The present invention broadly lies in the field of recombinant protein expression. More
particularly, the present invention relates to expression of difficult to express proteins in a
recombinant expression platform; constructs, methods and kits involved in expressing such
DTE-Ps through the said system.
BACKGROUND OF THE ART
Successful recombinant expression of proteins is a key requirement of the biotech industry to
aid in drug discovery and in the production of bio-therapeutics and vaccines. This entails not
only successful expression but also high quality and large-scale production of the target
proteins. Host cells such as E. coli, yeast, mammalian and insect cell-based expression
systems are generally selected to achieve successful expression of well-folded and active
form of such proteins in recombinant mode. However, there are numerous proteins which do
not express efficiently due to their inherent nature such as hydrophobicity, higher cysteineproline residues, repetitive amino acids, protein half-life, mRNA turnover, stable RNA
production etc. Suitability of host cells also plays an important role in achieving the desired
expression. Hence, a robust and consistent platform and host system is required for
expression of such proteins along with commercial scalability and cost-effective production
for supply in the industry in required amounts and cost.
Many proteins which are important for various applications are categorized under difficult to
express category and may pose a challenge in expression and manufacturing of these proteins
for industrial scale production. This could be ascribed to lower or no expression due to
sequence complexities or problems like, manufacturing scale ups, product recovery or
purification. Further, applying varied host systems and methodologies and cumbersome
optimizations, make it a very unpredictable, laborious, costly and time-consuming affair.
Expression of few challenging proteins using various vectors and hosts, is reported in the
prior art. Indian patent application numbers 1017/DEL/2009 and 1018/DEL/2009, disclose
heterologous over-expression of one such protein, i.e., human cytochrome P450 reductase.
However, these documents focus on the expression of cytochrome P450 specifically with a
different construct.
Another patent document CN1757745A relates to a method of high efficiency expression of
exogenic protein using methanol yeast system.
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US7910364B2 discloses rapidly cleavable sumo fusion protein expression system for difficult
to express proteins.
Saccardo et al., 2016 have shed some light on the general techniques and tools to cope with
expression of difficult to express proteins.
Thoring et al., 2017 reported high-yield production of difficult to express proteins in a
continuous exchange cell-free system based on CHO cell lysates.
However still, there are a large number of difficult to express proteins which remain a
challenge for researchers globally. Additionally, not many successes have been achieved in
the aspect of commercial scalability for over-expression and purification with enhanced
quality and scalable amount. Furthermore, there is a big lacuna in the art to have a stable,
consistent and robust platform for expression of difficult to express recombinant proteins
across varied origin and families, with scalability as well as quality of expressed target
protein.
For instance, more than 50% of known and novel drug target receptors and vaccine targets
are recognized as membrane proteins. It is also well understood that over-expression of
membrane proteins in their full length including all the domains is not an easy task.
Recombinant expression of proteins with transmembrane domain/s in heterologous systems is
also very challenging due to their high hydrophobic nature and sequence complexities which
lead to aggregation, precipitation, incorrect protein folding of proteins are difficult to
solubilize and refold (Lundstrom et al. 2006). These proteins are usually expressed as
modified proteins with deletion of the transmembrane domain, using E. coli host system to
avoid insoluble expression. The drawback with this approach is that this results in lack of
full-length sequence expression and consequently, a lack of full-length protein for analysis.
Eukaryotic host systems are recommended for their expression in their natural form, as intact
and full-length for their biochemical and structural characterization requirements. As per
available literature, there are no universal solutions for membrane protein production, and
this continues to remain a considerable obstacle (Elizabeth Massey-Gendel et al. 2009).
4
Expressing nascent polypeptide chain and intimate interactions and insertion in membrane is
one important parameter to study a particular protein with respect to its analysis as drug
target or vaccine candidate.
There is a need in the art for providing a stable and versatile expression system for expressing
multiple full-length transmembrane proteins. As an example, the inventors have demonstrated
the expression of full-length and functionally active Neuraminidase (NA), a transmembrane
DTE-P. Other than the difficulty of expressing Neuraminidase as membrane anchored
protein, its sequence is also found to have high number cysteine residues and hydrophobicity
which further describes its tendency towards insoluble expression and aggregation in
expression systems like E. coli. The presence of high proline residues also adds instability
being helix breaker for stable product generation. Limited literature for recombinant
expression of full-length NA expression is available in yeast host expression system.
Currently there is an unmet need to develop a universal effective vaccine which elicits
immune response against influenza virus and subtypes. Full length NA expression is of
utmost importance to analyse the immune response including conformation epitopes. To
perform such functions, it is required to express neuraminidase efficiently.
Similarly, expression of structural proteins, which are considered difficult to express, do not
emerge in fully soluble, well-folded, and active form in a heterologous expression system.
Their major characteristic is the tendency to aggregate and form inclusion bodies. Viral
surface glycoproteins and other capsid proteins, which belong to the family of structural
proteins, have been long recognized as functional targets for vaccines. Vaccine candidates
like capsid protein could be an attractive strategy to induce protection against severe viral
diseases.
The versatile platform described in this application is able to express some of the difficult to
express structural proteins. The inventors have demonstrated, that the claimed platform
allows for the stable and enhanced expression of capsid glycoprotein viral protein VP7 which
is a 347 amino acids long glycosylated protein with added His- tag. The sequence contains
high number of cysteine and proline residues along with high hydrophobicity and is hence a
difficult to express protein. The features describe that the protein has tendency towards
insoluble expression and aggregation when overexpressed, which recommends its expression
using a eukaryote system. Additionally, high number of proline residues, which is a structure
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breaker, puts the protein in unstable category. The neutralizing antibodies against the protein
may provide both serotype specific and cross-reactive protection and hence considered
important for vaccine development in human healthcare stream.
The inventors have also demonstrated that the platform of the present invention is capable of
expressing enzyme proteins, such as fatty acid enzymes. Few fatty acids and derivatives
known as polyunsaturated fatty acids (PUFA) are very essential and have functions including
inflammatory response, controlling lipid metabolism and also have function in signaling
pathways (Hoshino et al., 1984). Desaturase enzymes, a key representative of such fatty acid
enzymes, are transmembrane proteins varying from being single pass to multi-pass and likely
to be localized in endoplasmic reticular membranes of plants, fungi and animals.
Progress on the study of desaturases has been constrained due to the complexity in membrane
protein extraction and crystallization of these enzymes. Consequently, the knowledge about
the structure and expression regulation of membrane-bound fatty acid desaturases is still
lacking and whether the transmembrane domain has any role in fatty acid desaturase
efficiency remains unknown (Wyatt et al., 1983). Destaurases have high hydrophobicity as
described by its membrane nature, high cysteine and proline content. The attributes keep it in
difficult to express category and describes tendency for insoluble expression if expressed in
bacterial host system. Hence, it is of utmost importance to have dependable and efficient
systems to express these difficult to express enzyme proteins, especially for producing them
on a commercial scale. The inventors have successfully demonstrated the expression of 4-
multi-pass membrane protein sequences of desaturase through the platform of the present
invention.
Further, fatty acid elongation is also a very crucial step, serving as an alternative pathway
of fatty acid production involved in lipid metabolism applications. Elongase proteins also
have high hydrophobicity, high cysteine and proline content. These attributes make them
difficult to express. Besides, there is a tendency for insoluble expression if expressed in
bacterial host system.
Many important proteins, such as ion pumps, ion channels, and transporters, span
the membrane multiple times. Each membrane-spanning α helix in these multi-pass
transmembrane proteins is thought to act as a topogenic sequence. In nature, transmembrane
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proteins mediate communication between cells, ferry molecules into and out of the cell, and
are common targets for drugs. Expression of nascent polypeptide chain and intimate
interactions and insertion in membrane is one important parameter to study a particular
protein with respect to its analysis as drug target or vaccine candidate. However, predicting
how a large, multi-pass transmembrane protein design might fold into shape and function
while spanning such different environments has been challenging.
Recombinant expression of proteins with transmembrane domain/s in heterologous systems is
also very challenging due to their high hydrophobic nature and sequence complexities which
leads to aggregation, precipitation, and incorrect protein folding of proteins and are difficult
to solubilize and refold. To produce well-ordered multi-pass transmembrane proteins from
scratch, several biophysical demands have to be balanced at the same time. Placing
hydrophobic swatches on alpha-helical structures is sufficient to generate membraneassociation, but the packing and orientation of hydrophobic helices are difficult to control.
Thereby expression of such multi transmembrane proteins in prokaryotes and then refolding
them to generate them in correct conformation is not an easy task.
The inventors have successfully demonstrated the expression of an ion channel receptor, a
multi-pass membrane protein Nav1.7 through the versatile platform of the present invention.
Nav1.7 is a voltage-gated sodium channel mediates the voltage-dependent sodium ion
permeability of excitable membranes and implicated in pain signaling. Nav1.7 is a validated
and promising drug target for pain treatment in humans.
Nav1.7 is glycosylated 1988 amino acids long, multi-pass membrane protein with 24 trans
membrane domains and is localized to cell/plasma membrane. The principal subunit of this
channel is a protein of >200 kDa, the alpha subunit. The sequence contains 4 internal repeats,
each with 5 hydrophobic segments (S1, S2, S3, S5, S6) and one positively charged segment
(S4).
The protein contain oligomeric conformation, very big in size, high and odd number of
cysteines, proline residues and high hydrophobicity. Observed parameter shows the over
expression of protein may yield in-soluble expression and aggregation in prokaryotes. Both
high size and prolines residues (a helix breaker) further can lead to degradation. The
instability index of this protein classifies this protein as unstable protein.
7
In some culture models, it was found that the receptor expression degrades or dysregulates,
creating difficulty in expression of Nav1.7 over time in culture. Inventors of the present
invention have successfully demonstrated the expression of the full-length membraneanchored alpha subunit of Nav1.7 by the recombinant expression platform of the present
invention.
Another important category of the DTE-Ps are drug target molecules (GPI anchored protein).
Major drug target classes belong to antineoplastics, G protein-coupled receptors (GPCR’s),
ion channels, kinases and proteases (Kubic et al, 2019). A broad range of protein expression
systems are currently available, mostly based on cellular organisms of prokaryotic and
eukaryotic origin. Limitations of prokaryotic systems occur when complex mammalian target
proteins requiring posttranslational modifications, cofactors and chaperones for correct
protein folding, assembly and activity need to be produced.
One such drug target protein is CD59 protein which is a glycosylphosphatidylinositolanchored (GPI anchored) membrane protein that acts as an inhibitor of the formation of the
membrane attack complex to regulate complement activation. Recent studies have shown that
CD59 is highly expressed in several cancer cell lines and tumor tissues. CD59 also regulates
the function, infiltration, and phenotypes of a variety of immune cells in the tumor
microenvironment. (Zhao et al, 2018). CD59 is being considered as a promising target in the
gene therapy of breast cancer. (Xu et al).
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 are only rarely reported as being successful. Generation of eukaryotic
stable cell lines for protein production purposes have been found to be quite laborious due to
slow cell growth, and low protein yields apart from high production time thereby leading to
costly protein production processes. (Kubic et al, 2019).
As discussed in the preceding paragraphs, it is a need of the hour to have an efficient
recombinant expression system/platform which is flexible and adaptable for the expression
and production of varied DTE-Ps. The desirable features demand optimum expression, quick
and time saving standardized methodologies and scalability ease with capability of producing
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large amount of material for analysis, diagnostic and therapeutic use amenable to cater the
large population with cost effectiveness.
Hence, the present invention addresses this need by presenting a versatile recombinant
protein expression platform comprising recombinant expression vectors with protease
deficient yeast cell host system capable of expressing the target DTE-P proteins from varied
origin and families, at a scalable and commercial level. The claimed recombinant expression
platform and methods overcome the shortcomings of the prior arts and provide significant
technical advance over the same.
OBJECTIVES OF THE INVENTION
The principal objective of the present invention is to provide 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.
Yet another objective of the present invention is to provide a method for the enhanced
expression of DTE-Ps using the recombinant expression platform of the present invention.
Yet another objective of the present invention is to provide a kit comprising the recombinant
expression platform of the present invention for producing the target DTE-Ps.
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 the desired
DTE-Ps with all their functions intact.
BRIEF DESCRIPTION OF FIGURES AND DRAWINGS
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.
Figure 1 shows the pYRI100 yeast integrative vector for expression of recombinant proteins
using inducible GAL promoter.
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Figure 2 shows the pYRI200 yeast integrative vector for expression of recombinant proteins
using ADH2 constitutive promoter.
Figure 3 shows pYRE100 yeast episomal vector for expression of recombinant proteins
using inducible GAL promoter.
Figure 4 shows pYRE200 yeast episomal vector for expression of recombinant proteins
using ADH2 constitutive promoter.
Figure 5 shows Western Blot analysis of Neuraminidase.
Figure 6 shows the Western Blot analysis of Neuraminidase at large scale.
Figure 7 shows the flow cytometry result of Neuraminidase.
Figure 8 shows IgM response against Neuraminidase.
Figure 9 shows IgG response against Neuraminidase.
Figure 10 Activity assay of Neuraminidase
Figure 11 shows Immunoblot analysis of expression of VP7.
Figure 12 shows SDS PAGE analysis of purified VP7 protein.
Figure 13 shows Immunoblot analysis confirming expression of fatty acid desaturase
protein.
Figure 14 shows Immunoblot analysis confirming the expression of fatty acid elongase
protein.
Figure 15 illustrates SDS PAGE and Immunoblot analysis of purified fatty acid elongase
protein from scale up culture.
Figure 16 depicts Immunoblot analysis showing expression of Nav1.7.
Figure 17 illustrates Confocal Microscopy showing surface expression of NaV1.7.
Figure 18 shows the Immunoblot analysis using Anti-His antibody and Anti-CD59 antibody
to confirm overexpression of CD59 protein.
Figure 19 shows the purification of CD59 protein.
SUMMARY
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:
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;
10
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;
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.
Constructs, methods and kits involved in expressing such DTE-Ps through the said system are
also described.
DETAILED DESCRIPTION
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:
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.
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.
The term “expression platform” defines a system to produce large amounts of proteins, sugars
or other compounds for research or industrial uses.
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The term “expression vectors” defines a plasmid or virus designed for gene expression in
cells.
The term “host cell” means a host cell used for generation of recombinant proteins.
The term “prokaryotic proteins” includes the proteins found in prokaryotic cells/organisms.
The term “eukaryotic proteins” includes the proteins found in eukaryotic cells/organisms.
The term “viral proteins” includes proteins generated by viruses including enzyme proteins as
well as structural proteins such as capsid and viral envelope.
The term “mammalian proteins” include proteins produced in mammals.
The term “plant protein” includes proteins produced in plants.
The term “algal proteins” include the proteins found in all class of algae.
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.
The term “proteins with multiple transmembrane” includes proteins predominantly with
nonpolar amino acid residues with possibility of traversing the bilayer once or several times.
The term “transmembrane proteins” includes type of integral membrane proteins that span
the entirety of the cell membrane.
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.
The term “ion channel receptors” includes multimeric proteins usually located in the plasma
membrane.
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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.
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.
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.
In the principal embodiment, the present invention provides 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 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;
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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.
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.
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
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.
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.
In still another embodiment, the present invention discloses a method of producing
membrane bound Neuraminidase by the recombinant expression platform, comprising the
steps of:
i. preparing the nucleic acid construct;
ii. transforming the said construct in protease deficient yeast host cell;
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iii. culturing the transformed host cell for enhanced expression of
Neuraminidase.
In yet another embodiment, said Neuraminidase protein elicits an immunogenic response and
is functionally active.
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.
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.
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:
i) preparing the nucleic acid construct;
ii) transforming the said construct in protease deficient yeast host cell;
iii) culturing the transformed host cell for enhanced expression of VP7.
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.
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
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expression vector comprising Ura3 auxotrophic selection marker, CYCT1 terminator, an
Ampicillin resistance marker, pUC ori along with Gal1 promoter.
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:
i) preparing the nucleic acid construct;
ii) transforming the said construct in protease deficient yeast host cell;
iii)culturing the transformed host cell for enhanced expression of Nav1.7.
iv)surface localization of expressed Nav1.7 using confocal microscopy.
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.
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.
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:
i) preparing the nucleic acid construct;
ii) transforming the said construct in protease deficient yeast host cell;
iii) culturing the transformed host cell for enhanced expression of fatty acid
desaturase.
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.
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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
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:
i) preparing the nucleic acid construct;
ii) transforming the said construct in protease deficient yeast host cell;
culturing the transformed host cell for enhanced expression of fatty acid
elongase.
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.
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.
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:
i) preparing the nucleic acid construct;
ii) transforming the said construct in protease deficient yeast host cell;
iii) culturing the transformed host cell for enhanced expression of CD59.
In still another embodiment, said platform is scalable and capable of producing proteins from
diverse origin and families at an industrial scale.
In yet another embodiment, the present invention provided a kit comprising the recombinant
expression platform 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.
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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
Representative S. Cerevisiae expression vectors as developed and used in the present
invention are designated as below:
a. pYRI100 yeast integrative vector comprising Leu auxotrophic selection marker,
CYCT1 terminator, resistance marker, pUC ori along with Gal promoter.
b. pYRI200 yeast integrative vector comprising Leu auxotrophic selection marker,
CYCT1 terminator, resistance marker, pUC ori along with ADH2 promoter.
c. pYRE100 yeast episomal vector comprises Ura3 auxotrophic selection marker,
CYCT1 terminator, resistance marker for selection, pUC ori along with Gal
promoter.
d. pYRE200 yeast episomal vector comprises Ura3 auxotrophic selection marker,
CYCT1 terminator, resistance marker for selection, pUC ori along with ADH2
promoter.
EXAMPLES
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
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working the invention and nothing in this section should be taken as a limitation of the
claims.
Example 1: Expression of Neuraminidase (NA):
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
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:
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 24th hr post induced (Induction at late log phase
A600~5.0 OD/ml; final concentration 2% galactose) time point samples using anti- His
antibody by Immuno-blot analysis.
Immuno Blot analysis using anti His antibody showed a specific band at higher size of
~52kda 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:
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
19
up to develop a robust expression system for large scale production of viral vaccine
candidates. The clone was further scaled up to 100X 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).
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
Yeast cells expressing plasma membrane localized NA equivalent to 1 OD600nm 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, resuspended and incubated in 1% BSA prepared in 1X PBS for 1 hr at 25 °C.
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 1X PBS only. Centrifugation at 4000 rpm 1 min. Resuspended the cells in secondary antibody (1:100 µl), and were incubated for 30 min at 25
°C. The cells were washed with of 1X PBS by centrifugation at 4000rpm for 1.5 minutes at 4
°C. Cells were re-suspended in 2 % FBS in 1 X PBS and readings were taken in FACS
Instruments ACEA Novo Cyte Flow Cytometer (Model: 3005). 6X His tag specific antibody
was used as primary antibody and anti-mouse Alexa flour 488 labelled secondary antibody
was used for the study.
Results
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).
20
Example 1.4: Animal studies
Analysis of immunogenic response in mice against recombinant Neuraminidase (human)
expressed using S. cerevisiae platform.
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.
Following are the host details for checking immune response:
Test system : Mice; Mus musculus
Strain : Balb/c
Sex : Male
Age : 6-8 weeks
Study design:
Antigen Route of
administration
Purpose Requirement
Immunogenic response studies
Balb/c Microsomal
preps
Intramuscular Study immune response against NA 3 mice study
Collection of blood samples for immune assessments
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 21st 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:
21
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
To assess the immune response, NA and control microsomes were coated at a concentration
of 100ng/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).
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:
Isolated healthy patched colonies were inoculated in 100 ml of YNB Glucose - URA media as
pre seed and were cultured in shaker incubator at 28ᵒC for 24 hr along with host strain
Protease deficient strain transformed with pYRE100 as host-vector control.
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 28ᵒC 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.
22
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):
Microsome concentration of 5nM, 10nM, 25nM and 50nM was used for the activity assay,
assuming 1% of total cell protein form the NA microsomes.10µl of respective concentration
sample was mixed with an equal volume of assay buffer (32.5mM 2-(N-morpholino) ethane
sulfonic acid (MES), pH 6.5, containing 4mM 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
460nm respectively. Blank control reactions contained substrate alone.
All reactions were conducted 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 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:
The results showed that the NA is active and saturating at the 50nM 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
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 10X 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
23
studies. Expression of His tagged rVP7 was confirmed using anti-His antibody in
Immunoblot analysis. The expressed platform was scaled up to 25X. Expressed protein was
purified using Ni NTA chromatography and quantitated against standard.
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 1XSDS 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:
Band was picked at 24h (Two clones- 5 and 6) with anti-His antibody at correct size of
38kda, 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 10X 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
The protein was expressed with a 10X His tag. The negligible expression of VP7 protein was
present at small scale (20ml) (the yields were in range of 60 to 100ng/ml). The clone was
further scaled up to 25X or 500ml scale. The expressed protein was purified using affinity
chromatography, e.g., Ni NTA chromatography. Expression was analyzed on reducing SDS
PAGE.
Result:
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 8mg/L, up
from 0.08 mg/ml measured basis densitometry analysis against known standard BSA.
24
The scalable process over a linear range of 25X 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)
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 10X his tag at C
terminus.
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:
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.
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 800bar 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, 20mM 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.
25
Expression was observed at expected size of ~41kda for supernatant fraction of fatty acid
desaturase for one of the clones ((Fig. 13). Fatty acid elongase showed the expression in
pellet at ~40kda (expected size of 33.4kda) 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:
Further scale-up to 10X 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 500ml scale analysis. The process showed
10X scale up and productions of Fatty acid desaturase enzyme.
0.5mL of pre seed culture was prepared in shaker incubator at 30°C for 15-20 hr to a cell
density (OD600) of 3.0-4.0. The initial fermentation process was started with inoculation of
media with 500 mL of seed culture. When OD600 reached till 7-8, the temperature of the
fermenter was kept at 25˚C and the culture was induced by the addition of 1L 5 X YPG (yeast
extract, peptone, and galactose) solutions. DO and pH was maintained at 20% and 5.6 to 6.0.
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.
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
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.
The expressed platform was scaled up 10X 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
26
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.
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
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 40ml 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:
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.
27
Example 4.2: Confocal microscopy analysis for membrane localization of Nav1.7
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): The protein
was expressed with a 6X His tag.
Example 5.1: Process for expression:
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 30ºC with shaking for 22-24hrs.
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
4ºC. The pellet was resuspended in Yeast Peptone (Galactose) media-YPG (5ml) for
induction and incubated at 30ºC with shaking for 8hrs. The culture was spun down at 3500
rpm for 15 min at 4ºC 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:
The expression of CD-59, a glycoprotein with a GPI anchor was confirmed at small scale,
5ml and was found to show optimum expression in 8hr induction sample as confirmed using
anti His antibody. These studies were performed at small scale and the clone was further
scaled up to 200X 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).
28
Example 6: Scale up process at 500ml scale
Synthetic minimal media containing 2% glucose and respective selection markers were
prepared in 250mL 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 (16h).
Large scale growth was performed using 2 x 200 mL YPD medium in 1L shake flask at 30 ±
1°C and 250 ± 10 rpm and culture was incubated to grow to OD600 = 4 -5 (24h)
followed by induction with 2% Galactose into 2 x 250 mL in 1L shake flask each 30 ± 1°C,
250± 10 rpm with OD600 for 12h/24hrs/36hrs as required. The entire culture in pre weighed
centrifuge bottle was centrifuged at 1000g (3000/4000rpm) for 10 minutes, 4 ºC 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.
Table 2 below provides an overview of the scale of expression of the representative proteins
through the platform of the present invention.
Table 2
Sr
No
Protein Type Scale Increase Yield Increase
1 VP7 Structural protein,
Viral
25X-50X 15X-25X
2 Neuraminidase Single pass
transmembrane
protein, Enzyme,
Antigen Viral
100X 5X-10X
3 Fatty acid desaturase Enzyme 10X – 50X 40X-50X
4 CD59 GPI-anchored protein 50X – 100X 4X - 10X
Some of the important features of present expression platform are as follows:
 Engineered for high expression of very low expressing proteins.
 Engineered for wide applicability in proteins of different origin.
29
 Engineered to be protease deficient.
 Array of expression vectors with designed upstream regulatory sequence for enhanced
expression.
 Multi-engineered expression strains for varied target proteins and its intrinsic
properties.
 Multi protein co-expression along with codon harmonization.
 Possibility of further strain optimization and engineering for increased expression of
proteins.
 The engineered strain can be scaled up to 500L fermentation scale.
The present invention offers the following advantages:
 Provides a versatile, robust, scalable platform for expression of conformationally
active protein expression.
 Can be utilized for a wide variety of proteins from different families and varied
origin.
 The technology can have applications in fields like vaccine development, drug
discovery, metabolism, diagnostics, therapeutics and healthcare.

We Claim:

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.
5. The recombinant expression platform as claimed in claim 4, wherein said viral protein
is a viral enzyme protein.
6. The recombinant expression platform as claimed in claim 5, wherein said viral enzyme
protein is a membrane bound single pass membrane protein, Neuraminidase.
7. A nucleic acid construct with SEQ ID NO 7 for expression of membrane bound
Neuraminidase as claimed in claim 6, 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.
8. A method of producing membrane bound Neuraminidase by the recombinant
expression platform as claimed in claim 1, comprising the steps of:
i. preparing the nucleic acid construct as claimed in claim 7;
ii. transforming the said construct in the host cell as claimed in claim 1;
iii. culturing the transformed host cell for enhanced expression of
Neuraminidase.
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 as claimed in claim 1, wherein said difficult to
express protein is highly hydrophobic viral protein.
11. The recombinant expression platform as claimed in claim 10, wherein said highly
hydrophobic viral protein is a structural protein.
12. The recombinant expression platform as claimed in claim 11, wherein said highly
hydrophobic viral structural protein is a capsid protein.
13. The recombinant expression platform as claimed in claim 12, wherein said highly
hydrophobic viral structural, capsid protein is a vaccine candidate.
14. The recombinant expression platform as claimed in claim 13, wherein said vaccine
candidate protein is VP7.
15. A nucleic acid construct with SEQID No 8 for expression of viral structural capsid
protein VP7 as claimed in claim 14, wherein said construct comprising 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.
16. A method of producing highly hydrophobic viral structural capsid protein VP7 by the
recombinant expression platform as claimed in claim 1, comprising the steps of:
i) preparing the nucleic acid construct as claimed in claim 15;
ii) transforming the said construct in the host cell as claimed in claim 1;
iii) culturing the transformed host cell for enhanced expression of VP7.
17. The recombinant expression platform as claimed in claim 1, wherein said difficult to
express protein is a multi-pass transmembrane protein.
18. 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.
19. The recombinant expression platform as claimed in claim 18, wherein said multi-pass
transmembrane protein is from ion channel receptor family.
20. The recombinant expression platform as claimed in claim 17, wherein said
transmembrane proteinis a drug target receptor protein.
21. The recombinant expression platform as claimed in claim 20, wherein said drug target
receptor protein is sodium ion channel receptor Nav1.7.
22. A nucleic acid construct with SEQ ID NO 9 for expression of transmembrane ion
channel receptor protein Nav1.7 as claimed in claim 21, 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.
23. A method of producing transmembrane Nav1.7 protein by the recombinant expression
platform as claimed in claim 1 comprising the steps of:
i) preparing the nucleic acid construct as claimed in claim 22;
ii) transforming the said construct in the host cell as claimed in claim 1;
iii) culturing the transformed host cell for enhanced expression of Nav1.7.
iv) surface localization of expressed Nav1.7 using confocal microscopy.
24. The recombinant expression platform as clamed in claim 1, wherein said difficult to
express protein is an enzyme protein.
25. The recombinant expression platform as claimed in claim 24, wherein said enzyme
protein is a transmembrane protein.
26. The recombinant expression platform as claimed in claim 25, wherein said
transmembrane protein is from lipid biosynthesis.
27. The recombinant expression platform as clamed in claim 26, wherein lipid biosynthesis
protein is fatty acid desaturase.
28. The recombinant expression platform as clamed in claim 27, wherein said desaturase is
from fungal origin.
29. A nucleic acid construct with SEQ ID NO 10 for expression of fatty acid desaturase as
claimed in claim 28, 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.
30. A method of producing fatty acid desaturase protein by the recombinant expression
platform as claimed in claim 1 comprising the steps of:
i) preparing the nucleic acid construct as claimed in claim 29;
ii) transforming the said construct in the host cell as claimed in claim 1;
iii) culturing the transformed host cell for enhanced expression of fatty acid
desaturase.
31. The recombinant expression platform as clamed in claim 1, wherein said difficult to
express protein is an enzyme protein.
32. The recombinant expression platform as claimed in claim 31, wherein said enzyme
protein is a transmembrane protein.
33. The recombinant expression platform as claimed in claim 32, wherein said
transmembrane protein is from lipid biosynthesis.
34. The recombinant expression platform as clamed in claim 33, wherein said
transmembrane protein from lipid biosynthesis is fatty acid elongase.
35. A nucleic acid construct with SEQ ID NO 11 for expression of fatty acid elongase as
claimed in claim 33, 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.
36. A method of producing fatty acid elongase protein by the recombinant expression
platform as claimed in claim 1, comprising the steps of:
i) preparing the nucleic acid construct as claimed in claim 35;
ii) transforming the said construct in the host cell as claimed in claim 1;
iii) culturing the transformed host cell for enhanced expression of fatty acid
elongase.
37. The recombinant expression platform as claimed in claim 1, wherein said difficult to
express protein is a glycosylphosphatidylinositol-anchored (GPI anchor) protein.
38. The recombinant expression platform as claimed in claim 37, wherein said GPI anchor
protein is a drug target protein.
39. The recombinant expression platform as claimed in claim 38, wherein said drug target
protein is CD59.
40. The recombinant expression platform as claimed in claim 1, wherein said CD59 is from
human origin.
41. A nucleic acid construct with SEQ ID NO 12 for expression of GPI anchor protein
CD59 as claimed in claim 40, 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.
42. A method of producing GPI anchor CD59 protein by the recombinant expression
platform as claimed in claim 1comprising the steps of:
i) preparing the nucleic acid construct as claimed in claim 41;
ii) transforming the said construct in the host cell as claimed in claim 1;
iii) culturing the transformed host cell for enhanced expression of CD59.
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.