blanks).Incubate the plate at room temperature in the dark for 5 minutes.

[0075] Using a multichannel pipette, transfer 160 μl from each well of the 96-well plate to the corresponding wells of a black 96-well plate.

[0076] The fluorescence of the samples was measured using a fluorescence microplate reader (e.g., Molecular Devices SpectraMax iD5) with an excitation wavelength of 480 nm and an emission wavelength of 520 nm.

[0077] Calculate the average of the fluorescence readings from the blank wells and subtract this average from all sample fluorescence readings to correct for background fluorescence.

[0078] The mean corrected fluorescence of the vehicle group was calculated. The fluorescence readings of the experimental groups were expressed as fold over control (FOC) by dividing the sample readings (ie, IGF-1 group) by the mean vehicle readings.

[0079] Results: IGF-1 stimulated the growth of MCF-7 cells in a dose-dependent manner

[0080] To verify that IGF-1 stimulates the growth of human MCF-7 cells, cells were treated with increasing doses of human IGF-1 (0 pg / ml-100 ng / ml) for 10 days, and then cell counts were performed. Figure 2 As shown, although 1-100 pg / ml IGF-1 did not significantly increase cell number, further increasing IGF-1 concentrations (1-100 ng / ml) promoted cell growth in a dose-dependent manner. Therefore, MCF-7 cells are suitable for evaluating the growth stimulatory activity of IGF-1.

[0081] Example 2 - Human IGF-1 promotes the growth of human MCF-7 cells, but mouse or fish IGF-1 does not

[0082] To investigate whether the growth stimulatory activity of IGF-1 depends on its species origin, MCF-7 cells were treated with 1.5 nM recombinant IGF-1 from different species, namely human, mouse, and fish (bream, tuna). After 7 days, cell growth was assessed by the CyQUANT assay ( Figure 3 While human IGF-1 obtained from a variety of sources consistently increased MCF-7 cell growth (by approximately 50-100%), mouse and fish IGF-1 did not ( Figure 3 ). These findings suggest that human IGF-1, which comes from the same species as human MCF-7 cells, is more effective than fish and mouse IGF-1 in promoting cell growth.

[0083] Example 3 - Fish IGF-1 expressed by recombinant yeast cells promotes the growth of swim bladder cells

[0084] To investigate the effects of recombinant fish growth factor produced by recombinant yeast cells on fish cell growth, fish IGF-1 was produced and obtained from a recombinant yeast strain genetically modified to carry and express the fish IGF-1 gene and then added to the culture medium for culturing fish swim bladder cells. Figure 4 A graph showing the relative fluorescence of swim bladder cells after treatment with 10 nM of three different recombinant fish IGF-1 clones (Fish IGF-1 (A), Fish IGF-1 (B), and Fish IGF-1 (C)), each of which has a nucleotide sequence that differs from each other and from the native gene for fish IGF-1. For example, cells were harvested on day 3 and subjected to the CyQUANT cell proliferation assay.

[0085] Figure 5 The relative fluorescence of fish bladder cells after treatment with 1% (v / v) of three different batches of yeast culture medium, each culture medium containing only one of the recombinant fish IGF-1 clones described above (i.e., fish IGF-1 (A), fish IGF-1 (B), and fish IGF-1 (C)). In this example, three batches of yeast culture medium containing IGF-1 were collected, centrifuged, and the resulting supernatant was directly added to fish bladder cells. The cultured cells were harvested on day 3 and subjected to the CyQUANT cell proliferation assay.

[0086] Figure 4 and Figure 5 The results show that adding fish IFG-1 obtained from recombinant yeast culture to the culture medium of fish bladder cells can promote the growth of fish bladder cells. It will be appreciated by those skilled in the art that in addition to IFG-1, other growth factors and cytokines may also be used in combination with these and other embodiments of the present invention, including but not limited to insulin, interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), interleukin 11 (IL-11), fibroblast growth factor (FGF), epidermal growth factor (EGF), transferrin, and the resulting recombinant growth factors or cytokines, whether in purified form or mixed with yeast culture medium, can be used to supplement cell culture medium to promote the growth of various cells. In addition, as shown in the present disclosure and the examples herein, growth factors that are the same or different from the source of the cultured cells may be used, and natural or modified growth factors may also be used to achieve maximum responsiveness in the cell type in question. Those skilled in the art will appreciate that cultured cells treated with such exogenous growth factors or cytokines are not limited to fish cells, but may be non-fish cells, including non-mammalian cells and mammalian cells, and that the genes of growth factors or cytokines introduced into recombinant yeast cells as an expression system by genetic engineering should preferably be (i) species genetically identical or similar to the target cells and / or (ii) genus genetically identical to the target cells.

[0087] In one embodiment, the concentration of the recombinant growth factor or cytokine to be added to the culture medium of the target isolated cells is in the range of 0.1%-1% (v / v) or 1nM-10nM. The present invention shows that it is more effective to use growth factors of (i) the same or similar species genetically as the cultured cell type or (ii) the same genus genetically as the cultured cell type. The use of these growth factors or protein factors can be reduced while achieving the same growth rate. Species-specific growth factors and / or genus-specific growth factors represent a promising direction for reducing culture medium costs, especially during large-scale cell production of cultured meat and other applications using cultured cell clusters. The use of more bioactive growth factors can also reduce processing time and improve the quality (e.g., texture, taste, nutritional value) of cultured meat or cell clusters.

[0088] The following examples will describe embodiments of genetically engineered yeast cells expressing recombinant fish IGF-1, and their use in promoting the growth of fish and human cells in vitro. It will be appreciated by those skilled in the art that other growth factors may be produced and used in a similar manner, based on the teachings of the present invention.

[0089] Example 4 - Cloning of grouper IGF-1 gene into yeast expression vector

[0090] The grouper IGF-1 gene having the nucleotide sequence shown in SEQ ID NO.1-5 (which encodes the amino acid sequence shown in SEQ ID NO.6-10) was cloned into PichiaPink according to the prescribed scheme. TM Vector (ThermoFisher), a grouper IGF-1 gene clone with the natural grouper IGF-1 gene sequence and four different modified grouper IGF-1 gene clones were obtained. The details of the clones are summarized in Table 1 below.

[0091] In some embodiments, the present invention encompasses a genetically modified yeast cell comprising a nucleic acid encoding a recombinant IGF-1 selected from SEQ ID NO. 1 to 5. In some embodiments, the resulting recombinant IGF-1 can be introduced into an in vitro cell culture to increase the growth rate or protein expression rate of cells in the in vitro cell culture.

[0092] In addition, the present invention also encompasses polypeptide fragments that are substantially similar in sequence to the polypeptide fragments selected from SEQ ID NOs: 6-10. In one embodiment, the polypeptide fragment may have at least 80% sequence identity with the polypeptide fragment selected from SEQ ID NOs: 6-10. In another embodiment, the polypeptide fragment may have at least 90% sequence identity with the polypeptide fragment selected from SEQ ID NOs: 6-10.

[0093] Table 1 - Grouper IGF-1 gene and protein sequence examples

[0094]

[0095]

[0096]

[0097]

[0098] Example 5 - From PichiaPink TM Strains expressing recombinant fish IGF-1 protein

[0099] A) Experimental expression of recombinant IGF-1 protein

[0100] Recombinant yeast cells expressing recombinant fish IGF-1 protein were constructed, and the experimental expression of recombinant fish IGF-1 protein in the obtained recombinant yeast cells was analyzed as follows:

[0101] 1. Transform yeast cells with one of the clones of the recombinant grouper IGF-1 gene (from Pichia Pink) according to the manufacturer's laboratory guidelines. TM Expression strain collection, ThermoFisher).

[0102] 2. Use the transformed PichiaPink TM A single colony of the strain was inoculated into 10 mL of BMGY medium (buffered glycerol complex medium) in a 50 mL centrifuge tube. The cells were placed in a shaking incubator at 24-30°C with the speed set to 200-300 rpm for 1-3 days.

[0103] 3. Centrifuge the yeast culture at 1,500 x g for 5 minutes at room temperature. Pour off the BMGY supernatant and resuspend the cell pellet in 1 mL of BMMY medium to induce expression.

[0104] 4. Place the cells in BMMY medium back into the 30°C shaking incubator and continue growing overnight.

[0105] 5. Take 100 μL from the sample and add 100 μL of 40% methanol every 24 hours to induce expression. Continue to culture the cells in a shaker at 24-30°C with the speed set to 200-300 rpm for 24-72 hours.

[0106] 6. Harvest the cells by centrifugation at 1,500 x g for 10 minutes.

[0107] 7. Transfer the supernatant to a separate tube and store both the supernatant and the cell pellet at -80°C until use.

[0108] 8. Analyze the supernatant by Western blotting to obtain the best expressing clone and the optimal induction time.

[0109] B) Large-scale expression of recombinant IGF-1 protein

[0110] The recombinant fish IGF-1 protein was expressed on a large scale in recombinant yeast cells and analyzed as follows:

[0111] 1. Use the transformed PichiaPink TM A single colony of the strain was inoculated into 400 mL of BMGY and cultured at 24°C-30°C in a shaking incubator (250-300 rpm) for 2-3 days.

[0112] 2. Harvest the cells by centrifugation in a sterile centrifuge bottle at 1,500-3,000 x g for 5 minutes at room temperature.

[0113] 3. To induce expression, pour off the supernatant and resuspend the cell pellet in 50 mL of BMMY medium. Continue to culture at 24°C-30°C with shaking.

[0114] 4. Add 100% methanol to 0.5% every 24 hours until the optimal induction time is reached.

[0115] 5. Harvest the cells by centrifugation at 1,500 x g for 5 minutes at 4°C.

[0116] 6. Save the supernatant and centrifuge again at 7,197 x g (max g) for 15 minutes at 4°C.

[0117] 7. Save the supernatant. Store at -80°C until ready for further processing.

[0118] C) Processing of yeast culture medium containing raw IGF-1 for direct use

[0119] After the fish IGF-1 protein is expressed in yeast cells, the obtained fish IGF-1 protein is collected together with the yeast culture medium by the following steps to obtain a yeast culture medium containing IGF-1:

[0120] 1. Repeat steps 1 to 4 in Part B above.

[0121] 2. Harvest the cells by centrifugation at 1,500 x g for 5 minutes at 4°C.

[0122] 3. Save the supernatant and centrifuge again at 7,197 x g (max g) for 15 minutes at 4°C.

[0123] 4. Keep the supernatant and filter it with a 0.22 μm filter.

[0124] 5. Store the filtered yeast culture medium containing IGF-1 at 4°C temporarily and at -80°C for a long term.

[0125] D) Purification of recombinant IGF-1 protein by immobilized metal affinity chromatography

[0126] As an optional additional step, immobilized metal affinity chromatography (HisPur TM Recombinant fish IGF-1 protein expressed in yeast cells was purified using cobalt resin (ThermoFisher) as follows:

[0127] 1. Add 1 mL of cobalt resin to a 10 mL purification column. Allow the storage buffer to drain from the resin by gravity flow. Discard the flow-through.

[0128] 2. Add 2 mL of equilibration / wash buffer and mix until the resin is completely suspended. Allow the equilibration / wash buffer to drain from the resin by gravity flow. Discard the flow-through.

[0129] 3. To purify recombinant IGF-1 protein (with a His tag) from the culture supernatant, first mix the culture supernatant with the equilibration buffer at a 1:1 ratio. Then load the clarified culture supernatant into the purification column. Allow the culture supernatant to drain from the purification column by gravity flow. Re-add the flow-through to the purification column once to maximize binding.

[0130] 4. Wash the resin with 10 mL of equilibration / wash buffer. Allow the equilibration / wash buffer to drain from the purification column by gravity flow. Repeat this wash step twice.

[0131] 5. Elute the bound His-tagged protein using 1 mL of elution buffer. Collect the flow-through. Repeat this step four times, saving each portion of the flow-through in a separate tube.

[0132] 6. The eluted protein was dialyzed against PBS, pH 7.4 for a total of 3 rounds.

[0133] 7. Store the dialyzed protein at -80°C until use.

[0134] E) Verification of recombinant IGF-1 protein expression by Western blotting

[0135] The expression of recombinant fish IGF-1 protein in recombinant yeast cells was verified as follows:

[0136] 1. Mix the recombinant fish IGF-1 protein sample with 2X Tris-Glycine SDS sample buffer supplemented with reducing agent. Denature the protein sample at 95°C for 5-10 minutes.

[0137] 2. Load the protein samples and prestained protein ladder onto a Tris-Glycine polyacrylamide gel.

[0138] 3. Place the polyacrylamide gel in 1X Tris-Glycine SDS electrophoresis buffer and run at 120V for 75 minutes.

[0139] 4. Transfer the protein to a 0.22um PVDF membrane overnight.

[0140] 5. Block the PVDF membrane in 5% milk in 1X TBST for 30 minutes.

[0141] 6. Incubate the PVDF membrane with mouse anti-6X His tag monoclonal antibody (1:2000 dilution in TBST) at room temperature for 1 hour with constant shaking.

[0142] 7. Wash the membrane three times, 5 minutes each time.

[0143] 8. Incubate the PVDF membrane with goat anti-mouse IgG, IgM (H+L) secondary antibody, HRP (1:2000 dilution in TBST) at room temperature for 1 hour with constant shaking.

[0144] 9. Wash the membrane three times, 5 minutes each time.

[0145] 10. Mix the ECL components in a 1:1 ratio. Incubate the PVDF membrane with the ECL mixture for 1 minute.

[0146] 11. Remove excess ECL mixture from the PVDF membrane. Capture the signal using the Azure C300 imaging system.

[0147] F) Results:

[0148] The expression of grouper IGF-1 in yeast culture was confirmed by Western blotting. To confirm the expression of recombinant IGF-1 protein, 20 μL of yeast culture medium was harvested and analyzed by Western blotting. Figure 6As shown, the expression of recombinant IGF-1 protein in this recombinant yeast strain was detected at ~10 kDa and confirmed by Western blotting using anti-6X His antibody. The band between 35 kDa and 55 kDa is probably an IGF-1 multimer, as it can be detected with anti-6X His antibody, indicating the presence of a His tag in the protein. IGF-1 was not detected in BMMY only (medium control) and yeast only culture (uninduced control).

[0149] Characterization of purified grouper IGF-1 by total protein staining and western blotting. To characterize native and engineered grouper IGF-1 after immobilized metal affinity chromatography (IMAC), total protein staining and western blotting were performed. Figure 7 As shown, the expression of recombinant IGF-1 protein in the present recombinant yeast strain was detected and confirmed at ~10 kDa in total protein staining (Panel a) and Western blotting using anti-6X His antibody (Panel b). In total protein staining (Panel a), all purified IGF-1 proteins had two bands between 35 kDa and 70 kDa, which may be yeast proteins because they could not be detected with anti-6X His antibody, indicating that there was no His tag in the protein. In Western blotting (Panel b), a band between 35 kDa and 55 kDa was detected, which may be an IGF-1 multimer because it could only be detected with anti-6XHis antibody.

[0150] Example 6 - Cell Growth Stimulation

[0151] A) MCF-7 human epithelial cell line

[0152] MCF-7 human epithelial cell line was obtained from ATCC and treated with recombinant fish IGF-1 obtained from recombinant yeast cells as described in the previous examples according to the following steps:

[0153] 1. The cells were cultured in a humidified incubator (34°C; 5% CO2; 95% air) with DMEM / F12 complete medium [DMEM / F12, 10% FBS]. Daily maintenance was performed in batches at a ratio of 1:4 to 1:8.

[0154] 2. When the cells reach about 80% confluence, detach the cells with trypsin. To study the effect of growth factors on cell growth, seed the cells at a density of 10,000 cells / well in a 96-well plate containing DMEM / F12 complete medium. Return the cells to the incubator.

[0155] 3. After 24 hours, prepare a 2x working concentration of growth factor (e.g., IGF-1) in serum-free medium [DMEM / F12, 0.1% human serum albumin]. Add 2x growth factor to the wells and treat the cells with 1x growth factor (e.g., add 50uL 2x growth factor to a well containing 50uL serum-free medium). Return the cells to the incubator.

[0156] 4. Observe the cells under a microscope every day for signs of cell growth. When there is a clear difference in cell confluence between experimental groups (usually detected between days 2 and 10), cell growth can be quantified by resazurin assay (as described in Section C of this example) or any other cell proliferation / death assay.

[0157] B) FHM fish epithelial cell line

[0158] FHM fish epithelial cell line was obtained from ATCC and processed according to the following steps:

[0159] 1. Upon arrival of the cell line, culture the cells in DMEM / F12 complete medium in a humidified incubator (34°C; 5% CO2; 95% air) for 1-3 passages (P1-P3). Split the cells at a ratio of 1:4 to 1:8. From passage 4 onwards, sequentially adapt the FHM cells to reduced serum conditions and finally to serum-free medium. Culture the cells in a humidified incubator (34°C; 5% CO2; 95% air) using our in-house serum-free medium formulation. Split the cells at a ratio of 1:4 to 1:8 for routine maintenance.

[0160] 2. When the cells reach about 80% confluence, detach the cells with trypsin. To study the effect of growth factors on cell growth, seed the cells at a density of 10,000 cells / well on a 96-well plate containing serum-free medium. Return the cells to the incubator.

[0161] 3. After 24 hours, prepare a 2x working concentration of growth factor (e.g., IGF-1) in treatment medium [DMEM / F12, 0.1% human serum albumin, 32 μM ethanolamine]. Add 2x growth factor to the wells and treat the cells with 1x growth factor (e.g., add 50 μl of 2x growth factor to a well containing 50 μl of serum-free medium). Return the cells to the incubator.

[0162] 4. Observe the cells under a microscope every day for signs of cell growth. When there is a clear difference in cell confluence between experimental groups (usually detected between days 2 and 10), cell growth can be quantified by resazurin assay (as described in Section C of this example) or any other cell proliferation / death assay.

[0163] C) Measurement of cell growth by resazurin assay

[0164] Resazurin assays are commonly used to quantify the number of viable cells in a sample and to measure the metabolic capacity of viable cells.

[0165] 1. Use a multichannel pipette to remove as much treatment medium as possible. Avoid scratching the bottom of the well with the pipette tip.

[0166] 2. Mix the 1:20 diluted resazurin solution with the culture medium corresponding to each cell line.

[0167] 3. Use a multichannel pipette to add 100 μL of the diluted resazurin solution to each well of the 96-well plate. Place the plate in a humidified incubator (34°C; 5% CO2; 95% air) and incubate.

[0168] 4. When the color of the Resazurin solution (from step 4) in the plate turns pink (usually takes 3-6 hours), transfer 75 μL of the contents of each well of the 96-well plate to the corresponding well of the black 96-well plate. Add 75 μL of the diluted Resazurin solution (from step 3) to the empty wells (blanks).

[0169] 5. Measure the sample fluorescence using a fluorescence microplate reader (e.g., Molecular Devices SpectraMax iD5). Set the excitation wavelength and emission wavelength to 550 nm and 590 nm, respectively.

[0170] 6. Calculate the average of the fluorescence readings from the blank wells and subtract this average reading from all sample fluorescence readings to correct for background fluorescence.

[0171] 7. Calculate the average corrected fluorescence of the control group. Express the experimental group fluorescence readings as a percentage of the control group by dividing the sample readings (i.e., IGF-1 group) by the average control readings.

[0172] D) Results

[0173] Purified grouper IGF-1 stimulates the growth of both mammalian and non-mammalian cells.

[0174] The stimulatory effect of grouper IGF-1 on cell growth was measured by the resazurin assay. Purified native and engineered grouper IGF-1 were treated to MCF7 cells (human) and FHM cells (fish) at 0 nM, 0.1 nM, 1 nM, and 10 nM, respectively. Figure 8 As shown, grouper IGF-1 stimulated cell growth in both cell types, indicating that it could be used after purification. The two cell types also showed different responsiveness to the engineered grouper IGF-1.

[0175] Yeast cultures containing grouper IGF-1 can be used directly to stimulate cell growth of both mammalian and non-mammalian cells.

[0176] To test whether grouper IGF-1 yeast culture could be used directly to stimulate cell growth, a resazurin assay was performed. Grouper IGF-1 yeast culture was treated with MCF7 cells (human) and FHM cells (fish) at dilutions of 1:100 and 1:1000, respectively, after centrifugation and filtration. Fig. 9 As shown, grouper IGF-1 yeast culture stimulated cell growth in both cell types, whereas BMMY alone (media control) and yeast alone (uninduced control) had no significant effect on cell growth. These results suggest that the growth stimulatory effect is due to the presence of grouper IGF-1 in the yeast culture and not to other components of the culture medium and yeast proteins. This result demonstrates a revolutionary approach to stimulating cell growth using grouper IGF-1 yeast culture with minimal purification steps.

[0177] Recombinant growth factors or proteins

[0178] In various embodiments, recombinant growth factors or proteins are expressed and used to support growth of in vitro cell cultures. As described herein, embodiments of genetically engineered yeast cells can be used to express recombinant growth factors from various sources, and the resulting growth factors can be used to supplement in vitro cultures to promote growth of cells of the same or similar species or genus.

[0179] In some embodiments, the growth factor is IGF-1, such as an IGF-1 having the sequence shown in Table 2 below.

[0180] In some embodiments, the present invention encompasses a genetically modified yeast cell comprising a nucleic acid encoding a recombinant IGF-1 selected from SEQ ID NO. 11 to 55. In some embodiments, the resulting recombinant IGF-1 can be introduced into an in vitro cell culture to increase the growth rate or protein expression rate of cells in the in vitro cell culture.

[0181] In addition, the present invention also encompasses polypeptide fragments that are substantially similar in sequence to a polypeptide fragment selected from SEQ ID NOs: 56 to 100. In one embodiment, the polypeptide fragment may have at least 80% sequence identity with a polypeptide fragment selected from SEQ ID NOs: 56 to 100. In another embodiment, the polypeptide fragment may have at least 90% sequence identity with a polypeptide fragment selected from SEQ ID NOs: 56 to 100.

[0182] Table 2 - Examples of IGF-1 gene and protein sequences

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191]

[0192]

[0193]

[0194]

[0195]

[0196]

[0197]

[0198]

[0199]

[0200] In some embodiments, the growth factor is fibroblast growth factor 2 (FGF-2), such as FGF2 having the sequence shown in Table 3 below.

[0201] In some embodiments, the present invention encompasses a genetically modified yeast cell comprising a nucleic acid encoding a recombinant FGF-2 selected from SEQ ID NO. 101-145. In some embodiments, the resulting recombinant FGF-2 can be introduced into an in vitro cell culture to increase the growth rate or protein expression rate of cells in the in vitro cell culture.

[0202] In addition, the present invention also encompasses polypeptide fragments that are substantially similar in sequence to the polypeptide fragments selected from SEQ ID NOs: 146-190. In one embodiment, the polypeptide fragment may have at least 80% sequence identity with the polypeptide fragment selected from SEQ ID NOs: 146-190. In another embodiment, the polypeptide fragment may have at least 90% sequence identity with the polypeptide fragment selected from SEQ ID NOs: 146-190.

[0203] Table 3 - FGF2 gene and protein sequence examples

[0204]

[0205]

[0206]

[0207]

[0208]

[0209]

[0210]

[0211]

[0212]

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229] In some embodiments, the growth factor is epidermal growth factor (EGF), such as an EGF having the sequence shown in Table 4 below.

[0230] In some embodiments, the present invention relates to a genetically modified yeast cell comprising a nucleic acid encoding a recombinant EGF selected from SEQ ID NO. 191-219. In some embodiments, the obtained recombinant FGF2 can be introduced into an in vitro cell culture to increase the growth rate or protein expression rate of cells in the in vitro cell culture.

[0231] In addition, the present invention also encompasses polypeptide fragments that are substantially similar in sequence to a polypeptide fragment selected from SEQ ID NOs: 220-248. In one embodiment, the polypeptide fragment may have at least 80% sequence identity with a polypeptide fragment selected from SEQ ID NOs: 220-248. In another embodiment, the polypeptide fragment may have at least 90% sequence identity with a polypeptide fragment selected from SEQ ID NOs: 220-248.

[0232] Table 4 - EGF gene and protein sequence examples

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241] The above description is illustrative and non-restrictive. After reading this disclosure, those skilled in the art can clearly see many changes of the embodiment. Therefore, the scope of the embodiment should not be determined with reference to the above description, but should be determined with reference to the pending claims and their full scope or equivalents.

[0242] One or more features in any embodiment may be combined with one or more features in any other embodiment without departing from the scope of the embodiment. Unless otherwise expressly stated, the expression "a", "an" or "the" is intended to mean "one or more". Unless otherwise expressly stated, the expression "and / or" is intended to mean the broadest meaning of the term.

[0243] While the disclosure may be embodied in many different forms, the drawings and discussion are presented with the understanding that the disclosure is illustrative of the principles of one or more inventions and is not intended to limit any one embodiment to the one shown.

[0244] Therefore, in a broader sense, the present disclosure is not limited to the above specific details, representative systems and methods, and illustrative examples. Various modifications and changes may be made to the above description without departing from the scope or spirit of the present disclosure, and the present disclosure is intended to cover all such modifications and changes as long as they fall within the scope of the following claims and their equivalents.

Claims

1. A recombinant growth factor having an amino acid sequence that is identical or substantially similar to a sequence selected from the group consisting of SEQ ID NOs: 6-10, 56-100, 146-190 and 220-248.

2. The recombinant growth factor according to claim 1, wherein the recombinant growth factor is encoded by a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-5, 11-55, 101-145 and 191-219.

3. A genetically modified yeast cell comprising a nucleic acid encoding a recombinant growth factor 1, wherein the nucleic acid has a sequence selected from the group consisting of SEQ ID NOs. 1-5, 11-55, 101-145 and 191-219.

4. The genetically modified yeast cell according to claim 3, wherein the recombinant growth factor is insulin growth factor 1 (IGF-1), fibroblast growth factor 2 (FGF-2) or epidermal growth factor (EGF).

5. The genetically modified yeast cell according to claim 3, wherein the amino acid sequence of the recombinant growth factor is optimized to improve stability or biological activity compared to the natural growth factor.

6. The genetically modified yeast cell of claim 3, wherein the yeast cell is Pichia pastoris.

7. A method for increasing the growth or protein expression rate of a plurality of cells in in vitro cell culture, comprising: a) cultivating a plurality of cells in a nutrient medium under growth conditions suitable for initiating cell division; as well as b) introducing at least one recombinant growth factor into the nutrient medium, The amino acid sequence of the recombinant growth factor is (a) optimized to increase the growth or protein expression rate of the plurality of cells, or (b) (i) is of the same or similar species genetically as the plurality of cells and / or (ii) is of the same genus genetically as the plurality of cells.

8. The method of claim 7, wherein the recombinant growth factor is selected from the group consisting of insulin growth factor 1 (IGF-1), insulin, interleukin 6 (IL-6), interleukin 6 receptor (IL-6R), interleukin 11 (IL-11), fibroblast growth factor 2 (FGF-2), epidermal growth factor (EGF) and transferrin.

9. The method of claim 7, wherein the recombinant growth factor is encoded by a nucleic acid having a sequence selected from the group consisting of SEQ ID NOs. 1-5, 11-55, 101-145 and 191-219.

10. The method according to claim 7, wherein the recombinant growth factor is produced by a genetically modified yeast cell comprising a recombinant growth factor gene and is obtained from a cell culture medium in which the genetically modified yeast cell is cultured.

11. The method of claim 10, wherein the recombinant growth factor is purified from the cell culture medium prior to introduction into the nutrient medium.

12. The method of claim 10, wherein the recombinant growth factor is introduced into the nutrient medium by introducing a cell culture medium containing the growth factor into the nutrient medium.

13. The method of claim 7, wherein the plurality of cells are mammalian cells or non-mammalian cells.

14. The method of claim 7, wherein the plurality of cells are fish cells.

15. The method according to claim 7, further comprising a step between step a) and step b): culturing a plurality of cells on a food-grade scaffold in a nutrient medium, thereby allowing the plurality of cells to grow into a solid or semi-solid structure that mimics an animal organ.

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