Recombinant expression platform, constructs and methods for expression of Difficult to Express Proteins (DTE-Ps)

Abstract

The present invention relates to expression of SARS-CoV like virus proteins [S, M and E] proteins; recombinant polynucleotides, polypeptides; constructs, virus-like particles (VLPs); immunogenic compositions or vaccines comprising Virus Like Particles (VLPs). Method of producing the VLPs/expressing the multi-subunit virus like proteins and method for co-expression of multi-subunit and virus like proteins (VLPs) are also provided. The present invention also provides strategies, methods, systems, kits and combinations for scalable expression, purification and enhanced production of the virus like proteins of SARS-CoV while maintaining their size range and composition. Such multi-subunit VLPs can be utilized to make immunogenic compositions or vaccines.

Claims

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

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

3. The recombinant expression platform as claimed in claim 1, wherein said difficult to express proteins are from diverse origin and families selected from, but not limited to, viral, prokaryotic, eukaryotic, mammalian, human, plant, virus, 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 family, G-protein coupled receptors (GPCRs), GPI anchored proteins, enzymes, TNFR family and those localized in plasma membrane, endoplasmic reticulum, Golgi compartment and cytosol localized proteins.

4. The recombinant expression platform as claimed in claim 1, wherein said difficult to express protein is a viral protein which is a viral enzyme protein and optionally further is a membrane bound single pass membrane protein such as Neuraminidase.

5-6. (canceled)

7. A nucleic acid construct with at least one SEQ ID NO selected from: 7, 8, 9, 10, 11, and 12 for expression of at least one protein selected from: a membrane bound Neuraminidase, a viral structural capsid protein VP7, a fatty acid desaturase, a fatty acid elongase, and a GPI anchor protein CD59, wherein said construct comprises a nucleic acid sequence with at least one SEQ ID NO selected from: 1, 2, 3, 4, 5, and 6 respectively, encoding for full length of the protein selected from: the membrane bound Neuraminidase, the VP7, the fatty acid desaturase, the fatty acid elongase, and the GPI anchor protein CD59, respectively, and an episomal expression vector comprising Ura3 auxotrophic selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal1 promoter.

8. A method of producing at least one protein selected from: a membrane bound Neuraminidase, a viral structural capsid protein VP7, a fatty acid desaturase, a fatty acid elongase, and a GPI anchor protein CD59, by the recombinant expression platform as claimed in claim 1, comprising the steps of: i. preparing the nucleic acid construct with at least one SEQ ID NO selected from: 7, 8, 9, 10, 11, and 12 for expression of at least one protein selected from: a membrane bound Neuraminidase, a viral structural capsid protein VP7, a fatty acid desaturase, a fatty acid elongase, and a GPI anchor protein CD59, wherein said construct comprises a nucleic acid sequence with at least one SEQ ID NO selected from: 1, 2, 3, 4, 5, and 6 respectively, encoding for full length of the protein selected from: the membrane bound Neuraminidase, the VP7, the fatty acid desaturase, the fatty acid elongase, and the GPI anchor protein CD59, respectively, and an episomal expression vector comprising Ura3 auxotrophic selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal1 promoter; ii. transforming the said construct in the host cell of claim 1; iii. culturing the transformed host cell for enhanced expression of at least one protein selected from: the membrane bound Neuraminidase, the VP7, the fatty acid desaturase, the fatty acid elongase, and the GPI anchor protein CD59.

9. The recombinant full length Neuraminidase protein expressed using the platform as claimed in claim 1, wherein said Neuraminidase protein elicits an immunogenic response and is functionally active.

10. The recombinant expression platform of claim 1, wherein said difficult to express protein is highly hydrophobic viral protein which is a structural protein which is a capsid protein that is a vaccine candidate protein such as VP7.

11-16. (canceled)

17. The recombinant expression platform as claimed in claim 1, wherein said difficult to express protein is a multi-pass transmembrane protein from ion channel receptor family and is a drug target receptor protein such as a sodium ion channel receptor Nav1.7.

18-22. (canceled)

23. The method according to claim 8 further comprising assessing surface localization of expressed protein using confocal microscopy.

24. The recombinant expression platform as clamed in claim 1, wherein said difficult to express protein is an enzyme protein such as a transmembrane protein from lipid biosynthesis and is a fatty acid desaturase of fungal origin.

25-30. (canceled)

31. The recombinant expression platform as clamed in claim 1, wherein said difficult to express protein is an enzyme protein such as a transmembrane protein from lipid biosynthesis and is a fatty acid elongase.

32-36. (canceled)

37. The recombinant expression platform as claimed in claim 1, wherein said difficult to express protein is a glycosylphosphatidylinositol-anchored (GPI anchor) protein which is a drug target protein such as CD59 and is of human origin.

38-42. (canceled)

43. The recombinant expression platform of claim 1, wherein said platform is scalable and capable of producing proteins from diverse origin and families at an industrial scale.

44. A kit comprising the recombinant expression platform as claimed in claim 1 comprising: i) nucleic acid constructs encoding for said difficult to express target proteins; ii) engineered protease deficient yeast host cells; iii) instruction manual for operating said kit.

Description

BRIEF DESCRIPTION OF FIGURES AND DRAWINGS

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

[0034] FIG. 1 shows the pYRI100 yeast integrative vector for expression of recombinant proteins using inducible GAL promoter.

[0035] FIG. 2 shows the pYRI200 yeast integrative vector for expression of recombinant proteins using ADH2 constitutive promoter.

[0036] FIG. 3 shows pYRE100 yeast episomal vector for expression of recombinant proteins using inducible GAL promoter.

[0037] FIG. 4 shows pYRE200 yeast episomal vector for expression of recombinant proteins using ADH2 constitutive promoter.

[0038] FIG. 5 shows Western Blot analysis of Neuraminidase.

[0039] FIG. 6 shows the Western Blot analysis of Neuraminidase at large scale.

[0040] FIG. 7 shows the flow cytometry result of Neuraminidase.

[0041] FIG. 8 shows IgM response against Neuraminidase.

[0042] FIG. 9 shows IgG response against Neuraminidase.

[0043] FIG. 10 Activity assay of Neuraminidase

[0044] FIG. 11 shows Immunoblot analysis of expression of VP7.

[0045] FIG. 12 shows SDS PAGE analysis of purified VP7 protein.

[0046] FIG. 13 shows Immunoblot analysis confirming expression of fatty acid desaturase protein.

[0047] FIG. 14 shows Immunoblot analysis confirming the expression of fatty acid elongase protein.

[0048] FIG. 15 illustrates SDS PAGE and Immunoblot analysis of purified fatty acid elongase protein from scale up culture.

[0049] FIG. 16 depicts Immunoblot analysis showing expression of Nav1.7.

[0050] FIG. 17 illustrates Confocal Microscopy showing surface expression of NaV1.7.

[0051] FIG. 18 shows the Immunoblot analysis using Anti-His antibody and Anti-CD59 antibody to confirm overexpression of CD59 protein.

[0052] FIG. 19 shows the purification of CD59 protein.

SUMMARY

[0053] The present invention relates to the expression of difficult to express proteins (DTE-Ps) in a recombinant expression platform and discloses a versatile recombinant expression platform comprising: [0054] i. an array of yeast based expression vectors, wherein the vectors are selected from one or more episomal or integrated yeast-based expression vectors operably linked with promoters selected from Gal1 promoter, ADH2 promoter or Gal10 promoter; wherein the promoters can be used singly or in combination; the said vector comprising an auxotrophic selection marker selected from Leu or Ura3, CYCT1 terminator; resistance marker Ampicillin; pUC ori; 2 micron origin; a specific upstream regulatory sequence and a sequence region comprising of multiple cloning sites, wherein desired target proteins could be incorporated; wherein the said vector directs insertion of full length or truncated polynucleotide sequence encoding the desired target protein into the host cell; [0055] ii. engineered protease deficient yeast host cell with disrupted endogenous genes encoding PRB1, PEP4, uracil, lysine, adenine and leucine auxotrophic markers and wherein said platform allows for enhanced expression of difficult to express proteins of diverse origin and families.

[0056] Constructs, methods and kits involved in expressing such DTE-Ps through the said system are also described.

DETAILED DESCRIPTION

[0057] The details of one or more embodiments of the invention are set forth in the accompanying description below including specific details of the best mode contemplated by the inventors for carrying out the invention, by way of example. It will be apparent to one skilled in the art that the present invention may be practiced without limitation to these specific details.

Definitions

[0058] The use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and this detailed description are exemplary and explanatory only and are not restrictive.

[0059] The term “difficult-to-express proteins (DTEPs)” defines the proteins that are difficult to or impossible to emerge in fully soluble, well-folded, and active form in a heterologous expression system.

[0060] The term “expression platform” defines a system to produce large amounts of proteins, sugars or other compounds for research or industrial uses.

[0061] The term “expression vectors” defines a plasmid or virus designed for gene expression in cells.

[0062] The term “host cell” means a host cell used for generation of recombinant proteins.

[0063] The term “prokaryotic proteins” includes the proteins found in prokaryotic cells/organisms.

[0064] The term “eukaryotic proteins” includes the proteins found in eukaryotic cells/organisms.

[0065] The term “viral proteins” includes proteins generated by viruses including enzyme proteins as well as structural proteins such as capsid and viral envelope.

[0066] The term “mammalian proteins” include proteins produced in mammals

[0067] The term “plant protein” includes proteins produced in plants.

[0068] The term “algal proteins” include the proteins found in all class of algae.

[0069] The term “highly hydrophobic proteins” includes proteins with side chains that do not like to reside in an aqueous environment and hence difficult to express and purify.

[0070] The term “proteins with multiple transmembrane” includes proteins predominantly with nonpolar amino acid residues with possibility of traversing the bilayer once or several times.

[0071] The term “transmembrane proteins” includes type of integral membrane proteins that span the entirety of the cell membrane.

[0072] The term “structural proteins” includes the proteins that have typical amino acid sequence which are repetitive and contributes to the framework and provides mechanical strength to the living organism or cell.

[0073] The term “ion channel receptors” includes multimeric proteins usually located in the plasma membrane.

[0074] Unless otherwise defined, scientific and technical terms used herein shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, nomenclatures utilized in connection with, and techniques of, cell and tissue culture, molecular biology, and protein and oligo- or polynucleotide chemistry and hybridization described herein are those well-known and commonly used in the art.

[0075] The present invention discloses recombinant yeast-based expression platform, for enhanced expression of difficult to express proteins (DTE-Ps) of various families and origin. The disclosed platform uses a recombinant yeast host-based system. The platform includes the array of vectors, both integrative and episomal, with designed upstream regulatory sequence; engineered protease deficient yeast host (Protease deficient strain) and codon harmonization for the robust and enhanced expression of sequence optimized proteins from different origin and families. Multiple engineered expression strains can be selected depending on target protein and its intrinsic properties.

[0076] The present invention further discloses the use of single recombinant expression platform for expression of several target proteins, including DTE-Ps of plant, human, animal, bacterial, fungal or viral origin, and with various levels of complexity, different sources, categories and families.

[0077] In the principal embodiment, the present invention provides a versatile recombinant expression platform comprising: [0078] i. an array of one or more episomal or integrated yeast based expression vectors operably linked with one or more promoters selected from Gal1 promoter, ADH2 promoter or Gal10 promoter; wherein the promoters can be used singly or in combination; the said vector comprising an auxotrophic selection marker selected from Ura3 or Leu; a terminator CYCT1; an Ampicillin resistance marker; an origin of replication site pUC ori; a 2 micron origin; one or more specific upstream regulatory sequences and a sequence region comprising of multiple cloning sites; wherein said vector directs insertion of full length or truncated polynucleotide sequence for desired target proteins into the host cell; [0079] ii. engineered protease deficient yeast host cell with disrupted endogenous genes encoding protease PRB1, protease PEP4 and auxotrophic markers uracil, lysine, adenine and leucine; and
wherein said platform allows for enhanced expression of difficult to express proteins of diverse origin and families.

[0080] In still another embodiment, engineered protease deficient yeast host cell with disrupted endogenous genes encoding protease PRB1, protease PEP4 and auxotrophic markers uracil, lysine, adenine and leucine is of Saccharomyces cerevisiae origin.

[0081] In yet another embodiment, said difficult to express proteins are from diverse origin and families and are selected from, but not limited to, viral, prokaryotic, eukaryotic, mammalian, human, plant, virus family, 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 family, G-protein coupled receptors (GPCRs), GPI anchored proteins, enzymes, TNFR family, plasma membrane and those found in endoplasmic reticulum, Golgi compartment and cytosol localized proteins

[0082] In still another embodiment, said difficult to express protein is a viral protein and the said viral protein could be a viral enzyme protein and in turn the said viral enzyme protein could be a membrane bound single pass membrane protein, such as a Neuraminidase.

[0083] In still another embodiment, the present invention proposes a nucleic acid construct with SEQ ID NO 7 for expression of membrane bound Neuraminidase wherein said construct comprises a nucleic acid sequence with SEQ ID NO 1 encoding for full length Neuraminidase, and an episomal expression vector comprising Ura3 auxotrophic selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal1 promoter.

[0084] In still another embodiment, the present invention discloses a method of producing membrane bound Neuraminidase by the recombinant expression platform, comprising the steps of: [0085] i. preparing the nucleic acid construct; [0086] ii. transforming the said construct in protease deficient yeast host cell; [0087] iii. culturing the transformed host cell for enhanced expression of Neuraminidase.

[0088] In yet another embodiment, said Neuraminidase protein elicits an immunogenic response and is functionally active.

[0089] In still another embodiment, said difficult to express protein could be a highly hydrophobic viral protein, the said highly hydrophobic viral protein being a structural protein, which could be a capsid protein, such as VP7. The said highly hydrophobic viral structural, capsid protein VP 7 being a vaccine candidate.

[0090] In yet another embodiment, the present invention discloses a nucleic acid construct with SEQ ID No 8 for expression of viral structural capsid protein VP7, wherein said construct comprises a nucleic acid sequence SEQ ID NO 2 encoding for full length VP7, and an episomal expression vector comprising Ura3 auxotrophic selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal1 promoter.

[0091] In yet another embodiment, the present invention proposes a method of producing highly hydrophobic viral structural capsid protein VP7 by the recombinant expression platform, comprising the steps of: [0092] i) preparing the nucleic acid construct; [0093] ii) transforming the said construct in protease deficient yeast host cell; [0094] iii) culturing the transformed host cell for enhanced expression of VP7.

[0095] In still another embodiment, said difficult to express protein could be a multi-pass transmembrane protein, said multi-pass transmembrane protein being from ion channel receptor family and such multi-pass transmembrane protein is from ion channel receptor family and the said protein can be a Nav 1.7 protein and the same could be a drug target receptor protein.

[0096] In yet another embodiment, A nucleic acid construct with SEQ ID NO 9 for expression of transmembrane ion channel receptor protein Nav1.7, wherein said construct comprising a nucleic acid sequence SEQ ID NO 3 encoding for full length Nav1.7, and an episomal expression vector comprising Ura3 auxotrophic selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal1 promoter.

[0097] In yet another embodiment, the present invention proposes a method of producing transmembrane Nav1.7 protein by the recombinant expression platform of the present invention comprising the steps of: [0098] i) preparing the nucleic acid construct; [0099] ii) transforming the said construct in protease deficient yeast host cell; [0100] iii) culturing the transformed host cell for enhanced expression of Nav1.7. [0101] iv) surface localization of expressed Nav1.7 using confocal microscopy.

[0102] In still another embodiment, said difficult to express protein could be an enzyme protein which being a transmembrane protein. Such transmembrane protein is from lipid biosynthesis cycle and being a fatty acid desaturase and can be from fungal origin.

[0103] In yet another embodiment, the present invention proposes a nucleic acid construct with SEQ ID NO 10 for expression of fatty acid desaturase, wherein said construct comprising a nucleic acid sequence SEQ ID NO 4 encoding for full length Nav1.7, and an episomal expression vector comprising Ura3 auxotrophic selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal1 promoter.

[0104] In still another embodiment, the present invention proposes a method of producing fatty acid desaturase protein by the recombinant expression platform comprising the steps of: [0105] i) preparing the nucleic acid construct; [0106] ii) transforming the said construct in protease deficient yeast host cell; [0107] iii) culturing the transformed host cell for enhanced expression of fatty acid desaturase.

[0108] In still another embodiment, said difficult to express protein is an enzyme protein and being a transmembrane protein from lipid biosynthesis cycle. Said transmembrane protein from lipid biosynthesis being a fatty acid elongase.

[0109] In still another embodiment, the present invention proposes a nucleic acid construct with SEQ ID NO 11 for expression of fatty acid elongase, wherein said construct comprising a nucleic acid sequence with SEQ ID NO 5 encoding for full length elongase, and an episomal expression vector comprising Ura3 auxotrophic selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal1 promoter

[0110] In yet another embodiment, the present invention discloses a method of producing fatty acid elongase protein by the recombinant expression platform, comprising the steps of: [0111] i) preparing the nucleic acid construct; [0112] ii) transforming the said construct in protease deficient yeast host cell; culturing the transformed host cell for enhanced expression of fatty acid elongase.

[0113] In still another embodiment, said difficult to express protein could be a glycosylphosphatidylinositol-anchored (GPI anchor) protein and could be a drug target protein, the said drug target protein being CD59 of human origin.

[0114] In yet another embodiment, the present invention proposes a nucleic acid construct with SEQ ID NO 12 for expression of GPI anchor protein CD59, wherein said construct comprising a nucleic acid sequence with SEQ ID NO 6 encoding for full length elongase, and an episomal expression vector comprising Ura3 auxotrophic selection marker, CYCT1 terminator, an Ampicillin resistance marker, pUC ori along with Gal1 promoter.

[0115] In still another embodiment, the present invention proposes a method of producing GPI anchor CD59 protein by the recombinant expression platform comprising the steps of: [0116] i) preparing the nucleic acid construct; [0117] ii) transforming the said construct in protease deficient yeast host cell; [0118] iii) culturing the transformed host cell for enhanced expression of CD59.

[0119] In still another embodiment, said platform is scalable and capable of producing proteins from diverse origin and families at an industrial scale.

[0120] In yet another embodiment, the present invention provided a kit comprising the recombinant expression platform comprising: [0121] i) nucleic acid constructs encoding for said difficult to express target proteins; [0122] ii) engineered protease deficient yeast host cells; [0123] iii) instruction manual for operating said kit.

TABLE-US-00001 TABLE 1 SEQ IDs corresponding certain target proteins and respective constructs SEQ IDs Target proteins/respective constructs SEQ ID NO 1 Neuraminidase SEQ ID NO 2 VP7 SEQ ID NO 3 Nav1.7 SEQ ID NO 4 Desaturase SEQ ID NO 5 Elongase SEQ ID NO 6 CD59 SEQ ID NO 7 Neuraminidase vector construct SEQ ID NO 8 VP7 vector construct SEQ ID NO 9 Nav1.7 vector construct SEQ ID NO 10 Desaturase vector construct SEQ ID NO 11 Elongase vector construct SEQ ID NO 12 CD59 vector construct

[0124] Representative S. cerevisiae expression vectors as developed and used in the present invention are designated as below: [0125] a. pYRI100 yeast integrative vector comprising Leu auxotrophic selection marker, CYCT1 terminator, resistance marker, pUC ori along with Gal promoter. [0126] b. pYRI200 yeast integrative vector comprising Leu auxotrophic selection marker, CYCT1 terminator, resistance marker, pUC ori along with ADH2 promoter. [0127] c. pYRE100 yeast episomal vector comprises Ura3 auxotrophic selection marker, CYCT1 terminator, resistance marker for selection, pUC ori along with Gal promoter. [0128] d. pYRE200 yeast episomal vector comprises Ura3 auxotrophic selection marker, CYCT1 terminator, resistance marker for selection, pUC ori along with ADH2 promoter.

EXAMPLES

[0129] The present invention is further described hereinbelow by way of illustration and more particularly, the following paragraphs are provided in order to describe the best mode of working the invention and nothing in this section should be taken as a limitation of the claims.

Example 1: Expression of Neuraminidase (NA)

[0130] The present example uses Influenza A virus (A/Hatay/2004/(H5N1) as study example for expression using recombinant expression platform. Variant was also expressed successfully using same methodologies showing the platform adaptability, time saving and cost-effective approach. Full length Amino acid sequence of NA (449aa) was utilized.

Example 1.2: Cloning and Construct Preparation

[0131] The gene was cloned using conventional cloning methodologies into expression plasmid pYRE100 (FIG. 3) such that the expression protein has a C-terminus His tag. The cloned gene was analyzed through restriction digestion. The construct was transformed into S. cerevisiae host of recombinant expression platform for expression studies using anti-His antibody immunoblots analysis (FIG. 5).

Example 1.2: Transformation in S. cerevisiae Protease Deficient Host Strain

[0132] The characterized recombinant construct was finalized for expression studies. The construct was transformed into protease deficient yeast strain using Lithium acetate/SS-DNA/PEG mediated protocol and transformants were selected over YNB Glucose—URA plates along with control (Protease deficient strain transformed with pYRE100 vector backbone). Few isolated healthy transformed colonies were inoculated in 10 ml of YNB Glucose—URA media and were analyzed for expression in 24.sup.th hr post induced (Induction at late log phase .sup.A600˜5.0 OD/ml; final concentration 2% galactose) time point samples using anti-His antibody by Immuno-blot analysis

[0133] Immuno Blot analysis using anti His antibody showed a specific band at higher size of ˜52 kda due to glycosylation of the protein in three colonies (Lane 2, 3 and 5) of induced cultures. However, no band was observed in control sample (FIG. 5).

Results:

[0134] The S. cerevisiae strain and vector combination disclosed herein is used to express a significant viral vaccine candidate like Neuraminidase. The expressed protein was also found to be membrane anchored as it purified in the microsomal preparations. The current study demonstrates robustness of the developed expression platform as NA has mostly been expressed without the membrane anchor domain Thus, the expressed platform can be scaled up to develop a robust expression system for large scale production of viral vaccine candidates. The clone was further scaled up to 100× of volumetric scale. From the membrane fraction; microsomes were prepared using protocol mentioned in example. Expression was analysed on using anti-His immunoblot. Band of interest was observed in both cell lysate and prepared microsomes fraction. The expression was verified against cell lysate and microsomes of control (FIG. 6).

[0135] Further the plasma membrane localization of protein was confirmed using Flow cytometry studies (FIG. 7).

Example 1.3: Plasma Membrane Localization of Expressed NA Analysis Using Flow Cytometry Studies

Sample Preparation

[0136] Yeast cells expressing plasma membrane localized NA equivalent to 1 OD.sub.600 nm were taken and fixed with paraformaldehyde (4% v/v) for 15 mins of with and without Triton X100 (0.25%) for with and without permeabilization cells sample analysis. S. cerevisiae cells without any gene of insert were taken as control. Cells were re-suspended, washed, re-suspended and incubated in 1% BSA prepared in 1×PBS for 1 hr at 25° C.

[0137] The cells were further washed, re-suspended and incubated with 1:50 dilution of primary antibody for 1 hour at 25° C. followed by secondary antibody incubation after three washings Control cells were incubated in 1×PBS only. Centrifugation at 4000 rpm 1 min. Re-suspended the cells in secondary antibody (1:100 μl), and were incubated for 30 min at 25° C. The cells were washed with of 1×PBS by centrifugation at 4000 rpm for 1.5 minutes at 4° C. Cells were re-suspended in 2% FBS in 1×PBS and readings were taken in FACS Instruments ACEA Novo Cyte Flow Cytometer (Model: 3005). 6×His tag specific antibody was used as primary antibody and anti-mouse Alexa flour 488 labelled secondary antibody was used for the study.

Results

[0138] The Flow cytometry data showed the shift in NA expression cells for both permeabilized and non-permeabilized samples. The 11.13% in non-permeabilized cells showed the expression of NA at surface while in permeabilized cells expression was found to be more (14.38%). The result suggests the protein localizes to the cell surface (FIG. 7).

Example 1.4: Animal Studies

[0139] Analysis of immunogenic response in mice against recombinant Neuraminidase (human) expressed using S. cerevisiae platform.

[0140] The NA protein was enriched, as microsomes and injected in mice intramuscularly for studying the immunogenic response. For immunization, BALB/c mice were injected intramuscularly (i.m) with NA microsomal formulations in a dose volume of (50 μl having 100 μg of NA microsomes) on day 0, 14 and 28. Mice were bled retro-orbitally/tail vein after administration of Anesthesia. Total IgG, and IgM response were measured using ELISA with pre-immune, 21- and 35-day sera.

[0141] Following are the host details for checking immune response:

[0142] Test system: Mice; Mus musculus

[0143] Strain: Balb/c

[0144] Sex: Male

[0145] Age: 6-8 weeks

Study Design:

[0146]

TABLE-US-00002 Route of Antigen administration Purpose Requirement Immunogenic response studies Balb/c Microsomal Intramuscular Study immune 3 mice study preps response against NA

Collection of Blood Samples for Immune Assessments

[0147] In order to assess the immune response generated against the NA microsomes blood samples were collected from mice by retro-orbital bleeding after administration of anesthesia. Control serum samples were collected a day prior to the initiation of immunization. Post immunization, blood samples were collected after second immunization at 21.sup.st day and on day 35. The blood samples collected were used for serum preparation. The serum from the samples was collected and used to measure IgM and IgG response (FIG. 8 and FIG. 9) (humoral immune response).

Evaluation of the Humoral Immune Response:

[0148] The serum samples were used to measure the antibody response against the NA microsomes using ELISA. Microsomes preparations from native S. cerevisiae strain were used as control. ELISA plates were coated with either microsomal preparation overnight at 4° C. The plates were blocked with 1% BSA. Subsequently the serum samples were diluted appropriately and applied in duplicate and incubated at 37° C. for 1 h. The plate were subsequently washed (PBS containing 0.01% of Tween 20). Followed by secondary anti-mouse antibodies conjugated with horseradish peroxidase incubation to estimate IgG and IgM (1 h at 37° C.). The plates were developed using TMB substrate solution for color development. The reaction was stopped with 2N H2S04, and absorbance determined at 450 nm.

Total IgG Levels at 21 and 35 Days: Sera Studies to Determine Immune Response

[0149] To assess the immune response, NA and control microsomes were coated at a concentration of 100 ng/well for ELISA. Serum samples were diluted at 1:1000 and 1:2500 for determination of IgM and IgG response respectively (FIG. 8 and FIG. 9).

[0150] These results show that the mice injected with the NA microsomal preparations showed a specific IgM and IgG response in comparison to the control yeast cell microsomes. Thus, suggesting the NA protein in the microsomal preparations is immunogenic and elicits an immune response in mice. Thus, the NA protein from the yeast platform could be potentially used as a vaccine candidate.

Example 1.5: Culture for Microsomes Preparation

[0151] Isolated healthy patched colonies were inoculated in 100 ml of YNB Glucose—URA media as pre seed and were cultured in shaker incubator at 28° C. for 24 hr along with host strain Protease deficient strain transformed with pYRE100 as host-vector control.

[0152] Scale up culture was prepared by re inoculation in 1 litre of YNB Glucose—URA media with ˜0.25 OD/ml as inoculum OD600 and were cultured in shaker incubator at 28° C. for 24 hr. The culture was harvested, and the cell pellet was induced with galactose at a final concentration of 2% in YNB—URA minimal medium. All the cultures were harvested at the 24 hr of post induction. Harvested cell pellets were used for Microsome preparation. The microsomes prepared were analyzed for expression of NA. Microsomes from the protease deficient strain were used as control.

[0153] Results depict the presence of expressed NA protein probed using anti His antibody in the Microsomes (lane 3; FIG. 6). However, no band was observed in control microsomes.

Example 1.6 Activity Assay of Neuraminidase (NA)

[0154] Microsome concentration of 5 nM, 10 nM, 25 nM and 50 nM was used for the activity assay, assuming 1% of total cell protein form the NA microsomes. 10 μl of respective concentration sample was mixed with an equal volume of assay buffer (32.5 mM 2-(N-morpholino) ethane sulfonic acid (MES), pH 6.5, containing 4 mM CaCl2). The enzymatic reaction was initiated by addition of 30 μl of 833 μM MUNANA substrate, followed by a 30 min incubation at 37° C. The reaction was terminated by the addition of 150 μl of stop solution (100 μM glycine, pH-10.7, in 25% ethanol). The amount of fluorescent product, 4-methylumbelliferone (4-MU) released was measured in a Spectra MAX Gemini EM (Molecular Devices) Fluorimeter with excitation and emission wavelengths of 355 and 460 nm respectively. Blank control reactions contained substrate alone.

[0155] All reactions were conducted in triplicate in 96-well flat-bottom opaque polystyrene plates (Corning Costar, Corning, N.Y., 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 one micromolar of 4-MU produced per min at 37° C. Microsome concentration was determined using Bradford's method with bovine serum albumin as standard (Bradford, 1976).

Results:

[0156] The results showed that the NA is active and saturating at the 50 nM concentration (FIG. 10). For the control, substrate without NA was used in the experiment.

Example 2: Expression of Viral Structural Capsid Protein

Example 2.1: Expression of VP7

[0157] Viral capsid protein VP7 glycoprotein a potential vaccine target was recombinantly expressed using yeast host expression platform. The gene for expression was codon biased and optimized for expression in yeast host. The protein was expressed along with a 10×His tag. The gene was cloned using conventional cloning methodologies into pYRE100 expression vector. The cloned gene was analysed through restriction digestion. The characterized construct was transformed into protease deficient S. cerevisiae host strain for expression studies. Expression of His tagged rVP7 was confirmed using anti-His antibody in Immunoblot analysis. The expressed platform was scaled up to 25×. Expressed protein was purified using Ni NTA chromatography and quantitated against standard.

[0158] The characterized recombinant construct was transformed in yeast host as mentioned in other examples. Few isolated healthy transformed colonies were inoculated in YNB Glucose—URA media and scaled up to culture of 475 ml was prepared and analysed for expression in 12 and 24th hr post induced (induction of late log phase; final concentration 2% galactose) time point samples. Cells were harvested and samples were prepared in 1×SDS reducing dye for expression analysis in cell pellet. The protein was characterized using anti-His antibodies immunoblot Immunoblot was developed using anti-His tag as primary antibody followed by incubation in HRP conjugated anti-mouse secondary antibody.

Results:

[0159] Band was picked at 24 h (Two clones—5 and 6) with anti-His antibody at correct size of 38 kda, Clone 6 showed a faint band at 12 hr induction sample as well, whereas no band was observed in backbone (BB) and before induction (BI) sample (FIG. 11). Using said yeast expression platform, recombinant 10×His tagged VP7 capsid protein was expressed effectually. The protein was purified using Ni NTA affinity chromatography and is described in the scale up example. Use of presently mentioned platform and similar expression and purification methodologies further showed the platform USER friendly, cost effective and timesaving approach.

Example 2.2 Scale Up of Structural Protein VP7

[0160] The protein was expressed with a 10×His tag. The negligible expression of VP7 protein was present at small scale (20 ml) (the yields were in range of 60 to 100 ng/ml). The clone was further scaled up to 25× or 500 ml scale. The expressed protein was purified using affinity chromatography, e.g., Ni NTA chromatography. Expression was analyzed on reducing SDS PAGE.

Result:

[0161] Band of interest was observed after purification. Yields were measured against BSA as standard (FIG. 12). Protein was purified with >90% purity. Obtained yield was 8 mg/L, up from 0.08 mg/ml measured basis densitometry analysis against known standard BSA.

[0162] The scalable process over a linear range of 25× volumetrically, was found to demonstrate increased yields which further describes and confirms the platform capability towards enhanced productions and suitability in producing large quantities required for various applications

Example 3: Expression of Enzyme Protein (Fatty Acid Proteins)

[0163] The enzyme gene sequence of both fatty acid desaturase and elongase was codon biased and optimized for expression in S. cerevisiae host. The gens were fused to 10× his tag at C terminus.

[0164] The genes were cloned using conventional cloning methodologies into proprietary expression plasmid pYRE100. The cloned genes were analysed through restriction digestion. The construct was transformed into S. cerevisiae host for expression studies using anti-His antibody immunoblot analysis.

Example 3.1: Process for the Expression

[0165] The characterized respective recombinant constructs were transformed using similar methodologies described for other examples. Two clones of each were expressed into proprietary protease deficient yeast expression host in rich YPD media. Expression was verified against control which was yeast transformed with episomal vector backbone.

[0166] Scale up culture of both proteins and both clones at 475 ml was produced in rich YPD media and induced using 2% galactose. 24 hrs induced cells were pelleted. The cells were resuspended in buffer and homogenized at 800 bar for 5 passes. Solution was centrifuged at 4000 rpm. The supernatant was collected without disturbing the pellet and the pellet was solubilized in same volume (as of supernatent) of urea buffer (8 M urea, 20 mM Tris, pH 8). Both proteins were expressed and are likely to be localized in ER membranes as nature. The expression was analyzed through immunoblotting analysis Immuno blot was developed using anti-His antibody as primary antibody and HRP conjugated anti-mouse secondary antibody.

[0167] Expression was observed at expected size of ˜41 kda for supernatant fraction of fatty acid desaturase for one of the clones ((FIG. 13). Fatty acid elongase showed the expression in pellet at ˜40 kda (expected size of 33.4 kda) for one of the clones (FIG. 14). Higher size could be due to post translational modification (glycosylation) in yeast host.

Example 3.2: Scale Up of Fatty Acid Desaturase

[0168] Further scale-up to 10× volumetric scale was performed using said platform. The scaled-up batch was set up at fermenter level with in YPD (yeast extract, peptone, and dextrose) media and induction by galactose, same as was used at 500 ml scale analysis. The process showed 10× scale up and productions of Fatty acid desaturase enzyme.

[0169] 0.5 mL of pre seed culture was prepared in shaker incubator at 30° C. for 15-20 hr to a cell density (OD.sub.600) of 3.0-4.0. The initial fermentation process was started with inoculation of media with 500 mL of seed culture. When OD.sub.600 reached till 7-8, the temperature of the fermenter was kept at 25° C. and the culture was induced by the addition of 1 L 5×YPG (yeast extract, peptone, and galactose) solutions. DO and pH was maintained at 20% and 5.6 to 6.0.

[0170] Pellet was lysed through homogenization, solubilized and purified using Ni NTA affinity chromatography. The purified protein was characterized through SDS PAGE and anti-His tag immunoblotting (FIG. 15) Immunoblot was developed using anti-His antibody as primary antibody and HRP conjugated anti-mouse secondary antibody.

[0171] Total protein amount of 2.85 mg protein was purified from the 5 litre scale up. This clearly demonstrates scope of further yield enhancement through process development.

Example 4: Expression of Ion Channel Receptor Protein

[0172] The yeast platform herein is used to express Nav1.7 multipass transmembrane protein localized to plasma membrane, a promising drug target candidate, using protease deficient S. cerevisiae host strain and episomal expression vector combination.

[0173] The expressed platform was scaled up 10× times and showed consistency in yields and localization of protein. Membrane fractions were purified and analyzed as full-length protein using protein specific antibody and confocal microscopy. These purified membrane fractions have been used in literature in developing screening assays for compound screening in 96 well and 384 well formats. The scale up gives a very significant advantage of eliminating batch to batch variation in assay set up and screening data as the entire compound library or a large number of compounds can be screened using the same batch of the recombinant protein.

[0174] The principal subunit of this channel is a protein of >200 kDa, the alpha subunit. The subunit consists of four large domains of internal homology with 24 transmembrane multipass domains. The gene for expression was codon biased and optimized for expression in yeast host. The gene was cloned using conventional cloning methodologies into proprietary expression plasmid pYRE100. The cloned gene was analyzed through restriction digestion. The construct was transformed into S. cerevisiae protease deficient host of recombinant expression platform using anti-His antibody immunoblots analysis.

Example 4.1: Process for the Expression of Nav1.7

[0175] The characterized recombinant construct was transformed in yeast host as mentioned in example 1. Few isolated healthy transformed colonies were inoculated in 20 ml of YNB Glucose—URA media and were analyzed for expression in 24 hr post induced (Induction at late log phase A600˜3.0 OD/ml; final concentration 2% galactose) time point samples using Nav1.7 protein specific antibody by Immuno-blot analysis. Selected clone was further expressed at 40 ml scale, microsomes were prepared and localization studies were done using confocal microscopy. Immunoblot was developed using Nav1.7 protein specific antibody as primary antibody followed by incubation in HRP conjugated anti-mouse secondary antibody.

Results:

[0176] Immuno Blot analysis using specific antibody showed a light band at 226 kda and higher size which could be due to glycosylation and oligomeric nature of the protein in membrane preparations (Lane 2). While minimal degradation is also seen. However, no band was observed in control (FIG. 16). It is demonstrated that complex and multipass membrane proteins which are very high impact and value drug targets can be expressed using the combination of vector and strain along with the methodologies described in this application. This allows for studying other target proteins as ion channel receptor family, GPCRs, kinases, phosphatases and so on.

Example 4.2: Confocal Microscopy Analysis for Membrane Localization of Nav1.7

[0177] Confocal microscopy confirmed the localization of Nav1.7 at the cell surface (FIG. 17). The purified protein (membranous and microsomes) could be used to screen Nav1.7 inhibitors and thus be advantageous for therapeutic purposes.

Example 5: Expression of Drug Target Molecules (GPI Anchored Protein)

[0178] The protein was expressed with a 6×His tag.

Example 5.1: Process for Expression

[0179] A single colony from the yeast selection plate was taken and put into 5 ml selection (Glucose) Media-SD with appropriate amino acids and Incubated at 30° C. with shaking for 22-24 hrs. The cultures were spun at 3500 rpm for 15 min at room temperature. The supernatant was poured off and pellets washed with sterile water. It was spun again at 3500 rpm for 15 min at 4° C. The pellet was resuspended in Yeast Peptone (Galactose) media-YPG (5 ml) for induction and incubated at 30° C. with shaking for 8 hrs. The culture was spun down at 3500 rpm for 15 min at 4° C. and pellets were lysed and analysed for protein expression. The clones were analysed using both Anti His antibody and Anti-CD 59 antibody to confirm for specific protein expression (FIG. 18). The protein was further solubilized and purified using NI-NTA column and was obtained at greater than 90% purity levels. This is an approach that has helped in establishing screening assays for compounds that bind to CD59 protein

Results:

[0180] The expression of CD-59, a glycoprotein with a GPI anchor was confirmed at small scale, 5 ml and was found to show optimum expression in 8 hr induction sample as confirmed using anti His antibody. These studies were performed at small scale and the clone was further scaled up to 200× or 1 litre scale. The expressed protein was purified using affinity chromatography, e.g. Ni NTA chromatography and was analysed on reducing SDS PAGE. Band of interest was observed after purification. Yields were measured against BSA as standard (FIG. 19).

Example 6: Scale Up Process at 500 ml Scale

[0181] Synthetic minimal media containing 2% glucose and respective selection markers were prepared in 250 mL and mixed with expression vectors pYRE100 and pYRI100 or both were incubated in shaker in case multiprotein components for expression (as per final genotype) where the parameters selected were 30±1° C., 250±10 rpm at OD600=1.5-2.0 for (16 h).

[0182] Large scale growth was performed using 2×200 mL YPD medium in 1 L shake flask at 30±1° C. and 250±10 rpm and culture was incubated to grow to OD600=4-5 (24 h) followed by induction with 2% Galactose into 2×250 mL in 1 L shake flask each 30±1° C., 250±10 rpm with OD600 for 12 h/24 hrs/36 hrs as required. The entire culture in pre weighed centrifuge bottle was centrifuged at 1000 g (3000/4000 rpm) for 10 minutes, 4° C. and cell pellet was weighed and stored at −80° C. till further processing to purify protein for analysis and characterization/analysis as per protein specific conditions and requirement using SDS PAGE, Immunoblot and Flow cytometry.

[0183] Table 2 below provides an overview of the scale of expression of the representative proteins through the platform of the present invention.

TABLE-US-00003 TABLE 2 Sr Scale Yield No Protein Type Increase Increase 1 VP7 Structural protein, 25X-50X 15X-25X Viral 2 Neuraminidase Single pass 100X 5X-10X transmembrane protein, Enzyme, Antigen Viral 3 Fatty acid desaturase Enzyme 10X-50X 40X-50X 4 CD59 GPI-anchored protein 50X-100X 4X-10X

[0184] Some of the important features of present expression platform are as follows: [0185] Engineered for high expression of very low expressing proteins. [0186] Engineered for wide applicability in proteins of different origin. [0187] Engineered to be protease deficient. [0188] Array of expression vectors with designed upstream regulatory sequence for enhanced expression. [0189] Multi-engineered expression strains for varied target proteins and its intrinsic properties. [0190] Multi protein co-expression along with codon harmonization. [0191] Possibility of further strain optimization and engineering for increased expression of proteins. [0192] The engineered strain can be scaled up to 500 L fermentation scale.

[0193] The present invention offers the following advantages: [0194] Provides a versatile, robust, scalable platform for expression of conformationally active protein expression. [0195] Can be utilized for a wide variety of proteins from different families and varied origin. [0196] The technology can have applications in fields like vaccine development, drug discovery, metabolism, diagnostics, therapeutics and healthcare.