Recombinant expression platform, constructs and methods for expression of difficult to express proteins

Abstract

To provide a recombinant expression platform for the enhanced expression of "Difficult to Express" proteins (DTE-Ps).SOLUTION: The present invention provides a versatile yeast-based recombinant expression platform for the enhanced expression of full length or truncated target "Difficult to Express" proteins (DTE-Ps) of diverse origin and families. Constructs, methods and kits involved in expressing such DTE-Ps are also provided. The recombinant expression platform of the present invention is robust, scalable and can have applications in fields like vaccine development, drug discovery, metabolism, diagnostics, therapeutics and healthcare.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention is broadly in the field of recombinant protein expression. In particular, the present invention relates to the expression of difficult to express proteins in recombinant expression platforms; compositions, methods, and kits involved in expressing such DTE-Ps via said systems. [Background technology]

[0002] To support drug discovery and the production of biotherapeutics and vaccines, successful recombinant expression of proteins is a key requirement in the biotechnology industry. This requires not only successful expression but also high-quality, large-scale production of target proteins. To achieve good expression of correctly folded, active forms of such proteins in recombinant format, host cells such as Escherichia coli, yeast, mammalian cells, and insect cell-based expression systems are commonly selected. However, many proteins do not express efficiently due to inherent properties such as hydrophobicity, higher cysteine ​​and proline residues, repetitive amino acids, protein half-life, mRNA turnover, and stable RNA production. Host cell adaptability also plays a key role in achieving the desired expression. Therefore, robust and consistent platforms and host systems are required for the expression of such proteins, along with commercial scalability and cost-effective production for industrial supply at the required quantities and costs.

[0003] Many proteins important for various applications fall under the category of difficult-to-express proteins, which may pose challenges in expressing and manufacturing these proteins for industrial-scale production. This can result in low or no expression due to sequence complexity or issues such as scaling up production, product recovery, or purification. Furthermore, the application of various host systems, methodologies, and laborious optimization can be highly unpredictable, laborious, expensive, and time-consuming.

[0004] The expression of a few challenging proteins using various vectors and hosts has been reported in the prior art. Indian Patent Applications Nos. 1017 / DEL / 2009 and 1018 / DEL / 2009 disclose the heterologous overexpression of one such protein, namely, human cytochrome P450 reductase. However, these documents specifically focus on the expression of cytochrome P450 with different constructs.

[0005] Another patent document, CN1757745A, relates to a method for highly efficient expression of exogenous proteins using a methylotrophic yeast system.

[0006] US7910364B2 discloses a rapidly cleavable sumo fusion protein expression system for difficult to express proteins.

[0007] Saccardo et al., 2016, clarified some general techniques and tools to address the expression of difficult-to-express proteins.

[0008] Thoring et al., 2017, reported high-yield production of difficult-to-express proteins in a continuous exchange cell-free system based on CHO cell lysate.

[0009] However, there are still many difficult-to-express proteins that remain a challenge for researchers worldwide. In addition, little success has been achieved in terms of commercial scalability for overexpression and purification with enhanced quality and scalable quantity. Furthermore, there is a significant gap in the art in having a stable, consistent, and robust platform for the expression of difficult-to-express recombinant proteins across various origins and families with scalability as well as the quality of the expressed target protein.

[0010] For example, over 50% of known and novel drug target receptors and vaccine targets are found as membrane proteins. It is also well understood that overexpression of full-length membrane proteins, including all domains, is not an easy task. Furthermore, recombinant expression of proteins with transmembrane domain(s) in heterologous systems is extremely challenging due to their high hydrophobicity and sequence complexity, which can lead to aggregation, precipitation, and protein misfolding, making them difficult to solubilize and refold (Lundstrom et al., 2006). These proteins are typically expressed as modified proteins with deleted transmembrane domains using E. coli host systems to avoid insoluble expression. The drawback of this approach is that it results in a lack of full-length sequence expression and, therefore, a lack of full-length proteins for analysis. Eukaryotic host systems are recommended for expression in their native form, as they are intact and full-length for biochemical and structural characterization requirements. As per the available literature, there is no universal solution for membrane protein production and this continues to remain a major obstacle (Elizabeth Massey-Gendel et al., 2009).

[0011] The expression, close interaction and insertion of nascent polypeptide chains into membranes is one important indicator for their analysis to study a particular protein as a drug target or vaccine candidate.

[0012] There is a need in the art for a stable, versatile expression system for expressing multiple full-length transmembrane proteins. As an example, the present inventors have demonstrated the expression of full-length, functionally active neuraminidase (NA), a transmembrane DTE-P. Besides the difficulty of expressing neuraminidase as a membrane-anchored protein, its sequence is also known to contain numerous cysteine ​​residues and hydrophobicity, further explaining its tendency for insoluble expression and aggregation in expression systems such as E. coli. The presence of proline-rich residues further adds instability, acting as a helix disruptor for stable product formation. Limited literature exists on recombinant expression of full-length NA in yeast host expression systems. Currently, there is an unmet need to develop a universally effective vaccine that elicits immune responses against influenza viruses and subtypes. Full-length NA expression is paramount for analyzing immune responses, including conformational epitopes. To fulfill such a function, efficient expression of neuraminidase is required.

[0013] Similarly, structural proteins are considered difficult to express; they do not appear fully soluble, correctly folded, and active in heterologous expression systems. Their primary characteristic is their tendency to aggregate and form inclusion bodies. Viral surface glycoproteins and other capsid proteins belong to the structural protein family and have long been recognized as functional targets for vaccines. Vaccine candidates such as capsid proteins could be an attractive strategy for inducing protection against severe viral disease.

[0014] The versatile platform described in this application is capable of expressing several difficult-to-express structural proteins. The inventors have demonstrated that the claimed platform enables stable and enhanced expression of the His-tagged, glycosylated, 347-amino acid capsid glycoprotein viral protein VP7. The sequence contains numerous cysteine ​​and proline residues, making it a difficult protein to express. These characteristics explain the protein's tendency toward insoluble expression and aggregation when overexpressed, favoring expression using eukaryotic systems. Additionally, the numerous proline residues are structural breakers, placing the protein in an unstable category. Neutralizing antibodies against the protein can provide both serotype-specific and cross-reactive protection and are therefore considered important for vaccine development in the human medical field.

[0015] The present inventors have further demonstrated that the platform of the present invention is capable of expressing enzyme proteins, such as fatty acid enzymes. Fatty acids and derivatives known as polyunsaturated fatty acids (PUFAs) are of great importance, with functions including regulating inflammatory responses and lipid metabolism, as well as in signal transduction pathways (Hoshino et al., 1984). Desaturase enzymes, a prime example of such fatty acid enzymes, are transmembrane proteins, ranging from single-pass to multi-pass, and can be localized in the endoplasmic reticulum membrane of plants, fungi, and animals.

[0016] Progress in research on desaturases has been hindered by the complexities involved in membrane protein extraction and crystallization of these enzymes. Consequently, knowledge regarding the structure and expression regulation of membrane-bound fatty acid desaturases is still lacking, and it remains unknown whether the transmembrane domain plays a role in fatty acid desaturase efficiency (Wyatt et al., 1983). Desaturases are highly hydrophobic, as explained by their membrane nature and high cysteine ​​and proline content. These characteristics place them in a difficult-to-express category and explain their tendency for insoluble expression when expressed in bacterial host systems. Therefore, it is of utmost importance to have a reliable and efficient system for expressing these difficult-to-express enzyme proteins, especially for producing them on a commercial scale. The present inventors have successfully demonstrated the expression of a four-pass membrane protein sequence of a desaturase via the platform of the present invention.

[0017] Furthermore, fatty acid elongation is a critical step that serves as an alternative pathway for fatty acid production associated with lipid metabolic applications. Elongase proteins also have high hydrophobicity and high cysteine ​​and proline contents. These characteristics make them difficult to express. Furthermore, when expressed in bacterial host systems, there is a tendency for insoluble expression.

[0018] Many important proteins, such as ion pumps, ion channels, and transporters, span membranes multiple times. Each transmembrane α-helix in these multi-pass transmembrane proteins is thought to act as a conformational sequence. Essentially, transmembrane proteins mediate cell-to-cell communication, transport molecules into and out of cells, and are common drug targets. The expression, close interaction, and insertion of nascent polypeptide chains into membranes are key indicators for analyzing and studying specific proteins as drug targets or vaccine candidates. However, while large multi-pass transmembrane protein designs span such diverse environments, it is difficult to predict how they will fold and function.

[0019] Furthermore, recombinant expression of proteins with transmembrane domain(s) in heterologous systems is extremely challenging due to their high hydrophobicity and sequence complexity, which can lead to aggregation, precipitation, and protein misfolding, as well as difficulty in solubilizing and refolding. To produce a well-formed multi-pass transmembrane protein from scratch, several biophysical requirements must be simultaneously balanced. While arranging the hydrophobic swatch in an alpha-helical conformation to generate membrane binding is sufficient, controlling the packing and orientation of the hydrophobic helix is ​​difficult. Therefore, expressing such multi-pass transmembrane proteins in prokaryotes and subsequently refolding them to generate the correct conformation is not an easy task.

[0020] The present inventors have successfully demonstrated the expression of the ion channel receptor, multi-pass membrane protein Nav1.7, via the versatile platform of the present invention. Nav1.7 is a voltage-gated sodium channel that mediates voltage-dependent sodium ion permeability in excitable membranes and is also involved in pain signaling. Nav1.7 is a promising drug target that has been validated for the treatment of human pain.

[0021] Nav1.7 is a glycosylated, 1988 amino acid long, multi-pass membrane protein with 24 transmembrane domains that is localized to the cell / plasma membrane. The major subunit of this channel is the alpha subunit, a >200 kDa protein. The sequence contains four internal repeats, each with five hydrophobic segments (S1, S2, S3, S5, S6) and one positively charged segment (S4).

[0022] The protein has a very large oligomeric structure, contains numerous and odd numbers of cysteine ​​and proline residues, and is highly hydrophobic. Observed indicators indicate that overexpression of the protein can lead to insoluble expression and aggregation in prokaryotes. Both the large size and the high proline residue content (helix breakers) can lead to further degradation. The instability index of this protein classifies it as an unstable protein.

[0023] In some culture models, receptor expression has been found to be degraded or dysregulated in culture, resulting in difficulties in Nav1.7 expression over time. The inventors of the present invention have successfully demonstrated the expression of the full-length membrane-anchored alpha subunit of Nav1.7 using the recombinant expression platform of the present invention.

[0024] Another important category of DTE-Ps is drug target molecules (GPI-anchored proteins). Major drug target classes are antitumor drugs, G protein-coupled receptors (GPCRs), ion channels, kinases, and proteases (Kubicet et al., 2019). A wide range of protein expression systems is currently available, many based on cellular organisms of prokaryotic and eukaryotic origin. The limitations of prokaryotic systems arise when complex mammalian target proteins need to be produced, which require post-translational modifications, cofactors, and chaperones for correct protein folding, assembly, and activity.

[0025] One such drug target protein is CD59, a glycosylphosphatidylinositol-anchored (GPI-anchored) membrane protein that acts as an inhibitor of membrane attack complex formation to regulate complement activation. Recent studies have shown that CD59 is highly expressed in several cancer cell lines and tumor tissues. CD59 further regulates the function, infiltration, and phenotype of various immune cells in the tumor microenvironment (Zhao et al., 2018). CD59 is considered a promising target in gene therapy for breast cancer (Xu et al.).

[0026] To circumvent these issues, eukaryotic cell-based expression systems, including yeast systems (Pichia pastoris, Saccharomyces cerevisiae, Kluyveromyces lactis) and mammalian systems (HEK293, Chinese hamster ovary cells (CHO cells)), have been proposed in the art. Mammalian systems have only rarely been reported as successful. The generation of eukaryotic stable cell lines for protein production has proven to be quite labor-intensive due to long production times, slow cell growth, and low protein yields, leading to costly protein production processes (Kubic et al., 2019).

[0027] As discussed in the previous paragraph, there is an urgent need to have an efficient recombinant expression system / platform that is flexible and adaptable for the expression and production of various DTE-Ps. Desired characteristics require optimal expression, a rapid and time-saving standardized method, and ease of scalability with the ability to produce large amounts of material for analytical, diagnostic, and therapeutic uses that can serve large populations cost-effectively.

[0028] Therefore, the present invention addresses this need by providing a versatile recombinant protein expression platform comprising a recombinant expression vector using a protease-deficient yeast cell host system, capable of expressing target DTE-P proteins from various origins and families at a scalable and commercial level. The claimed recombinant expression platform and method overcome the shortcomings of the prior art and represent a significant technological advance.

[0029] Object of the invention The primary objective of the present invention is to provide a yeast-based, versatile recombinant expression platform for the enhanced expression of full-length or truncated target "difficult to express" proteins (DTE-Ps) of diverse origins and families.

[0030] Furthermore, another object of the present invention is to provide methods for enhanced DTE-P expression using the recombinant expression platform of the present invention.

[0031] Furthermore, another object of the present invention is to provide a kit comprising the recombinant expression platform of the present invention for producing the target DTE-P.

[0032] Another important objective of the present invention is to provide a versatile recombinant yeast-based platform, method, and kit for enhanced expression and scalability of desired DTE-Ps with all functions intact. [Brief explanation of the drawings]

[0033] The accompanying drawings illustrate some of the embodiments of the invention and, together with the description, explain the invention. These drawings are provided by way of example, not limitation.

[0034] [Figure 1] 1 shows the pYRI100 yeast integration vector for expression of recombinant proteins using the inducible GAL promoter. [Figure 2] 1 shows the pYRI200 yeast integration vector for expression of recombinant proteins using the ADH2 constitutive promoter. [Figure 3] 1 shows the pYRE100 yeast episomal vector for expression of recombinant proteins using the inducible GAL promoter. [Figure 4] 1 shows the pYRE200 yeast episomal vector for expression of recombinant proteins using the ADH2 constitutive promoter. [Figure 5] Western blot analysis of neuraminidase is shown. [Figure 6] Large scale Western blot analysis of neuraminidase is shown. [Figure 7] 1 shows the flow cytometry results for irraminidase. [Figure 8]IgM responses to neuraminidase are shown. [Figure 9] IgG response to neuraminidase is shown. [Figure 10] Neuraminidase activity assay. [Figure 11] Immunoblot analysis of VP7 expression is shown. [Figure 12] SDS-PAGE analysis of purified VP7 protein is shown. [Figure 13] Immunoblot analysis confirming the expression of fatty acid desaturase proteins is shown. [Figure 14] 1 shows immunoblot analysis confirming the expression of fatty acid elongase proteins. [Figure 15] 1 shows SDS-PAGE and immunoblot analysis of fatty acid elongase protein purified from scale-up cultures. [Figure 16] 1 depicts immunoblot analysis showing the expression of Nav1.7. [Figure 17] 1 illustrates confocal microscopy showing surface expression of NaV1.7. [Figure 18] To confirm the overexpression of CD59 protein, immunoblot analysis using anti-His and anti-CD59 antibodies is shown. [Figure 19] Purification of CD59 protein is shown. Summary of the Invention

[0035] The present invention relates to expression of difficult to express proteins (DTE-Ps) in a recombinant expression platform and discloses a versatile recombinant expression platform, wherein the recombinant expression platform comprises: i. an array of yeast-based expression vectors, wherein the vectors are selected from one or more episomal or integrative yeast-based expression vectors operably linked to one or more promoters selected from a Gal promoter, an ADH2 promoter, or a Gal10 promoter; wherein the promoters may be used alone or in combination; said vectors comprising an auxotrophic selection marker selected from Leu or Ura3, a CYCT1 terminator; an ampicillin resistance marker; a pUC ori; a 2 micron origin; one specific upstream regulatory sequence, and one sequence region comprising a multiple cloning site, wherein a desired target protein can be integrated; wherein said vectors direct the insertion of a full-length or truncated polynucleotide sequence encoding the desired target protein into a host cell; and said recombinant expression platform comprises: ii. Engineered protease-deficient yeast host cells with disrupted endogenous genes encoding auxotrophic markers for PRB1, PEP4, uracil, lysine, adenine, and leucine; wherein the platform allows for enhanced expression of difficult-to-express proteins of diverse origins and families.

[0036] Constructs, methods and kits involved in expressing such DTE-P via said system are also described. DETAILED DESCRIPTION OF THE INVENTION

[0037] The details of one or more embodiments of the present invention are set forth, by way of example, in the accompanying description below, including specific details of the best mode contemplated by the inventors for carrying out the invention. It will be apparent to those skilled in the art that the present invention can be practiced without limitation to these specific details.

[0038] definition The use of "comprise," "comprises," "comprising," "contain," "contains," "containing," "include," "includes," and "including" is not intended to be limiting. It is understood that the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.

[0039] The term "difficult to express proteins (DTEPs)" describes proteins that are difficult or impossible to express in a fully soluble, well-folded, and active form in a heterologous expression system.

[0040] The term "expression platform" describes a system for producing large amounts of proteins, sugars, or other compounds for research or industrial use.

[0041] The term "expression vector" describes a plasmid or virus designed for the expression of genes in cells.

[0042] The term "host cell" refers to a host cell used for the production of a recombinant protein.

[0043] The term "prokaryotic protein" includes proteins found in prokaryotic cells / organisms.

[0044] The term "eukaryotic protein" includes proteins found in eukaryotic cells / organisms.

[0045] The term "viral protein" includes proteins produced by viruses, including enzymatic proteins as well as structural proteins such as capsids and viral envelopes.

[0046] The term "mammalian protein" includes proteins produced in mammals.

[0047] The term "plant protein" includes proteins produced in plants.

[0048] The term "algal protein" includes proteins found in all classes of algae.

[0049] The term "highly hydrophobic protein" includes proteins with side chains that do not like to exist in an aqueous environment and are therefore difficult to express and purify.

[0050] "Multi-pass transmembrane protein" refers to a protein containing primarily non-polar amino acid residues that may traverse the bilayer membrane one or several times.

[0051] The term "transmembrane protein" includes a type of integral membrane protein that spans the entire cell membrane.

[0052] The term "structural protein" includes proteins that have a typical amino acid sequence that is repeating and contributes to the framework and provides mechanical strength to an organism or cell.

[0053] The term "ion channel receptor" includes multimeric proteins that are usually located in the plasma membrane.

[0054] Unless otherwise defined, scientific and technical terms used herein shall have the meanings commonly understood by those skilled in the art. Furthermore, unless otherwise required by context, singular terms shall include plural terms, and plural terms shall include singular terms. Generally, the nomenclature used in connection with cell and tissue culture, molecular biology, and protein and oligonucleotide or polynucleotide chemistry and hybridization, and the techniques described herein, are those well known and commonly used in the art.

[0055] The present invention discloses a recombinant yeast-based expression platform for enhancing the expression of difficult-to-express proteins (DTE-Ps) from various families and origins. The disclosed platform uses a recombinant yeast host-based system. The platform includes an array of both integrative and episomal vectors with designed upstream regulatory sequences; an engineered protease-deficient yeast host (protease-deficient strain); and codon harmonization for robust and enhanced expression of optimized sequences for proteins from various origins and families. Multiple engineered expression strains can be selected depending on the target protein and its intrinsic properties.

[0056] The present invention further discloses the use of a single recombinant expression platform to express several target proteins, including DTE-Ps of plant, human, animal, bacterial, fungal, or viral origin, and of various levels of complexity, different sources, categories, and families.

[0057] In a main embodiment, the present invention provides a universal recombinant expression platform, said recombinant expression platform comprising: i. an array of one or more episomal or integrative yeast-based expression vectors operably linked to one or more promoters selected from the Gal1 promoter, the ADH2 promoter, or the Gal10 promoter; wherein the promoters can be used alone or in combination; the vectors comprising an auxotrophic selection marker selected from Ura3 or Leu; the terminator CYCT1; an ampicillin resistance marker; an origin of replication pUC ori; a 2 micron origin; one or more specific upstream regulatory sequences; and a sequence region consisting of a multiple cloning site; wherein the vectors direct the insertion of a full-length or truncated polynucleotide sequence for a desired target protein into a host cell; and ii. Engineered protease-deficient yeast host cells with disrupted endogenous genes encoding protease PRB1, protease PEP4, and auxotrophic markers for uracil, lysine, adenine, and leucine; wherein the aforementioned platform allows for enhanced expression of difficult-to-express proteins of diverse origins and families.

[0058] In yet another embodiment, the engineered protease-deficient yeast host cell with disrupted endogenous genes encoding protease PRB1, protease PEP4, and auxotrophic markers for uracil, lysine, adenine, and leucine is of Saccharomyces cerevisiae origin.

[0059] In yet another embodiment, the difficult-to-express proteins are from a variety of origins and families and are selected from, but not limited to, those found in viruses, prokaryotes, eukaryotes, mammals, humans, plants, virus families, algae proteins, toxins, highly hydrophobic proteins, proteins with multiple transmembrane domains, transmembrane proteins, structural proteins, non-structural proteins, drug target receptors such as ion channel families, G protein-coupled receptors (GPCRs), GPI-anchored proteins, enzymes, TNFR families, those found in the plasma membrane and endoplasmic reticulum, proteins localized in the Golgi compartment and cytosol.

[0060] In yet another embodiment, the difficult-to-express protein is a viral protein, which may be a viral enzyme protein, which may be a membrane-bound single-pass membrane protein such as neuraminidase.

[0061] In yet another embodiment, the present invention proposes a nucleic acid construct having SEQ ID NO: 7 for the expression of membrane-bound neuraminidase, wherein said nucleic acid construct comprises a nucleic acid sequence having SEQ ID NO: 1 encoding full-length neuraminidase and an episomal expression vector comprising an auxotrophic selection marker Ura3, a CYCT1 terminator, an ampicillin resistance marker, pUC ori together with a Gal1 promoter.

[0062] In yet another embodiment, the present invention discloses a method for producing membrane-bound neuraminidase by a recombinant expression platform, the method comprising: i. preparing a nucleic acid construct; ii. transforming the construct into a protease-deficient yeast host cell; iii. Culturing the transformed host cells to enhance expression of neuraminidase.

[0063] In yet another embodiment, the neuraminidase protein elicits an immunogenic response and is functionally active.

[0064] In yet another embodiment, the difficult-to-express protein can be a highly hydrophobic viral protein, which is a structural protein that can be a capsid protein, such as VP7. The highly hydrophobic viral protein, structural capsid protein VP7, is a vaccine candidate.

[0065] In yet another embodiment, the present invention discloses a nucleic acid construct having SEQ ID NO: 8 for expression of viral structural capsid protein VP7, wherein said nucleic acid construct comprises the nucleic acid sequence of SEQ ID NO: 2 encoding full-length VP7 and an episomal expression vector comprising the auxotrophic selection marker Ura3, the CYCT1 terminator, an ampicillin resistance marker, and pUC ori with a Gal1 promoter.

[0066] In yet another embodiment, the present invention proposes a method for producing the highly hydrophobic viral structural capsid protein VP7 by a recombinant expression platform, said method comprising: i) preparing a nucleic acid construct; ii) transforming the construct into a protease-deficient yeast host cell; iii) culturing the transformed host cells to enhance expression of VP7.

[0067] In yet another embodiment, the difficult-to-express protein may be a multi-pass transmembrane protein, and the multi-pass transmembrane protein may be from the ion channel receptor family, and such a multi-pass transmembrane protein may be from the ion channel receptor family, and the protein may be a Nav1.7 protein, and the protein may be a drug target receptor protein.

[0068] In yet another embodiment, the present invention discloses a nucleic acid construct having SEQ ID NO: 9 for expressing the transmembrane ion channel receptor protein Nav1.7, wherein the nucleic acid construct comprises the nucleic acid sequence of SEQ ID NO: 3 encoding the full-length Nav1.7, and an episomal expression vector comprising the auxotrophic selection marker Ura3, the CYCT1 terminator, an ampicillin resistance marker, and pUC ori together with the Gal1 promoter.

[0069] In yet another embodiment, the present invention proposes a method for producing a transmembrane Nav1.7 protein by a recombinant expression platform, said method comprising: i) preparing a nucleic acid construct; ii) transforming the construct into a protease-deficient yeast host cell; iii) culturing the transformed host cells to enhance expression of Nav1.7; iv) Using confocal microscopy to localize the surface of expressed Nav1.7.

[0070] In yet another embodiment, the difficult-to-express protein may be an enzyme protein that is a transmembrane protein. Such transmembrane proteins may be from the lipid biosynthesis cycle and may be fatty acid desaturases and may be of fungal origin.

[0071] In yet another embodiment, the present invention provides a nucleic acid construct having SEQ ID NO: 10 for expression of a fatty acid desaturase, wherein said nucleic acid construct comprises the nucleic acid sequence of SEQ ID NO: 4 encoding full-length Nav1.7, and an episomal expression vector comprising the auxotrophic selection marker Ura3, the CYCT1 terminator, an ampicillin resistance marker, and pUC ori with a Gal1 promoter.

[0072] In yet another embodiment, the present invention provides a method for producing a fatty acid desaturase protein by a recombinant expression platform, said method comprising: i) preparing a nucleic acid construct; ii) transforming the construct into a protease-deficient yeast host cell; and iii) culturing the transformed host cell to enhance expression of the fatty acid desaturase.

[0073] In yet another embodiment, the difficult-to-express protein is an enzymatic protein and a transmembrane protein derived from the lipid biosynthesis cycle, wherein said transmembrane protein from lipid biosynthesis is a fatty acid elongase.

[0074] In yet another embodiment, the present invention provides a nucleic acid construct having SEQ ID NO: 11 for expression of a fatty acid elongase, wherein said nucleic acid construct comprises a nucleic acid sequence having SEQ ID NO: 5 encoding the full-length elongase and an episomal expression vector comprising an auxotrophic selection marker Ura3, a CYCT1 terminator, an ampicillin resistance marker, pUC ori with a Gal1 promoter.

[0075] In yet another embodiment, the present invention discloses a method for producing a fatty acid elongase protein by a recombinant expression platform, said method comprising: i) preparing a nucleic acid construct; ii) transforming the construct into a protease-deficient yeast host cell; and culturing the transformed host cell to enhance expression of the fatty acid elongase.

[0076] In yet another embodiment, the difficult-to-express protein may be a glycosylphosphatidylinositol-anchored (GPI-anchored) protein and may be a drug target protein, wherein the drug target protein is CD59 of human origin.

[0077] In yet another embodiment, the present invention provides a nucleic acid construct having SEQ ID NO: 12 for expression of the GPI-anchored protein CD59, wherein said nucleic acid construct comprises a nucleic acid sequence having SEQ ID NO: 6 encoding the full-length elongase, and an episomal expression vector comprising the auxotrophic selection marker Ura3, the CYCT1 terminator, an ampicillin resistance marker, pUC ori with a Gal1 promoter.

[0078] In yet another embodiment, the present invention provides a method for producing GPI-anchored CD59 by a recombinant expression platform, said method comprising: i) preparing a nucleic acid construct; ii) transforming the construct into a protease-deficient yeast host cell; iii) culturing the transformed host cells to enhance expression of CD59.

[0079] In yet another embodiment, the platform is scalable and capable of producing proteins from diverse origins and families on an industrial scale.

[0080] In yet another embodiment, the present invention provides a kit comprising the recombinant expression platform, wherein the recombinant expression platform comprises: i) a nucleic acid construct for encoding the difficult to express protein of interest; ii) an engineered protease-deficient yeast host cell; and iii) a manual for operating the kit.

[0081] [Table 1]

[0082] Representative S. cerevisiae expression vectors developed and used in the present invention are designated as follows: a, pYRI100 yeast integration vector containing a Leu auxotrophic selection marker, a CYCT1 terminator, a resistance marker, and a pUC ori with a Gal promoter. b. pYRI200 yeast integration vector containing a Leu auxotrophic selection marker, a CYCT1 terminator, a resistance marker, and pUC ori with the ADH2 promoter. c. pYRE100 yeast episomal vector containing the Ura3 auxotrophic selection marker, the CYCT1 terminator, a resistance marker for selection, and the pUC ori with the Gal promoter. d. pYRE200 yeast episomal vector containing the Ura3 auxotrophic selection marker, the CYCT1 terminator, a resistance marker for selection, and the pUC ori with the ADH2 promoter. [Example]

[0083] The present invention is further described below by way of example, and more particularly, the following paragraphs are provided to illustrate the best mode of carrying out the invention, and nothing in this section should be taken as limiting the scope of the claims.

[0084] Example 1: Expression of Neuraminidase (NA): In this example, influenza A virus (A / Hatay / 2004 / H5N1) was used as a study example for expression using a recombinant expression platform. Mutants were also successfully expressed using the same method, demonstrating a platform-adaptable, time-saving, and cost-effective approach. The full-length amino acid sequence of NA (449 aa) was utilized.

[0085] Example 1.2: Cloning and preparation of constructs The gene was cloned into the expression plasmid pYRE100 using conventional cloning methods so that the expressed protein had a C-terminal His tag (Figure 3). The cloned gene was analyzed through restriction digestion. The construct was transformed into the S. cerevisiae host of the recombinant expression platform for expression studies using anti-His antibody immunoblot analysis (Figure 5).

[0086] Example 1.2: Transformation of S. cerevisiae into a protease-deficient host strain: The characterized recombinant constructs were then finalized for expression studies. The constructs were transformed into a protease-deficient yeast strain using a lithium acetate / SS-DNA / PEG-mediated protocol, and transformants were selected on YNB glucose-URA plates along with a control (a protease-deficient strain transformed with the pYRE100 vector backbone). A small number of isolated, normal transformed colonies were inoculated into 10 ml of YNB glucose-URA medium, and expression was analyzed by immunoblot analysis 24 hours after induction with an anti-His antibody (induction A600 ~ 5.0 OD / ml in late logarithmic growth; final concentration 2% galactose).

[0087] Immunoblot analysis using anti-His antibody showed a specific band at a size greater than 52 kDa due to glycosylation of the protein in three colonies of the induced culture (lanes 2, 3, and 5), but no band was observed in the control sample (Fig. 5).

[0088] result: The S. cerevisiae strain and vector combination disclosed herein was used to express important viral vaccine candidates, such as neuraminidase. The expressed protein was found to be membrane-anchored, as it was purified in microsome preparations. This study demonstrates the robustness of the developed expression platform, as the majority of NA was expressed without the membrane-anchoring domain. Therefore, the expression platform can be scaled up to develop a robust expression system for large-scale production of viral vaccine candidates. Clones were further expanded up to 100X volumetric scale. Microsomes were prepared from the membrane fraction using the protocol described in the Examples. Expression was analyzed using anti-His immunoblot. The desired band was observed in both the cell lysate and the prepared microsomal fraction. This expression was verified against cell lysates and control microsomes (Figure 6).

[0089] Furthermore, plasma membrane localization of the protein was confirmed using flow cytometry studies (Figure 7).

[0090] Example 1.3: Plasma membrane localization of expressed NAs analysis using flow cytometry studies Sample preparation: Yeast cells expressing plasma membrane-localized NA equivalent to 1 OD600nm were obtained and fixed with paraformaldehyde (4% v / v) for 15 minutes with and without Triton X100 (0.25%), and samples were analyzed for permeabilized and non-permeabilized cells. S. cerevisiae cells lacking the inserted gene served as a control. Cells were resuspended, washed, resuspended in 1% BSA prepared in 1X PBS, and incubated for 1 hour at 25°C.

[0091] The cells were resuspended, washed, and incubated with a 1:50 dilution of primary antibody for 1 hour at 25°C, followed by three washes and secondary antibody incubation. Control cells were incubated with 1x PBS alone and centrifuged at 4000 rpm for 1 minute. Cells were resuspended in secondary antibody (1:100 μl) and incubated at 25°C for 30 minutes. The cells were washed with 1x PBS by centrifugation at 4000 rpm for 1.5 minutes at 4°C. The cells were resuspended in 2% FBS in 1x PBS, and readings were taken on a FACS Instruments ACEA NovoCyte Flow Cytometer (Model: 3005). For this study, a 6xHis tag-specific antibody was used as the primary antibody, and an anti-mouse Alexa 488-labeled secondary antibody, designated Alexa 488, was used.

[0092] result Flow cytometry data showed a shift in NA-expressing cells for both permeabilized and non-permeabilized samples. 11.13% of non-permeabilized cells showed NA expression on the surface, while expression in permeabilized cells was found to be higher (14.38%). The results suggest that the protein is localized to the cell surface (Figure 7).

[0093] Example 1.4: Animal studies Analysis of immunogenic responses in mice to recombinant neuraminidase (human) expressed using the S. cerevisiae platform.

[0094] For immunogen response studies, NA protein was concentrated as microsomes and injected intramuscularly into mice. For immunization, BALB / c mice were intramuscularly injected with a dose (50 μl containing 100 μg of NA microsomes) of the NA microsome preparation on days 0, 14, and 28. Mice were anesthetized and bled via the retroorbital / tail vein. Total IgG and IgM responses were measured using ELISA with preimmune sera on days 21 and 35.

[0095] Below are the host details to check the immune response: Test system: mice; Mouse strain: Balb / c Gender: Male Age: 6-8 weeks old

[0096] Study Design:

[0097] [Table 2]

[0098] Blood sampling for immune assessment To evaluate the immune response to NA microsomes, blood samples were collected from mice by retro-orbital bleeding after anesthesia administration. Control serum samples were collected one day before the start of immunization. Blood samples were collected after immunization, on days 21 and 35 after the second immunization, and on days 11 and 15 after the second immunization. The collected blood samples were used for serum preparation. Serum was collected from the samples and used to measure IgM and IgG responses (Figures 8 and 9). (Humoral immune response).

[0099] Assessment of humoral immune response: Serum samples were used to measure antibody responses to NA microsomes by ELISA. A microsome preparation from a native S. cerevisiae strain was used as a control. ELISA plates were coated overnight with either microsome preparation at 4°C. The plates were blocked with 1% BSA. Serum samples were then appropriately diluted, applied in duplicate, and incubated for 1 h at 37°C. The plates were then washed (PBS containing 0.01% Tween 20). IgG and IgM were then assessed by incubation with a secondary anti-mouse antibody conjugated with horseradish peroxidase (37°C, 1 h). The plates were developed using TMB substrate solution. The reaction was stopped with 2N H2SO4, and absorbance was determined at 450 nm.

[0100] Total IgG levels on days 21 and 35: A serum study measuring immune response To assess immune responses, NA and control microsomes were coated at a concentration of 100 ng / well for ELISA. Serum samples were diluted 1:1000 and 1:2500 for measurement of IgM and IgG responses, respectively (Figures 8 and 9).

[0101] These results demonstrated that mice injected with the NA microsome preparation exhibited specific IgM and IgG responses compared with control yeast cell microsomes. Therefore, the microsome preparation of NA protein is immunogenic and elicits an immune response in mice. Therefore, the NA protein derived from the yeast platform could potentially be used as a vaccine candidate.

[0102] Example 1.5: Cultures for microsome preparation: As a pre-seed, an isolated normal patched colony was inoculated into 100 ml of YNB Glucose-URA medium and cultured for 24 hours in a shaker incubator at 28°C together with the host strain protease deletion strain transformed with pYRE100 as a host vector control.

[0103] Scaled-up cultures were prepared by reinoculating 1 liter of YNB Glucose-URA medium with an inoculum OD600 of ~0.25 OD / ml and grown for 24 hours in a shaker incubator at 28°C. The cultures were harvested, and the cell pellets were induced with 2% final concentration of galactose in YNB-URA minimal medium. 24 hours after induction, all cultures were harvested. The harvested cell pellets were used to prepare microsomes. The prepared microsomes were analyzed for NA expression. Microsomes from a protease-deficient strain were used as a control.

[0104] The results depict the presence of expressed NA protein, detected using anti-His antibody in microsomes (lane 3; Figure 6), whereas no band was observed in control microsomes.

[0105] Example 1.6 Neuraminidase (NA) Activity Assessment: Microsome concentrations of 5 nM, 10 nM, 25 nM, and 50 nM were used for the activity assay, estimating 1% of the total cellular protein forming NA microsomes. 10 μl of each concentration sample was mixed with assay buffer (32.5 mM 2-(N-morpholino)ethanesulfonic acid (MES), pH 6.5, containing 4 mM CaCl2). The enzymatic reaction was initiated by adding 30 μl of 833 μM MUNANA substrate and then incubated at 37°C for 30 min. The reaction was terminated by adding 150 μl of stop solution (100 μM glycine in 25% ethanol, pH 10.7). The amount of released fluorescent product, 4-methylumbelliferone (4-MU), was measured using a Spectra MAX Gemini EM (Molecular Devices) fluorometer at excitation and emission wavelengths of 355 nm and 460 nm, respectively. Blank control reactions contained substrate only.

[0106] All reactions were performed in triplicate in 96-well flat-bottom opaque polystyrene plates (Corning Costar, Corning, NY, USA). A standard curve was generated by plotting relative fluorescence intensity against the amount of free 4-MU. One unit of NA was defined as 1 micromolar concentration of 4-MU produced per minute at 37°C. Microsome concentrations were determined using Bradford's method (Bradford, 1976) using bovine serum albumin as a standard.

[0107] result: The results showed that NA was active and saturated at a concentration of 50 nM (Figure 10). In this experiment, the substrate without NA was used as a control.

[0108] Example 2: Expression of viral structural capsid proteins Example 2.1: Expression of VP7 The viral capsid protein VP7 glycoprotein, a potential vaccine target, was recombinantly expressed using a yeast host expression platform. The gene for expression was codon-biased and optimized for expression in the yeast host. The protein was expressed with a 10x His tag. The gene was cloned into the pYRE100 expression vector using conventional cloning methods. The cloned gene was analyzed via restriction digestion. For expression studies, characterized constructs were transformed into a protease-deficient S. cerevisiae host strain. Expression of the His-tagged rVP7 was confirmed using an anti-His antibody in immunoblot analysis. The expression platform was scaled up to 25x. The expressed protein was purified using Ni-NTA chromatography and quantified against standards.

[0109] Characterized recombinant constructs were transformed into yeast hosts as described in other examples. A small number of isolated, healthy transformed colonies were inoculated into YNB Glucose-URA medium and expanded to prepare 475 ml cultures. Samples were analyzed for expression at 12 and 24 hours post-induction (induction in late logarithmic growth; final concentration of 2% galactose). Cells were harvested, and samples were prepared in 1x SDS to reduce the dye for expression analysis in cell pellets. Proteins were characterized using anti-His antibody immunoblots. Immunoblots were developed using anti-His tag as the primary antibody, followed by incubation in HRP-conjugated anti-mouse secondary antibody.

[0110] result: Using an anti-His antibody, bands at the precise size of 38 kDa were obtained at 24 h (two clones—(5) and (6)). Clone (6) also showed a weak band in the induced sample at 12 h, whereas no band was observed in the backbone (BB) and pre-induction (BI) samples (Figure 11). Using the previously described yeast expression platform, recombinant 10xHis-tagged VP7 capsid protein was efficiently expressed. The protein was purified using Ni-NTA affinity chromatography and the method described in the scale-up example. The use of this platform and similar expression and purification methods further demonstrated that the platform is user-friendly, cost-effective, and time-saving.

[0111] Example 2.2: Scaling up of the structural protein VP7 The protein was expressed with a 10x His tag. At small scale (20 ml), VP7 protein expression was negligible (yields ranged from 60 to 100 ng / ml). This clone was further expanded to 25x or 500 ml scale. The expressed protein was purified using affinity chromatography, e.g., Ni-NTA chromatography. Expression was analyzed by reducing SDS-PAGE.

[0112] result: After purification, the desired bands were observed. Yields were determined using BSA as a standard (Figure 12). Proteins were purified to >90% purity. Yields obtained ranged from 0.08 mg / ml to 8 mg / L as determined by densitometry analysis against a known BSA standard.

[0113] The scalable process, spanning a 25x linear volumetric range, demonstrates yield improvements, demonstrating and confirming the platform's ability to increase productivity and its suitability for the large-scale manufacturing required for a variety of applications.

[0114] Example 3: Expression of enzyme protein (fatty acid protein) The fatty acid desaturase and elongase enzyme gene sequences were codon-biased and optimized for expression in the S. cerevisiae host, and the genes were fused with a 10xhis tag at the C-terminus.

[0115] These genes were cloned into the proprietary expression plasmid pYRE100 using conventional cloning methods. The cloned genes were analyzed by restriction digestion. The constructs were transformed into S. cerevisiae hosts, and expression was examined by immunoblot analysis using an anti-His antibody.

[0116] Example 3.1: Process for Expression: Each recombinant construct characterized was transformed using methods similar to those described for other examples. Two clones of each were expressed in a unique protease-deficient yeast expression host in rich YPD medium. Expression was verified against a control transformed into yeast with the episomal vector backbone.

[0117] Scaled-up cultures of both proteins and both clones were grown in rich YPD medium (475 ml) and induced with 2% galactose. After 24 hours of induction, the cells were pelleted. The cells were resuspended in buffer and homogenized five times at 800 bar. The solution was centrifuged at 4000 rpm. The supernatant was collected without disturbing the pellet, and the pellet was solubilized in an equal volume of urea buffer (8 M urea, 20 mM Tris, pH 8). Both proteins were expressed and likely naturally localized to the ER membrane. Their expression was analyzed by immunoblot analysis. Immunoblots were developed using anti-His antibody as the primary antibody and HRP-conjugated anti-mouse antibody as the secondary antibody.

[0118] Fatty acid desaturase was observed in the supernatant fraction at ~41 kda per clone (Figure 13). Fatty acid elongase showed pellet expression of ~40 kda (expected size 33.4 kda) per clone (Figure 14). The larger size may be due to post-translational modification (glycosylation) in the yeast host.

[0119] Example 3.2: Scale-up of fatty acid desaturase: Further scale-up to 10x volumetric scale was performed using the platform described above. Scale-up batches were prepared at fermentation levels in YPD (yeast extract, peptone, and glucose) medium and induced with the same galactose as used in the 500 ml-scale analysis. The process demonstrated 10x scale-up and production of fatty acid desaturase enzymes.

[0120] A 0.5 mL pre-seed culture was prepared and incubated in a shaker incubator at 30°C for 15-20 hours to a cell density (OD600) of 3.0-4.0. 500 mL of the seed culture was inoculated into the medium to initiate initial fermentation. When the OD600 reached 7-8, the fermenter temperature was maintained at 25°C and 1 L of 5x YPG (yeast extract, peptone, and galactose) solution was added to induce the culture. The DO was maintained at 20% and the pH at 5.6-6.0.

[0121] The pellet was lysed by homogenization, solubilized, and purified using Ni NTA affinity chromatography. The purified protein was characterized by SDS PAGE and anti-His tag immunoblot (Figure 15). Immunoblots were developed using a His antibody as the primary antibody and an HRP-conjugated anti-mouse antibody as the secondary antibody.

[0122] A total of 2.85 mg of protein was purified from the 5 liter scale-up, clearly demonstrating further yield improvements with process development.

[0123] Example 4: Expression of ion channel receptor proteins We used the yeast platform herein to express the plasma membrane-localized Nav1.7 multi-pass transmembrane protein, a promising drug target candidate, using a combination of a protease-deficient S. cerevisiae host strain and an episomal expression vector.

[0124] The expression platform was scaled up 10-fold, demonstrating consistency in yield and protein localization. Membrane fractions were purified and analyzed as full-length proteins using protein-specific antibodies and confocal microscopy. These purified membrane fractions have been used in the development of compound screening assays in 96-well and 384-well formats. This scale-up has the significant advantage of eliminating batch-to-batch variability in assay setup and screening data, as the same batch of recombinant protein can be used to screen entire compound libraries or large numbers of compounds.

[0125] The major subunit of this channel is the α subunit, a >200 kDa protein. This subunit consists of four large domains with high internal homology, including 24 transmembrane multipath domains. The expression genes were codon-biased and optimized for expression in a yeast host. Using conventional cloning methods, these genes were cloned into the proprietary expression plasmid pYRE100. The cloned genes were analyzed by restriction digestion. The construct was transformed into the S. cerevisiae host of a recombinant expression platform and subjected to immunoblot analysis using an anti-His antibody.

[0126] Example 4.1: Process for the expression of Nav1.7 The characterized recombinant constructs were transformed into the yeast host described in Example 1. A small number of isolated, normal transformed colonies were inoculated into 20 ml of YNB Glucose-URA medium, and expression was analyzed by immunoblot analysis using a Nav1.7 protein-specific antibody 24 hours after induction (induction A600 ~ 3.0 OD / ml in late logarithmic growth; final concentration 2% galactose). Selected clones were further expressed at a 40 ml scale, and microsomes were prepared and localization was examined using confocal microscopy. Immunoblots were developed using a protein-specific antibody as the primary antibody, followed by an HRP-conjugated anti-mouse antibody as the secondary antibody.

[0127] result: Immunoblot analysis using specific antibodies revealed bright bands of 226 kDa and larger, which may be due to the glycosylation and oligomeric nature of the protein in the membrane preparation (lane 2). Minimal degradation was also observed. However, no bands were observed in the control (Figure 16). Using the vector and strain combinations described in this application, we demonstrated that complex and multi-path membrane proteins, which are highly impactful and valuable drug targets, can be expressed. This will also enable the study of other target proteins, such as ion channel receptor families, GPCRs, kinases, and phosphatases.

[0128] Example 4.2: Confocal laser microscopy analysis for membrane localization of Nav1.7 Confocal microscopy confirmed the localization of Nav1.7 to the cell surface (Figure 17). The purified protein (membrane and microsomal) can be used to screen for Nav1.7 inhibitors and may therefore also be advantageous for therapeutic purposes.

[0129] Example 5: Expression of drug target molecule (GPI-anchored protein): The protein was expressed with a 6xHis tag.

[0130] Example 5.1: Process for Expression: A single colony was picked from the yeast selection plate and placed in 5 ml of selective medium SD (glucose) containing the appropriate amino acid and incubated at 30°C for 22–24 hours with shaking. The culture was spun at 3500 rpm for 15 minutes at room temperature. The supernatant was removed, and the pellet was washed with sterile water. The mixture was again spun at 3500 rpm for 15 minutes at 4°C. The pellet was resuspended in 5 ml of induction yeast peptone (galactose) medium (YPG) and incubated at 30°C for 8 hours with shaking. The culture was spun down at 3500 rpm for 15 minutes at 4°C, the pellet was dissolved, and protein expression was analyzed. Clones were analyzed using both anti-His and anti-CD59 antibodies to confirm specific protein expression (Figure 18). The protein was further solubilized and purified using an NI-NTA column, achieving a purity level of over 90%. This approach is useful for establishing a screening assay for compounds that bind to the CD59 protein.

[0131] result: Expression of CD-59, a GPI-anchored glycoprotein, was confirmed at a small scale of 5 ml and confirmed using an anti-His antibody, showing optimal expression in 8-hour induction samples. These studies were performed at a small scale, and clones were further scaled up to 200x or 1 liter scale. The expressed protein was purified using affinity chromatography, such as Ni-NTA chromatography, and analyzed by reducing SDS-PAGE. After purification, the desired band was observed. Yields were measured using BSA as a standard (Figure 19).

[0132] Example 6: 500 ml scale-up process 250 mL of synthetic selective medium containing 2% glucose and each selection marker was prepared and mixed with the expression vector pYRE100, pYRI100, or both in the case of multiprotein components (depending on the final genotype). The expression was incubated in a shaker at 30 ± 1°C, 250 ± 10 rpm, with an OD600 of 1.5-2.0 for 16 hours.

[0133] Large-scale growth was performed using 2 x 200 mL of YPD medium in 1 L flasks at 30 ± 1°C, 250 ± 10 rpm, and incubated until OD600 = 4-5 (24 h). Cultures were then induced with 2 x 250 mL of 2% galactose in 1 L shake flasks and grown for 12 / 24 / 36 h as needed. The entire culture in pre-weighed centrifuge bottles was centrifuged at 1000 g (3000 / 4000 rpm) for 10 min at 4°C, and the cell pellet was weighed and stored at -80°C until further processing to purify the protein for analysis and characterization using SDS-PAGE, immunoblot, and flow cytometry per the specific conditions and requirements.

[0134] Table 2 below provides an overview of the scale of expression of representative proteins by the platform of the present invention.

[0135] [Table 3]

[0136] Some key features of this expression platform are: >Engineered to produce high expression of proteins with very low expression. >Engineered for broad applicability to proteins of different origins. > Engineered to be protease deficient. > Enhance expression through an array of expression vectors with engineered upstream regulatory sequences. >Multiple engineered expression lines for various target proteins and their unique properties. >Multiple protein co-expression through codon harmonization. >Potential for further optimization and engineering of strains to enhance protein expression. This engineered strain can be scaled up to a 500 L fermentation scale.

[0137] The present invention provides the following advantages: >Provides a versatile, robust, and scalable platform for conformationally active protein expression. > It can be used for a wide variety of proteins from different families and various sources. This technology can be applied to areas such as vaccine development, drug discovery, metabolism, diagnostics, therapeutics, and healthcare.

Claims

1. A versatile recombinant expression platform, comprising: i. an array of one or more episomal or integrative yeast-based expression vectors operably linked to one or more promoters selected from a Gal1 promoter, an ADH2 promoter, or a Gal10 promoter, wherein the promoters can be used alone or in combination, and the vectors comprise an auxotrophic selectable marker selected from Ura3 or Leu2, a terminator CYCT1, an ampicillin resistance marker, a pUC origin of replication site, a 2-micron origin, one or more specific upstream regulatory sequences, and a sequence region consisting of a multiple cloning site, wherein the vectors direct the insertion of a full-length or truncated polynucleotide sequence for a desired target protein into a host cell; ii. A recombinant expression platform comprising an engineered protease-deficient yeast host cell with disrupted endogenous genes encoding protease PRB1, protease PEP4, and auxotrophic markers for uracil, lysine, adenine, and leucine, wherein the platform allows for enhanced expression of difficult to express proteins of diverse origins and families.

2. 2. The recombinant expression platform of claim 1, wherein the engineered protease-deficient yeast host cell with disrupted endogenous genes encoding protease PRB1, protease PEP4, and auxotrophic markers for uracil, lysine, adenine, and leucine is Saccharomyces cerevisiae.

3. 2. The recombinant expression platform of claim 1, wherein the difficult-to-express proteins are from diverse origins and families and are selected from, but not limited to, viral, prokaryotic, eukaryotic, mammalian, human, plant, viral, algal proteins, toxins, highly hydrophobic proteins, proteins with multiple transmembrane domains, transmembrane proteins, structural proteins, non-structural proteins, drug target receptors such as ion channel families, G protein-coupled receptors (GPCRs), GPI-anchored proteins, enzymes, TNFR families, and those localized to the plasma membrane, endoplasmic reticulum, Golgi compartment, and cytosol.

4. 10. The recombinant expression platform of claim 1, wherein the difficult-to-express protein is a viral protein.

5. The recombinant expression platform of claim 4 , wherein the viral protein is a viral enzyme protein.

6. The recombinant expression platform of claim 5 , wherein the viral enzyme protein is a membrane-bound single-pass membrane protein, neuraminidase.

7. 7. A nucleic acid construct having SEQ ID NO: 7 for expressing the membrane-bound neuraminidase of claim 6, wherein the nucleic acid construct comprises a nucleic acid sequence having SEQ ID NO: 1 encoding the full-length neuraminidase, and an episomal expression vector comprising an auxotrophic selection marker Ura3, a CYCT1 terminator, an ampicillin resistance marker, and a pUC ori with a Gal1 promoter.

8. 10. A method for producing membrane-bound neuraminidase by the recombinant expression platform of claim 1, said method comprising: i. preparing the nucleic acid construct of claim 7; ii. Transforming the construct into the host cell of claim 1; iii. A method comprising culturing the transformed host cell to enhance expression of neuraminidase.

9. 10. A recombinant full-length neuraminidase protein expressed using the recombinant expression platform of claim 1, wherein the neuraminidase protein elicits an immunogenic response and is functionally active.

10. 2. The recombinant expression platform of claim 1, wherein the difficult-to-express protein is a highly hydrophobic viral protein.

11. The recombinant expression platform of claim 10, wherein the highly hydrophobic viral protein is a structural protein.

12. The recombinant expression platform of claim 11 , wherein the highly hydrophobic viral structural protein is a capsid protein.

13. The recombinant expression platform of claim 12, wherein the highly hydrophobic viral protein, the structural capsid protein, is a vaccine candidate.

14. The recombinant expression platform of claim 13, wherein the vaccine candidate protein is VP7.

15. 15. A nucleic acid construct having SEQ ID NO:8 for expressing the viral structural capsid protein VP7 of claim 14, wherein the nucleic acid construct comprises the nucleic acid sequence of SEQ ID NO:2 encoding full-length VP7, and an episomal expression vector comprising an auxotrophic selection marker Ura3, a CYCT1 terminator, an ampicillin resistance marker, and a pUC ori with a Gal1 promoter.

16. 10. A method for producing highly hydrophobic viral structural capsid proteins by the recombinant expression platform of claim 1, said method comprising: i) preparing the nucleic acid construct of claim 15; ii) transforming said construct into a host cell according to claim 1; iii) culturing the transformed host cell to enhance expression of VP7.

17. 2. The recombinant expression platform of claim 1, wherein the difficult-to-express protein is a multi-transmembrane protein.

18. 2. The recombinant expression platform of claim 1, wherein the difficult-to-express protein is a multi-transmembrane protein from the ion channel receptor family.

19. 19. The recombinant expression platform of claim 18, wherein the multi-transmembrane protein is from the ion channel receptor family.

20. The recombinant expression platform of claim 17, wherein the transmembrane protein is a drug target receptor protein.

21. The recombinant expression platform of claim 20, wherein the drug target receptor protein is the sodium ion channel receptor Nav1.

7.

22. A nucleic acid construct having SEQ ID NO: 9 for expressing the transmembrane ion channel receptor protein Nav1.7 described in claim 21, wherein the nucleic acid construct comprises an episomal expression vector containing the nucleic acid sequence of SEQ ID NO: 3 encoding full-length Nav1.7, and an auxotrophic selection marker Ura3, a CYCT1 terminator, an ampicillin resistance marker, and pUC ori together with a Gal1 promoter.

23. 10. A method for producing a transmembrane Navl.7 protein by a recombinant expression platform according to claim 1, said method comprising: i) preparing the nucleic acid construct of claim 22; ii) transforming said construct into a host cell according to claim 1; iii) culturing the transformed host cells to enhance expression of Navl.7; iv) using confocal microscopy to determine the surface localization of expressed Navl.

7.

24. 2. The recombinant expression platform of claim 1, wherein the difficult-to-express protein is an enzymatic protein.

25. 25. The recombinant expression platform of claim 24, wherein the enzyme protein is a transmembrane protein.

26. 26. The recombinant expression platform of claim 25, wherein the transmembrane protein is derived from lipid biosynthesis.

27. 27. The recombinant expression platform of claim 26, wherein the lipid biosynthesis protein is a fatty acid desaturase.

28. 28. The recombinant expression platform of claim 27, wherein the desaturase is of fungal origin.

29. A nucleic acid construct having sequence number 10 for expressing the fatty acid desaturase described in claim 28, comprising the nucleic acid sequence of sequence number 4 encoding full-length Nav1.7, and an episomal expression vector comprising an auxotrophic selection marker Ura3, a CYCT1 terminator, an ampicillin resistance marker, and pUC ori together with a Gal1 promoter.

30. 10. A method for producing a fatty acid desaturase protein by the recombinant expression platform of claim 1, said method comprising: i) preparing the nucleic acid construct of claim 29; ii) transforming said construct into a host cell according to claim 1; iii) culturing the transformed host cell to enhance expression of the fatty acid desaturase.

31. 2. The recombinant expression platform of claim 1, wherein the difficult-to-express protein is an enzymatic protein.

32. 32. The recombinant expression platform of claim 31 , wherein the enzyme protein is a transmembrane protein.

33. 33. The recombinant expression platform of claim 32, wherein the transmembrane protein is derived from lipid biosynthesis.

34. 34. The recombinant expression platform of claim 33, wherein said transmembrane protein from lipid biosynthesis is a fatty acid elongase.

35. 34. A nucleic acid construct having SEQ ID NO: 11 for expressing the fatty acid elongase of claim 33, comprising a nucleic acid sequence having SEQ ID NO: 5 encoding the full-length elongase, and an episomal expression vector comprising an auxotrophic selection marker Ura3, a CYCT1 terminator, an ampicillin resistance marker, and pUC ori with a Gal1 promoter.

36. 10. A method for producing a fatty acid elongase protein by the recombinant expression platform of claim 1, said method comprising: i) preparing the nucleic acid construct of claim 35; ii) transforming said construct into a host cell according to claim 1; iii) culturing the transformed host cell to enhance expression of the fatty acid elongase.

37. 2. The recombinant expression platform of claim 1, wherein the difficult-to-express protein is a glycosylphosphatidylinositol-anchored (GPI-anchored) protein.

38. 38. The recombinant expression platform of claim 37, wherein the GPI-anchored protein is a drug target protein.

39. 39. The recombinant expression platform of claim 38, wherein the drug target protein is CD59.

40. The recombinant expression platform of claim 1 , wherein the CD59 is of human origin.

41. 41. A nucleic acid construct having SEQ ID NO: 12 for expressing the GPI-anchored protein CD59 of claim 40, comprising a nucleic acid sequence having SEQ ID NO: 6 encoding the full-length elongase, and an episomal expression vector comprising an auxotrophic selection marker Ura3, a CYCT1 terminator, an ampicillin resistance marker, and pUC ori with a Gal1 promoter.

42. 10. A method for producing a GPI-anchored CD59 protein by the recombinant expression platform of claim 1, said method comprising: i) preparing the nucleic acid construct of claim 41; ii) transforming said construct into a host cell according to claim 1; iii) culturing the transformed host cell to enhance expression of CD59.

43. 10. The recombinant expression platform of claim 1, wherein the platform is scalable and capable of producing proteins from diverse origins and families on an industrial scale.

44. 10. A kit comprising the recombinant expression platform of claim 1, the kit comprising: i) a nucleic acid construct for encoding said difficult-to-express target protein; ii) engineered protease-deficient yeast host cells; iii) A kit comprising an instruction manual for operating said kit.

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