DESC:TECHNICAL FIELD
[001] The present disclosure generally relates to the field of peptide production. The present disclosure particularly relates to expression cassette(s), vector(s) and recombinant host cell(s) for increased expression and secretion of biologically active peptides.

BACKGROUND OF THE DISCLOSURE
[002] Recombinant expression of peptides has several obstacles to overcome to be a cost-effective means of production. The obstacles are usually related to low expression levels of the recombinant protein or the destruction of the expressed polypeptide by proteolytic enzymes within the cells. Additionally, purification can be difficult, resulting in poor yields depending on the nature of the protein or peptide of interest.
[003] Polypeptides are useful for the treatment of disease in humans and animals. Examples of such polypeptides include insulin for the treatment of diabetes, interferon for treating viral infections, interleukins for modulating the immune system, erythropoietin for stimulating red blood cell formation, and growth factors that act to mediate both prenatal and postnatal growth.
[004] Many bioactive polypeptides can be produced through use of chemical synthesis methods. However, such production methods are often inefficient and labor intensive, leading to increased cost and reduced availability of therapeutically useful polypeptides. An alternative to chemical synthesis is provided by recombinant technology which allows the high yield production of bioactive polypeptides in microbes. Such production permits a greater number of people to be treated at relatively lower cost.
[005] While great strides have been made in recombinant technology, expression of proteins and peptides in cells can be problematic. This can be due to low expression levels or through destruction of the expressed polypeptide by proteolytic enzymes contained within the cells. This is especially problematic when short proteins and peptides are being expressed.
[006] Therefore, the need of the hour is means and methods to enable efficient recombinant expression of peptides, ensuring high yields while maintaining low cost of production.

STATEMENT OF THE DISCLOSURE
[007] The present disclosure provides an expression cassette for enhanced expression and secretory production of a protein of interest comprising:
a. a polynucleotide encoding a signal peptide wherein the signal peptide is selected from a group comprising sequence represented by SEQ ID No. 1 and SEQ ID No. 21 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer wherein the expression enhancer is selected from a group comprising sequence represented by SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 23 or a sequence bearing at least 90% similarity thereto; and
c. a polynucleotide encoding the protein(s) of interest.
[008] In some embodiments, the expression cassette comprises nucleotides encoding signal peptide and expression enhancer sequences represented by SEQ ID No. 1 and SEQ ID No. 3, SEQ ID No. 1 and SEQ ID No. 4, SEQ ID No. 1 and SEQ ID No. 5 or SEQ ID No. 21 and SEQ ID No.23, respectively.
[009] In some embodiments, the expression cassette further comprises a polynucleotide encoding a stretch of amino acids to facilitate affinity purification.
[010] In some embodiments, the expression cassette further comprises a polynucleotide encoding protease cleavage site, preferably preceding the protein of interest.
[011] In a non-limiting embodiment, the protein of interest is a peptide hormone that is less than 200 amino acids long.
[012] The present disclosure also provides an expression vector comprising the expression cassette as described above.
[013] The present disclosure also provides a bacterial host cell comprising the expression cassette or the expression vector as described above.
[014] The present disclosure further provides a method for producing a protein of interest in a bacterial host cell, the said method comprising steps of:
i. culturing the bacterial host cell as described above under suitable conditions to secrete the protein of interest into culture medium; and
ii. purifying the protein of interest from the culture medium.
[015] Further envisaged herein is use of the expression cassette or the expression vector of the present disclosure for the expression of bioactive peptides.
[016] The present disclosure also provides a kit comprising the expression cassette or the expression vector described above and instructions for their use.

BRIEF DESCRIPTION OF THE ACCOMPANYING FIGURES
[017] In order that the disclosure may be readily understood and put into practical effect, reference will now be made to exemplary embodiments as illustrated with reference to the accompanying figures. The figures together with detailed description below, are incorporated in and form part of the specification, and serve to further illustrate the embodiments and explain various principles and advantages, where:
[018] Figure 1 provides a schematic representation of the design of the expression cassette of the present disclosure, with reference to the different expression enhancers and signal peptides that were studied as part of the experiments conducted to arrive at the expression cassette of the present disclosure.
[019] Figure 2 (A-C) provides a schematic representation of the vector comprising the expression cassette of the present disclosure.
[020] Figure 3 depicts results of analysis of expression of Lirapeptide employing different combinations of signal peptides and expression enhancers.
[021] Figure 4 depicts 112 different signal peptide-expression enhancer combinations formed on the basis of 16 signal peptides and 7 expression enhancers that were subjected to analysis.
[022] Figure 5a depicts results of analysis of expression of Lirapeptide employing expression enhancer without signal peptide in a high copy plasmid.
[023] Figure 5b depicts results of analysis of expression of Lirapeptide employing expression enhancer without signal peptide in a low copy plasmid.
[024] Figure 6 depicts results of analysis of expression of Lirapeptide employing signal peptide without expression enhancer.

DETAILED DESCRIPTION OF THE DISCLOSURE
[025] In view of general limitations in the art associated with low yield of recombinant proteins, the present disclosure provides expression cassette(s), vector(s), host cell(s) and method(s) employing the same for the expression of recombinant proteins.
[026] However, before describing the invention in greater detail, it is important to take note of the common terms and phrases that are employed throughout the present disclosure for better understanding of the technology provided herein.

Definitions
[027] As used herein, the term ‘expression enhancer’ refers to the stretch of amino acids or peptide sequences genetically grafted onto a recombinant protein in the process of protein expression to improve its expression or solubility.
[028] As used herein, the term ‘biologically active peptide’ or ‘bioactive peptide’ is intended to convey the ordinary conventional meaning of the term readily understood by a person skilled in the art and refers to protein fragments that can influence one or more bodily functions.
[029] As used herein, the term ‘signal peptide’, is intended to convey the ordinary or conventional meaning of the term which is also interchangeably referred to as ‘secretion signal’. The said term would be readily understood by a person skilled in the art to refer to short peptides directing newly synthesized proteins toward the secretory pathway or sequences that function to prompt a cell to translocate the protein. These sequences, thus, control protein secretion and/or translocation.
[030] The term ‘gIII(+A)’ refers to a signal peptide by the said name from filamentous phage fd, which has been modified in the context of the present disclosure to include an additional amino acid Alanine (A) to the known sequence. Said sequence is represented in the present disclosure by SEQ ID No. 1.
[031] The term ‘OmpT’ implies reference to the signal peptide of the E. coli outer membrane protein T (OmpT) protein. Said sequence is represented in the present disclosure by SEQ ID No. 21.
[032] The terms ‘YebFN13’ and ‘YebFN20’ refer to truncated versions of the YebF protein, without the signal peptide. N13 and N20 refer to the first 13 and first 20 amino acids of the YebF protein, respectively. Said sequences are represented in the present disclosure by SEQ ID Nos. 3 and 23, respectively.
[033] The term ‘CelCDN20’ refers to the N-Terminus portion of the cellulase-CD gene. Said sequence is represented in the present disclosure by SEQ ID No. 5.
[034] As used herein, the term ‘comprising’ when placed before the recitation of steps in a method means that the method encompasses one or more steps that are additional to those expressly recited, and that the additional one or more steps may be performed before, between, and/or after the recited steps. For example, a method comprising steps a, b, and c encompasses a method of steps a, b, x, and c, a method of steps a, b, c, and x, as well as a method of steps x, a, b, and c. Furthermore, the term “comprising” when placed before the recitation of steps in a method does not (although it may) require sequential performance of the listed steps, unless the content clearly dictates otherwise. For example, a method comprising steps a, b, and c encompasses, for example, a method of performing steps in the order of steps a, c, and b, the order of steps c, b, and a, and the order of steps c, a, and b, etc. Said term, when placed before a product means that the product may comprise one or more components in addition to those recited in the claim.
[035] With respect to the use of substantially any plural and/or singular terms herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. The various singular/plural permutations may be expressly set forth herein for sake of clarity. The suffix ‘(s)’ at the end of any term in the present disclosure envisages in scope both the singular and plural forms of said term.
[036] As used in this specification and the appended claims, the singular forms ‘a’, ‘an’ and ‘the’ includes both singular and plural references unless the content clearly dictates otherwise.
[037] Numerical ranges stated in the form ‘from x to y’ include the values mentioned and those values that lie within the range of the respective measurement as known to the skilled person. If several preferred numerical ranges are stated in this form, of course, all the ranges formed by a combination of the different end points are also included.
[038] The use of the expression ‘at least’ or ‘at least one’ suggests the use of one or more elements or ingredients or quantities, as the use may be in the embodiment of the disclosure to achieve one or more of the desired objects or results. As such, the terms “a” (or “an”), “one or more”, and “at least one” can be used interchangeably herein.
[039] The terms ‘about’ or ‘approximately’ as used herein when referring to a measurable value such as a parameter, an amount, a temporal duration, and the like, are meant to encompass variations of and from the specified value, such as variations of +/-10% or less, +/-5% or less, +/-1% or less, and +/-0.1% or less of and from the specified value, insofar such variations are appropriate to perform in the disclosed invention. It is to be understood that the value to which the modifier ‘about’ or ‘approximately’ refers is itself also specifically, and preferably, disclosed.
[040] As used herein, the terms ‘include’, ‘have’, ‘comprise’, ‘contain’ etc. or any form of said terms such as ‘having’, ‘including’, ‘containing’, ‘comprising’ or ‘comprises’ are inclusive and will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
[041] As regards the embodiments characterized in this specification, it is intended that each embodiment be read independently as well as in combination with another embodiment. For example, in case of an embodiment 1 reciting 3 alternatives A, B and C, an embodiment 2 reciting 3 alternatives D, E and F and an embodiment 3 reciting 3 alternatives G, H and I, it is to be understood that the specification unambiguously discloses embodiments corresponding to combinations A, D, G; A, D, H; A, D, I; A, E, G; A, E, H; A, E, I; A, F, G; A, F, H; A, F, I; B, D, G; B, D, H; B, D, I; B, E, G; B, E, H; B, E, I; B, F, G; B, F, H; B, F, I; C, D, G; C, D, H; C, D, I; C, E, G; C, E, H; C, E, I; C, F, G; C, F, H; C, F, I, unless specifically mentioned otherwise.
[042] Reference throughout this specification to “some embodiments”, “one embodiment”, “an embodiment”, “a preferred embodiment”, “a non-limiting embodiment” or “an exemplary embodiment” means that a particular feature, structure or characteristic described in connection with the embodiment may be included in at least one embodiment of the present disclosure. Thus, the appearances of the phrases “in some embodiments”, “in one embodiment”, “in an embodiment”, “a preferred embodiment”, “a non-limiting embodiment” or “an exemplary embodiment” in various places throughout this specification may not necessarily all refer to the same embodiment. It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.
[043] Throughout this specification, the term ‘a combination thereof’, ‘combinations thereof’ or ‘any combination thereof’ or ‘any combinations thereof’ are used interchangeably and are intended to have the same meaning, as regularly known in the field of patent disclosures.

Disclosure
[044] The present disclosure provides an expression cassette for expressing a protein of interest, comprising a polynucleotide encoding a signal peptide, a polynucleotide encoding an expression enhancer, and a polynucleotide encoding the protein of interest.
[045] In some embodiments, the expression cassette enables enhanced expression and secretory production of the protein of interest.
[046] In some embodiments, the signal peptide is selected from a group comprising DsbA, EOX, gIII(+A), LamB, mal(A), MglB, MmAp, OmpA, OmpC, OmpT, PelB, phoA, SfmC, STII, TolB and torT(+A).
[047] In an exemplary embodiment, the signal peptide is selected from gIII(+A) and OmpT.
[048] In a non-limiting embodiment, the signal peptide may be represented by SEQ ID No. 1 (gIII(+A)) or SEQ ID No. 21 (OmpT) or a sequence bearing at least 90% similarity thereto.
[049] In some embodiments, the signal peptide may be represented by SEQ ID No. 1 (gIII(+A)) or SEQ ID No. 21 (OmpT) or a sequence bearing at least 95% similarity thereto.
[050] In some embodiments, the signal peptide may be represented by SEQ ID No. 1 (gIII(+A)) or SEQ ID No. 21 (OmpT) or a sequence bearing 95%, 96%, 97%, 98%, 99% or 100% similarity thereto.
[051] In some embodiments, the signal peptide may be represented by SEQ ID No. 1 (gIII(+A)) or SEQ ID No. 21 (OmpT).
[052] In some embodiments, the expression enhancer is selected from a group comprising YebFN13, CelCDN20, LP2, YebFN33, YebFN20, LP2 with T7 and LP8 without T7.
[053] In an exemplary embodiment, the expression enhancer is selected from a group comprising YebFN13, YebFN20, CelCDN20 and LP2.
[054] In a non-limiting embodiment, the expression enhancer may have a sequence selected from a group comprising SEQ ID No. 3 (YebFN13), SEQ ID No. 4 (LP2), SEQ ID No. 5 (CelCDN20) or SEQ ID No. 23 (YebFN20) or a sequence bearing at least 90% similarity to the said sequences.
[055] In some embodiments, the expression enhancer may have a sequence selected from a group comprising SEQ ID No. 3 (YebFN13), SEQ ID No. 4 (LP2), SEQ ID No. 5 (CelCDN20) or SEQ ID No. 23 (YebFN20) or a sequence bearing at least 95% similarity to the said sequences.
[056] In some embodiments, the expression enhancer may have a sequence selected from a group comprising SEQ ID No. 3 (YebFN13), SEQ ID No. 4 (LP2), SEQ ID No. 5 (CelCDN20) or SEQ ID No. 23 (YebFN20) or a sequence bearing 95%, 96%, 97%, 98%, 99% or 100% similarity to the said sequences.
[057] In some embodiments, the expression enhancer may have a sequence selected from a group comprising SEQ ID No. 3 (YebFN13), SEQ ID No. 4 (LP2), SEQ ID No. 5 (CelCDN20) or SEQ ID No. 23 (YebFN20).
[058] It is submitted that the above sequences envisage some non-limiting exemplary embodiments of the present disclosure and further sequences of expression enhancers and/or signal peptides that are functionally or structurally similar are envisaged in the scope of the present disclosure.
[059] Accordingly, the present disclosure provides an expression cassette for expressing a protein of interest comprising:
a. a polynucleotide encoding a signal peptide wherein the signal peptide is selected from a group comprising sequence represented by SEQ ID No. 1 and SEQ ID No. 21 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer wherein the expression enhancer is selected from a group comprising sequence represented by SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 23 or a sequence bearing at least 90% similarity thereto; and
c. a polynucleotide encoding the protein(s) of interest.
[060] In some embodiments, envisaged herein is an expression cassette for expressing a protein of interest, comprising: a polynucleotide encoding signal peptide, wherein the signal peptide is represented by SEQ ID No. 1 (gIII(+A)); a polynucleotide encoding an expression enhancer, wherein the expression enhancer is represented by SEQ ID No. 3 (YebFN13); and a polynucleotide encoding the protein of interest.
[061] In some embodiments, envisaged herein is an expression cassette for expressing a protein of interest, comprising: a polynucleotide encoding signal peptide, wherein the signal peptide is represented by SEQ ID No. 1 (gIII(+A)); a polynucleotide encoding an expression enhancer, wherein the expression enhancer is represented by SEQ ID No. 5 (CelCDN20); and a polynucleotide encoding the protein of interest.
[062] In some embodiments, envisaged herein is an expression cassette for expressing a protein of interest, comprising: a polynucleotide encoding signal peptide, wherein the signal peptide is represented by SEQ ID No. 1 (gIII(+A)); a polynucleotide encoding an expression enhancer, wherein the expression enhancer is represented by SEQ ID No. 4 (LP2); and a polynucleotide encoding the protein of interest.
[063] In some embodiments, envisaged herein is an expression cassette for expressing a protein of interest, comprising: a polynucleotide encoding signal peptide, wherein the signal peptide is represented by SEQ ID No. 21 (OmpT); a polynucleotide encoding an expression enhancer, wherein the expression enhancer is represented by SEQ ID No. 23 (YebFN20); and a polynucleotide encoding the protein of interest.
[064] In a non-limiting embodiment, the signal peptide may be encoded by SEQ ID No. 2 (gIII(+A)) or SEQ ID No. 22 (OmpT) or a sequence bearing at least 90% similarity thereto.
[065] In some embodiments, the signal peptide may be encoded by SEQ ID No. 2 (gIII(+A)) or SEQ ID No. 22 (OmpT) or a sequence bearing at least 95% similarity thereto.
[066] In some embodiments, the signal peptide may be encoded by SEQ ID No. 2 (gIII(+A)) or SEQ ID No. 22 (OmpT) or a sequence bearing 95%, 96%, 97%, 98%, 99% or 100% similarity thereto.
[067] In some embodiments, the signal peptide may be encoded by SEQ ID No. 2 (gIII(+A)) or SEQ ID No. 22 (OmpT).
[068] In a non-limiting embodiment, the expression enhancer may be encoded by a sequence selected from a group comprising SEQ ID Nos. 6, 7, 8 or 24 (YebFN13, LP2, CelCDN20 or YebFN20, respectively) or a sequence bearing at least 90% similarity to the said sequences.
[069] In some embodiments, the expression enhancer may be encoded by a sequence selected from a group comprising SEQ ID Nos. 6, 7, 8 or 24 (YebFN13, LP2, CelCDN20 or YebFN20, respectively) or a sequence bearing at least 95% similarity to the said sequences.
[070] In some embodiments, the expression enhancer may be encoded by a sequence selected from a group comprising SEQ ID Nos. 6, 7, 8 or 24 (YebFN13, LP2, CelCDN20 or YebFN20, respectively) or a sequence bearing 95%, 96%, 97%, 98%, 99% or 100% similarity to the said sequences.
[071] In some embodiments, the expression enhancer may be encoded by a sequence selected from a group comprising SEQ ID Nos. 6, 7, 8 or 24 (YebFN13, LP2, CelCDN20 and YebFN20, respectively).
[072] Thus, in some embodiments, the polynucleotide encoding the signal peptide has sequence selected from a group comprising SEQ ID No. 2 and SEQ ID No. 22 or a sequence bearing at least 90% similarity thereto and the polynucleotide encoding the expression enhancer has sequence selected from a group comprising SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 24 or a sequence bearing at least 90% similarity thereto.
[073] Accordingly, in some embodiments the expression cassette of the present disclosure comprises:
a. a polynucleotide encoding a signal peptide wherein the polynucleotide has sequence selected from a group comprising SEQ ID No. 2 and SEQ ID No. 22 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer wherein the polynucleotide has sequence selected from a group comprising a group comprising SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 24 or a sequence bearing at least 90% similarity thereto; and
c. a polynucleotide encoding the protein(s) of interest.
[074] In some embodiments, the expression cassette of the present disclosure further comprises a polynucleotide encoding a stretch of amino acids to facilitate affinity purification.
[075] Accordingly, envisaged herein is an expression cassette comprising the polynucleotide encoding the signal peptide as described above, the polynucleotide encoding the expression enhancer as described above, and a polynucleotide encoding a protein of interest, along with a stretch of amino acids to facilitate affinity purification.
[076] Thus, in some embodiments, the present disclosure provides an expression cassette for expressing a protein of interest comprising:
a. a polynucleotide encoding a signal peptide wherein the signal peptide is selected from a group comprising sequence represented by SEQ ID No. 1 and SEQ ID No. 21 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer wherein the expression enhancer is selected from a group comprising sequence represented by SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 23 or a sequence bearing at least 90% similarity thereto;
c. a polynucleotide encoding a stretch of amino acids to facilitate affinity purification; and
d. a polynucleotide encoding the protein(s) of interest.
[077] In some embodiments, the polynucleotide encoding the expression enhancer and the polynucleotide encoding the stretch of amino acids to facilitate affinity purification, may be present in the expression cassette in any order.
[078] Accordingly, in some embodiments, the expression cassette comprises the polynucleotide encoding the signal peptide as described in any one of the above embodiments, followed by the polynucleotide encoding the expression enhancer as described in any one of the above embodiments, and the polynucleotide encoding the stretch of amino acids to facilitate affinity purification in any order, followed by the polynucleotide encoding the protein of interest.
[079] In some non-limiting embodiments, the expression cassette comprises the polynucleotide encoding the signal peptide as described in any one of the above embodiments, followed by the polynucleotide encoding the expression enhancer as described in any one of the above embodiments, followed by the polynucleotide encoding the stretch of amino acids to facilitate affinity purification, followed by the polynucleotide encoding the protein of interest.
[080] In some non-limiting embodiments, the expression cassette comprises the polynucleotide encoding the signal peptide as described in any one of the above embodiments, followed by the polynucleotide encoding the stretch of amino acids to facilitate affinity purification, followed by the polynucleotide encoding the expression enhancer as described in any one of the above embodiments, followed by the polynucleotide encoding the protein of interest.
[081] In some embodiments, the stretch of amino acids to facilitate the affinity purification of the expressed protein of interest may be specifically designed or selected depending upon the protein of interest.
[082] In some embodiments, the stretch of amino acids to facilitate affinity purification is selected from a group comprising poly-histidine tag and poly-arginine tag.
[083] In some embodiments, the stretch of amino acids to facilitate the affinity purification is a 6XHis tag has a sequence represented by SEQ ID No. 9 or a sequence bearing at least 90% similarity thereto.
[084] In some embodiments, the stretch of amino acids to facilitate the affinity purification is a 6XHis tag encoded by SEQ ID No. 10 or a sequence bearing at least 90% similarity thereto.
[085] Accordingly, in some embodiments, the present disclosure provides an expression cassette for expressing a protein of interest comprising:
a. a polynucleotide encoding a signal peptide wherein the signal peptide is selected from a group comprising sequence represented by SEQ ID No. 1 and SEQ ID No. 21 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer wherein the expression enhancer is selected from a group comprising sequence represented by SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 23 or a sequence bearing at least 90% similarity thereto;
c. a polynucleotide encoding a stretch of amino acids to facilitate affinity purification, wherein the stretch of amino acids to facilitate affinity purification has sequence represented by SEQ ID No. 9 or a sequence bearing at least 90% similarity thereto; and
d. a polynucleotide encoding the protein(s) of interest.
[086] In some embodiments, the expression cassette of the present disclosure:
a. a polynucleotide encoding a signal peptide wherein the signal peptide is selected from a group comprising sequence represented by SEQ ID No. 1 and SEQ ID No. 21 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer wherein the expression enhancer is selected from a group comprising sequence represented by SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 23 or a sequence bearing at least 90% similarity thereto;
c. a polynucleotide encoding a stretch of amino acids to facilitate affinity purification, wherein the stretch of amino acids to facilitate affinity purification has sequence represented by SEQ ID No. 9 or a sequence bearing at least 90% similarity thereto; and
d. a polynucleotide encoding the protein(s) of interest,
wherein (b) and (c) may be present in any order in the expression cassette.
[087] In some embodiments, the polynucleotide encoding the signal peptide has sequence selected from a group comprising SEQ ID No. 2 and SEQ ID No. 22 or a sequence bearing at least 90% similarity thereto; the polynucleotide encoding the expression enhancer has sequence selected from a group comprising SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 24 or a sequence bearing at least 90% similarity thereto; and the polynucleotide encoding the stretch of amino acids to facilitate affinity purification has sequence represented by SEQ ID No. 10 or a sequence bearing at least 90% similarity thereto.
[088] Accordingly, in some embodiments, the present disclosure provides an expression cassette for expressing a protein of interest comprising:
a. a polynucleotide encoding a signal peptide wherein the polynucleotide has sequence selected from a group comprising SEQ ID No. 2 and SEQ ID No. 22 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer wherein the polynucleotide has sequence selected from a group comprising a group comprising SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 24 or a sequence bearing at least 90% similarity thereto;
c. a polynucleotide encoding a stretch of amino acids to facilitate affinity purification, wherein the said polynucleotide has sequence represented by SEQ ID No. 10 or a sequence bearing at least 90% similarity thereto; and
d. a polynucleotide encoding the protein(s) of interest.
[089] In some embodiments, the expression enhancer and the signal peptide act in synergy in yielding the high expression levels of proteins.
[090] In some embodiments, the expression cassette may further comprise polynucleotide sequence(s) encoding protease cleavage site(s), preferably preceding the protein of interest.
[091] Accordingly, in some embodiments, the present disclosure provides an expression cassette for expressing a protein of interest comprising:
a. a polynucleotide encoding a signal peptide, wherein the signal peptide is selected from a group comprising sequence represented by SEQ ID No. 1 and SEQ ID No. 21 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer, wherein the expression enhancer is selected from a group comprising sequence represented by SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 23 or a sequence bearing at least 90% similarity thereto;
c. a polynucleotide encoding a stretch of amino acids to facilitate affinity purification;
d. a polynucleotide encoding a protease cleavage site; and
e. a polynucleotide encoding the protein(s) of interest.
[092] In some embodiments, the present disclosure provides an expression cassette for expressing a protein of interest comprising:
a. a polynucleotide encoding a signal peptide, wherein the signal peptide is selected from a group comprising sequence represented by SEQ ID No. 1 and SEQ ID No. 21 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer, wherein the expression enhancer is selected from a group comprising sequence represented by SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 23 or a sequence bearing at least 90% similarity thereto;
c. a polynucleotide encoding a stretch of amino acids to facilitate affinity purification, wherein the stretch of amino acids to facilitate affinity purification has sequence represented by SEQ ID No. 9 or a sequence bearing at least 90% similarity thereto;
d. a polynucleotide encoding a protease cleavage site; and
e. a polynucleotide encoding the protein(s) of interest.
[093] In some embodiments, the present disclosure provides an expression cassette for expressing a protein of interest comprising:
a. a polynucleotide encoding a signal peptide wherein the polynucleotide has sequence selected from a group comprising SEQ ID No. 2 and SEQ ID No. 22 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer wherein the polynucleotide has sequence selected from a group comprising a group comprising SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 24 or a sequence bearing at least 90% similarity thereto;
c. a polynucleotide encoding a stretch of amino acids to facilitate affinity purification, wherein the said polynucleotide has sequence represented by SEQ ID No. 10 or a sequence bearing at least 90% similarity thereto;
d. a polynucleotide encoding a protease cleavage site; and
e. a polynucleotide encoding the protein(s) of interest.
[094] In some non-limiting embodiments, the protease cleavage site is selected from a group comprising TEV cleavage site, Enterokinase cleavage site and Factor Xa cleavage site.
[095] In some exemplary non-limiting exemplary embodiments, the protease cleavage site is a TEV cleavage site and has a sequence represented by SEQ ID No. 11 or a sequence bearing at least 90% similarity thereto.
[096] In some embodiments, the protease cleavage site is a TEV cleavage site and has a sequence represented by SEQ ID No. 11 or a sequence bearing at least 95% similarity thereto.
[097] In some embodiments, the protease cleavage site is a TEV cleavage site and has a sequence represented by SEQ ID No. 11 or a sequence bearing 95%, 96%, 97%, 98%, 99% or 100% similarity thereto.
[098] In some embodiments, the protease cleavage site is a TEV cleavage site and has a sequence represented by SEQ ID No. 11.
[099] In some embodiments, the protease cleavage site is encoded by SEQ ID No. 12 or a sequence bearing at least 90% similarity thereto.
[0100] In some embodiments, the protease cleavage site is encoded by SEQ ID No. 12 or a sequence bearing at least 95% similarity thereto.
[0101] In some embodiments, the protease cleavage site is encoded by SEQ ID No. 12 or a sequence bearing 95%. 96%, 97%, 98%, 99% or 100% similarity thereto.
[0102] In some embodiments, the protease cleavage site is encoded by SEQ ID No. 12.
[0103] Accordingly, in some embodiments, the present disclosure provides an expression cassette for expressing a protein of interest comprising:
a. a polynucleotide encoding a signal peptide, wherein the signal peptide is selected from a group comprising sequence represented by SEQ ID No. 1 and SEQ ID No. 21 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer, wherein the expression enhancer is selected from a group comprising sequence represented by SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 23 or a sequence bearing at least 90% similarity thereto;
c. a polynucleotide encoding a stretch of amino acids to facilitate affinity purification, wherein the stretch of amino acids to facilitate affinity purification has sequence represented by SEQ ID No. 9 or a sequence bearing at least 90% similarity thereto;
d. a polynucleotide encoding a protease cleavage site, wherein the protease cleavage site has a sequence represented by SEQ ID No. 11 or a sequence bearing at least 90% similarity thereto; and
e. a polynucleotide encoding the protein(s) of interest.
[0104] In some embodiments, the polynucleotide encoding the signal peptide has sequence selected from a group comprising SEQ ID No. 2 and SEQ ID No. 22 or a sequence bearing at least 90% similarity thereto; the polynucleotide encoding the expression enhancer has sequence selected from a group comprising SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 24 or a sequence bearing at least 90% similarity thereto; the polynucleotide encoding the stretch of amino acids to facilitate affinity purification has sequence represented by SEQ ID No. 10 or a sequence bearing at least 90% similarity thereto; and the polynucleotide encoding the protease cleavage site has sequence represented by SEQ ID No. 12 or a sequence bearing at least 90% similarity thereto.
[0105] Thus, in some embodiments, the present disclosure provides an expression cassette for expressing a protein of interest comprising:
a. a polynucleotide encoding a signal peptide, wherein the polynucleotide has sequence selected from a group comprising SEQ ID No. 2 and SEQ ID No. 22 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer, wherein the polynucleotide has sequence selected from a group comprising SEQ ID No. 6, SEQ ID No. 7, SEQ ID No. 8 and SEQ ID No. 24 or a sequence bearing at least 90% similarity thereto;
c. a polynucleotide encoding a stretch of amino acids to facilitate affinity purification, wherein the polynucleotide has sequence represented by SEQ ID No. 10 or a sequence bearing at least 90% similarity thereto;
d. a polynucleotide encoding a protease cleavage site, wherein the polynucleotide has sequence represented by SEQ ID No. 12 or a sequence bearing at least 90% similarity thereto; and
e. a polynucleotide encoding the protein(s) of interest.
[0106] In some embodiments, the protein of interest may be any recombinant protein preferably having amino acid chain length less than 200. In some embodiments, the protein of interest is a bioactive peptide. In some embodiments, the protein of interest is a peptide hormone that is less than 200 amino acids long. Examples of such proteins include but are not limited to Lirapeptide, Teriparatide, Exenatide, Lixisenatide, Teduglutide, insulin and its analogues, or Semaglutide.
[0107] Accordingly, in some embodiments, the protein of interest is a therapeutic peptide selected from a group comprising Lirapeptide, Teriparatide, Exenatide, Lixisenatide, Teduglutide, insulin and its analogues and Semaglutide. While sequences of the said proteins are known in the art, the said proteins have been provided for the purposes of exemplification and the choice of the protein of interest may be varied as per the requirements of the user of the expression cassette of the present disclosure. Accordingly, the expression cassette of the present disclosure may be employed for the expression of any protein of interest.
[0108] Accordingly, in a non-limiting embodiment, for the purposes of exemplification, the expression cassette may comprise a polynucleotide sequence encoding Lirapeptide as the protein of interest and thus, may have sequence selected from a group comprising SEQ ID Nos. 13, 14, 15 and 25, or sequences bearing at least 90% similarity thereto.
[0109] In some embodiments, the expression cassette of the present disclosure, encoding Lirapeptide as the protein of interest, may have sequence selected from a group comprising SEQ ID Nos. 13, 14, 15 and 25, or sequences bearing at least 95% similarity thereto.
[0110] In some embodiments, the expression cassette of the present disclosure, encoding Lirapeptide as the protein of interest, may have sequence selected from a group comprising SEQ ID Nos. 13, 14, 15 and 25, or sequences bearing 95%, 96%, 97%, 98%, 99% or 100% similarity thereto.
[0111] In some embodiments, the expression cassette of the present disclosure, encoding Lirapeptide as the protein of interest, may have sequence selected from a group comprising SEQ ID Nos. 13, 14, 15 and 25.
[0112] In a non-limiting embodiment, the said expression cassette of the present disclosure encodes a peptide sequence represented by SEQ ID Nos. 16, 17, 18 and 26, or sequences bearing at least 90% similarity thereto.
[0113] In some embodiments, the said expression cassette of the present disclosure encodes a peptide sequence represented by SEQ ID Nos. 16, 17, 18 and 26, or sequences bearing at least 95% similarity thereto.
[0114] In some embodiments, the expression cassette encodes a peptide sequence represented by SEQ ID Nos. 16, 17, 18 and 26, or sequences bearing 95%, 96%, 97%, 98%, 99% or 100% similarity thereto.
[0115] In some embodiments, the expression cassette encodes a peptide sequence represented by SEQ ID Nos. 16, 17, 18 and 26.
[0116] In a non-limiting embodiment, the order of elements in the expression cassette is such that the polynucleotide sequence encoding the signal peptide is positioned first in the cassette followed by the polynucleotide sequence encoding the expression enhancer, followed by the polynucleotide sequence encoding the protease cleavage site and the polynucleotide sequence encoding the protein of interest.
[0117] In a non-limiting exemplary embodiment, the order of elements in the expression cassette is such that the polynucleotide sequence encoding signal peptide is positioned first in the cassette, followed by the polynucleotide sequence encoding the expression enhancer and the polynucleotide sequence encoding the stretch of amino acids to facilitate the affinity purification in any order. In a further non-limiting embodiment, the expression cassette subsequently comprises the polynucleotide sequence encoding the protein of interest, preferably preceded by the polynucleotide sequence encoding the protease cleavage site.
[0118] Accordingly, in some embodiments, the order of elements in the expression cassette is polynucleotide sequence encoding: signal peptide-expression enhancer-protease cleavage site-protein of interest.
[0119] In a non-limiting exemplary embodiment, the order of elements in the expression cassette is polynucleotide sequence encoding: signal peptide-expression enhancer-stretch of amino acids to facilitate affinity purification-protease cleavage site-protein of interest.
[0120] In yet another non-limiting exemplary embodiment, the order of elements in the expression cassette is polynucleotide sequence encoding: signal peptide-stretch of amino acids to facilitate affinity purification-expression enhancer-protease cleavage site-protein of interest.
[0121] In some embodiments, the expression cassette described in any of the previous embodiments is operably linked to a promoter.
[0122] In some embodiments, the promoter may be any promoter that is capable of inducing expression of the protein of interest.
[0123] In some embodiments, the promoter may vary depending on the specific protein of interest being expressed.
[0124] In some non-limiting embodiments, the promoter is an inducible promoter or a constitutive promoter.
[0125] The above-described expression cassette may be created through use of recombinant methods that are well known. Those of skill in the art will recognize that many techniques pertaining to recombinant technology such as digestion and ligation reactions which may be employed for producing recombinant nucleic acids can be used to produce the expression cassette of the present disclosure. Such methods employing the above discussed elements/sequences are envisaged in the scope of the present disclosure.
[0126] The present disclosure also provides an expression vector for the expression of protein(s) of interest, wherein the vector comprises the expression cassette as described above.
[0127] Although the subsequent embodiments relate to a vector comprising the expression construct, the expression construct per se including its various components, such as the polynucleotide sequences forming the expression construct as well as the peptide sequences encoded by said polynucleotide sequences, such as the signal peptide, the expression enhancer, the stretch of amino acids to facilitate the affinity purification, the protease cleavage site and the protein of interest are as described in the previous embodiments and are not being reiterated for the sake of brevity but fall wholly within the scope of the present embodiments.
[0128] In some embodiments, the vector comprises the expression cassette as described above, in combination with one or more promoter sequence(s), and an open reading frame coding for a protein of interest. In some embodiments, the vector may comprise further regulatory sequences capable of increasing or decreasing the expression of specific genes within an organism.
[0129] In some embodiments, the promoter may be any promoter that is capable of inducing expression of the protein of interest.
[0130] In some embodiments, the promoter may vary depending on the specific protein of interest being expressed.
[0131] In some non-limiting embodiments, the promoter is an inducible promoter or a constitutive promoter.
[0132] Without intending to be limited by theory, the various elements of the expression cassette and/or the vector may be operably linked for enhanced expression and secretion of recombinant protein.
[0133] In a non-limiting embodiment, examples of vector backbones employable to obtain the above-defined vector include but are not limited to pD451.SR, pD431.SR, pET28, pET36, pGEX, pBAD, pQE9, pD861.sskt and pRSET.
[0134] Methods to form a vector comprising the expression cassette of the present disclosure inserted into the vector backbone are well known in the art. Such methods employing the above discussed expression cassette and individual elements /sequences are envisaged in the scope of the present disclosure.
[0135] Further envisaged in the present disclosure is a host cell comprising the expression cassette or the expression vector as described in any of the above embodiments.
[0136] Although the subsequent embodiments relate to a host cell comprising the expression construct/expression vector, the expression construct and the expression vector per se including their various components, such as the polynucleotide sequences forming the expression construct and the expression vector as well as the peptide sequences encoded by said polynucleotide sequences, such as the signal peptide, the expression enhancer, the stretch of amino acids to facilitate the affinity purification, the protease cleavage site and the protein of interest, the elements of the vector, including the promoter, the open reading frame and the further regulatory sequences are as described in the previous embodiments and are not being reiterated for the sake of brevity but fall wholly within the scope of the present embodiments.
[0137] In some embodiments, the host cell is a prokaryotic or eukaryotic cell.
[0138] In exemplary embodiments of the present disclosure, the host cell is a prokaryotic cell.
[0139] In some embodiments of the present disclosure, the host cell is a bacterial cell. Examples of bacteria include, but are not limited to, Gram-negative and Gram-positive organisms.
[0140] In a non-limiting exemplary embodiment, the host cell is E.coli.
[0141] In some embodiments, the host cell may comprise one or more genetic modifications that improve the expression of the protein of interest.
[0142] In a non-limiting embodiment, when the host cell is E.coli, the E.coli is a strain that comprises one more mutations in the lipoprotein (lpp) gene to inhibit or reduce its expression. Deletion of the lpp gene makes the cells leaky, and aids in enhanced secretion of recombinant proteins expressed using the expression cassette of the present disclosure.
[0143] Accordingly, in a non-limiting embodiment, the host cell is an lpp mutant of E.coli. In a non-limiting exemplary embodiment, the host cell is an lpp deletion mutant of E.coli.
[0144] Thus, in some embodiments, envisaged herein is an lpp mutant of E.coli comprising the expression cassette or the expression vector as described in any of the embodiments above.
[0145] In some embodiments, envisaged herein is an lpp deletion mutant of E.coli comprising the expression cassette or the expression vector as described in any of the embodiments above.
[0146] In some exemplary, non-limiting embodiments, envisaged herein is an lpp deletion mutant of E.coli comprising the expression cassette or the expression vector as described above which help in enhanced expression and secretory production of Lirapeptide as the protein of interest and has sequence selected from a group comprising SEQ ID Nos. 13, 14, 15 and 25, or sequences bearing at least 90% similarity thereto.
[0147] Methods for introducing exogenous DNA such as the expression cassette or vector as defined above into host cells such as bacteria are well known in the art. In some embodiments, such methods may include either the transformation of the host cell treated with CaCl2 or other agents, such as divalent cations and DMSO. Exogenous DNA may also be introduced into bacterial cells by electroporation, use of a bacteriophage, or ballistic transformation. Such methods employing the above discussed expression cassette, vector and sequences are envisaged in the scope in the present disclosure.
[0148] The expression cassette, the vector and the host cell comprising the same find application in the recombinant expression and secretion of any protein of interest such as but not limited to bioactive peptides.
[0149] Accordingly, envisaged in the present disclosure is a method of producing proteins comprising culturing the host cell as described above. Although the following embodiments relate to method of producing proteins, the expression construct and the expression vector per se including their various components, such as the polynucleotide sequences forming the expression vector as well as the peptide sequences encoded by said polynucleotide sequences, such as the signal peptide, the expression enhancer, the stretch of amino acids to facilitate the affinity purification, the protease cleavage site and the protein of interest; the elements of the vector, including the promoter, the open reading frame and the further regulatory sequences; and the host cell are as described in the previous embodiments and are not being reiterated for the sake of brevity but fall wholly within the scope of the present embodiments.
[0150] As mentioned above, in some embodiments, the method of producing proteins comprises culturing the host cell of the present disclosure as described above. In a non-limiting embodiment, the said culture is performed in a fermentation tank by carefully adjusting parameters such as but not limited to dissolved oxygen, temperature control, pH, agitation and aeration.
[0151] In some embodiments, provided herein is a method for producing a protein of interest in a bacterial host cell, the said method comprising steps of:
- culturing the bacterial host cell described above under suitable conditions to secrete the protein of interest into culture medium; and
- purifying the protein of interest from the culture medium.
[0152] In some embodiments, the method for enhanced expression and secretory production of proteins in a bacterial host cell, comprises:
- transforming a bacterial host cell to express the expression cassette or the expression vector as defined above to obtain a transformed host;
- culturing the transformed bacterial host cell under suitable conditions to secrete the protein of interest into culture medium; and
- purifying the protein of interest from the culture medium.
[0153] The expression of the protein of interest is achieved by growth or culture of the above-described host cells. Methods and materials for the growth and maintenance of many types of cells are well known and are available commercially. Such methods can be easily adapted for culturing the host cell of the present disclosure for the production of proteins such as bioactive peptides at relatively high yields. Such methods are envisaged in the scope of the present disclosure.
[0154] In a non-limiting embodiment, broadly, the method of producing a protein of interest comprises:
construction of the expression cassette or the expression vector as described in any of the embodiments above;
transformation of the expression construct or the expression vector into a host cell;
evaluation of transformed clone(s) and selection thereof;
culturing the selected clones to secrete the protein of interest into culture medium; and
isolation and purification of the protein of interest from the culture medium.
[0155] In some embodiments, the method of producing a protein of interest consists of:
construction of the expression cassette or the expression vector as described in any of the embodiments above;
transformation of the expression construct or the expression vector into a host cell;
evaluation of transformed clone(s) and selection thereof;
subjecting the selected clones to fermentation process; and
isolation and purification of the protein of interest.
[0156] In a non-limiting embodiment, the present disclosure provides a method of producing a protein of interest in a bacterial host cell, the said method comprising the steps of:
i. Obtaining an expression cassette comprising:
a. a polynucleotide sequence encoding a signal peptide;
b. a polynucleotide sequence encoding an expression enhancer;
c. a polynucleotide sequence encoding a protease cleavage site; and
d. a polynucleotide sequence encoding the protein of interest,
wherein the polynucleotide sequences of the expression cassette are operably linked to an inducible or constitutive promoter,
ii. transforming a bacterial host cell with the expression cassette to obtain a transformed host;
iii. culturing the transformed bacterial host cell under suitable conditions to secrete the protein of interest into culture medium; and
iv. purifying the protein of interest from the culture medium.
[0157] In a further non-limiting embodiment, the present disclosure provides a method of producing a protein of interest in a bacterial host cell, the said method comprising the steps of:
i. Obtaining an expression vector having an expression cassette comprising:
e. a polynucleotide sequence encoding a signal peptide;
f. a polynucleotide sequence encoding an expression enhancer;
g. a polynucleotide sequence encoding a protease cleavage site; and
h. a polynucleotide sequence encoding the protein of interest,
wherein the polynucleotide sequences of the expression cassette are operably linked to an inducible or constitutive promoter,
ii. transforming a bacterial host cell with the expression vector to obtain a transformed host;
iii. culturing the transformed bacterial host cell under suitable conditions to secrete the protein of interest into culture medium; and
iv. purifying the protein of interest from the culture medium.
[0158] In some embodiments, the signal peptide encoded by the expression cassette of the above-mentioned method for expression of protein of interest has sequence selected from SEQ ID No: 1 and SEQ ID No: 21 or a sequence bearing at least 90% similarity to the sequences thereto.
[0159] In some embodiments, the expression enhancer encoded by the expression cassette of the above-mentioned method for expression of protein of interest has sequence selected from the group comprising SEQ ID Nos: 3, 4, 5 or 23, or a sequence having at least 90% similarity to the said sequences.
[0160] In some embodiments, the stretch of amino acids to facilitate the affinity purification is a 6XHis tag has a sequence represented by SEQ ID No. 9 or a sequence bearing at least 90% similarity thereto.
[0161] In some embodiments, the protease cleavage site is a TEV cleavage site and has a sequence represented by SEQ ID No. 11 or a sequence bearing at least 90% similarity thereto.
[0162] Thus, in some embodiments, the method of producing a protein of interest in a bacterial host cell, comprises:
i. Obtaining an expression cassette comprising:
a. a polynucleotide encoding a signal peptide, wherein the signal peptide is selected from a group comprising sequence represented by SEQ ID No. 1 and SEQ ID No. 21 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer, wherein the expression enhancer is selected from a group comprising sequence represented by SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 23 or a sequence bearing at least 90% similarity thereto;
c. a polynucleotide encoding a stretch of amino acids to facilitate affinity purification, wherein the stretch of amino acids to facilitate affinity purification has sequence represented by SEQ ID No. 9 or a sequence bearing at least 90% similarity thereto;
d. a polynucleotide encoding a protease cleavage site, wherein the protease cleavage site has a sequence represented by SEQ ID No. 11 or a sequence bearing at least 90% similarity thereto; and
e. a polynucleotide encoding the protein(s) of interest.
wherein the polynucleotide sequences of the expression cassette are operably linked to an inducible or constitutive promoter,
ii. transforming a bacterial host cell with the expression cassette to obtain a transformed host;
iii. culturing the transformed bacterial host cell under suitable conditions to secrete the protein of interest into culture medium; and
iv. purifying the protein of interest from the culture medium.
[0163] In some embodiments, the method of producing a protein of interest in a bacterial host cell, comprises:
i. Obtaining an expression vector having an expression cassette comprising:
f. a polynucleotide encoding a signal peptide, wherein the signal peptide is selected from a group comprising sequence represented by SEQ ID No. 1 and SEQ ID No. 21 or a sequence bearing at least 90% similarity thereto;
g. a polynucleotide encoding an expression enhancer, wherein the expression enhancer is selected from a group comprising sequence represented by SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 23 or a sequence bearing at least 90% similarity thereto;
h. a polynucleotide encoding a stretch of amino acids to facilitate affinity purification, wherein the stretch of amino acids to facilitate affinity purification has sequence represented by SEQ ID No. 9 or a sequence bearing at least 90% similarity thereto;
i. a polynucleotide encoding a protease cleavage site, wherein the protease cleavage site has a sequence represented by SEQ ID No. 11 or a sequence bearing at least 90% similarity thereto; and
j. a polynucleotide encoding the protein(s) of interest.
wherein the polynucleotide sequences of the expression cassette are operably linked to an inducible or constitutive promoter,
ii. transforming a bacterial host cell with the expression vector to obtain a transformed host;
iii. culturing the transformed bacterial host cell under suitable conditions to secrete the protein of interest into culture medium; and
iv. purifying the protein of interest from the culture medium.
[0164] In a non-limiting embodiment, the method of producing a protein of interest comprises:
construction of an expression cassette or an expression vector comprising the expression cassette having sequence selected from a group comprising SEQ ID Nos. 13, 14, 15 and 25, or sequences bearing at least 90% similarity thereto;
transformation of the vector into an lpp mutant E.coli;
evaluation of the transformed clones and selection thereof;
subjecting the selected clones to fermentation process; and
isolation and purification of the protein of interest.
[0165] Methodologies for expression cassette and vector construction, transformation, clone evaluation, fermentation, protein isolation and purification are widely described in the art and in some embodiments, such methodologies may be suitably adapted to employ the combination of elements and sequences described in any of the previous embodiments that characterize the present disclosure.
[0166] The above-described method of the present disclosure improves the expression and secretion of the protein of interest into the extra cellular fermentation broth.
[0167] Therefore, in another embodiment, the present disclosure provides a method of enhancing the expression of any protein of interest comprising culturing the host cell as described above.
[0168] Further envisaged in the present disclosure, is use of the expression cassette or the expression vector as described above for the expression of any protein of interest.
[0169] In another embodiment, the present disclosure provides use of the host cell as described above for the expression of any protein of interest.
[0170] In some embodiments, the protein of interest is a bioactive peptide.
[0171] Accordingly, envisaged herein is use of the expression cassette as described above for the expression of bioactive peptides.
[0172] In another embodiment, the present disclosure provides use of the expression vector as described above for the expression of bioactive peptides.
[0173] In another embodiment, the present disclosure provides use of the host cell as described above for the expression of bioactive peptides.
[0174] In some embodiments, the bioactive peptides are therapeutic peptides preferably composed of less than 200 amino acids. In some embodiments, the protein of interest is a peptide hormone. In a non-limiting embodiment, the bioactive peptides may be therapeutic peptides including but not limited to Lirapeptide, Teriparatide, Exenatide, Lixisenatide, Teduglutide, insulin and its analogues, or Semaglutide.
[0175] Further envisaged in the present disclosure is a protein encoded by the expression cassette or the expression vector as described in any of the embodiments above.
[0176] Also envisaged herein is a protein produced by the above-described method of protein production.
[0177] The present disclosure further envisages a kit comprising the expression cassette and/or the vector of the present disclosure and instructions for their use. In some embodiments, the expression cassette and/or vector incorporated in the kit may be designed to comprise a spacer sequence in place of a sequence encoding the protein such that the spacer may be replaced by a sequence encoding a protein of interest to the user.
[0178] In some embodiments, the kit may further comprise means for manipulation of the expression cassette or the vector, wherein the manipulation may include but is not limited to modification of the expression cassette or vector for introducing sequence(s) of a protein of interest. In a non-limiting embodiment, means for manipulation may include one or more components such as but not limited to restriction enzyme(s), dNTPs and buffer(s).

Advantages:
The advantages of the present disclosure include but are not limited to:
• Typically, expression cassettes are large in size, decreasing the potential yield of a peptide of interest. This is particularly problematic in situations where the peptide of interest is small. The expression cassette of the present disclosure is relatively short and provides high expression levels of small peptides such as Lira-peptide while improving the stability of the peptide, which is helpful in post expression purification.
• The means and method of the present disclosure provides a cost-effective way of protein production.
• The present disclosure overcomes the obstacles that are usually related to low expression levels of protein, more particularly recombinant proteins or the destruction of expressed polypeptide by proteolytic enzymes within the cytoplasm of the cells. The expression cassette ensures relatively high expression levels of the protein interest.
• Ease of purification and continuous fermentation are possible as the recombinant proteins are secreted into the culture medium.
[0179] In an embodiment, the foregoing descriptive matter is illustrative of the disclosure and not a limitation. While the present disclosure is susceptible to various modifications and alternative forms, specific aspects thereof have been shown by way of examples and drawings and are described in detail below. However, it should be understood that it is not intended to limit the invention to the particular forms disclosed, but on the contrary, the intention is to cover all modifications, equivalents, and alternatives falling within the spirit and the scope of the invention as defined by the embodiments of the disclosure.

EXAMPLES:
EXAMPLE 1: Construction and evaluation of high-copy Lirapeptide expression clones:
[0180] The nucleotide sequence encoding Lirapeptide (SEQ ID NO. 20) was PCR amplified with the signal peptide, the expression enhancer, 6X-his tag and a TEV cleavage recognition site, using suitable primers, and the amplified products were digested with SapI restriction enzyme. Different combinations of the signal peptide (16 nos.) and expression enhancer (7 nos.) were employed for preparing each amplified product. The design of the expression cassette containing the signal peptide and secretion enhancer combinations is depicted in Figure 1. Restriction-digested fragments were ligated with the pD861.sskt linear plasmids containing different signal peptides. About 112 plasmid constructs were designed having different combinations of signal peptide and expression enhancers formed on the basis of 16 signal peptides and 7 expression enhancers containing Lirapeptide. Resultant plasmids containing Lirapeptide expression cassettes were confirmed by nucleotide sequencing.
[0181] The sequence confirmed by high copy vector constructs was transformed into E. coli JW1667?Kan?LPP by calcium chloride heat-shock transformation method. The transformed E. coli cells were cultured in about 3 ml salt media containing about 25 µg/ml Kanamycin in 24 deep well plates overnight at about 37°C in a shaker incubator, followed by diluting the culture with new media at about 1:100 ratio and allowing the cultured cells to grow until OD reaches ~0.6. Then rhamnose was added to a final concentration of about 6.6mM and the cultures were incubated in a shaker incubator for about 5 hours at about 30°C. After centrifuging the cultures, the supernatant and whole cell samples were run on the tris-tricine gel. Clones were selected based on higher densities of Lirapeptide secretion bands on the gel. Of the 112 combinations tested, only 15 combinations secreted Lirapeptide in E.coli at promising levels while using a high copy plasmid (Figure 4). It was observed that most of the peptide accumulated intracellularly as an insoluble fraction. Usually, protein accumulation happens when the expression rate exceeds the secretion capacity. Hence, the selected 15 combinations were subsequently tested in a low-copy plasmid, in anticipation of a lower expression rate.

EXAMPLE 2: Construction and evaluation of low-copy Lirapeptide expression clones:
[0182] The high-copy constructs (15 nos.) that showed positive for Lirapeptide secretion were selected to create their low-copy version. The expression cassettes containing nucleotide sequence encoding promoter, signal peptide, expression enhancer, Lirapeptide and a terminator from their respective high copy plasmids were isolated through restriction digestion using XbaI & NotI enzymes and ligated with a low copy plasmid pD431. The resultant vector constructs containing Lirapeptide expression cassettes were confirmed by nucleotide sequencing.
[0183] The various elements of the vector construct were operably linked for enhanced expression and secretion of recombinant protein. Figures 2A-2C depict the representative expression vector constructs, comprising the expression cassettes of the present disclosure.
[0184] The sequence confirmed low copy vector constructs containing Lirapeptide with the expression cassettes of the present disclosure, were transformed into E. coli JW1667?Kan?LPP by calcium chloride heat-shock transformation method and the transformed cells were tested for Lirapeptide secretion using the same protocol mentioned in the above example. Clones were selected based on higher densities of Lirapeptide secretion bands on the gel and were confirmed using a specific antibody (anti-GLP-1) by Western blotting. The tris-tricine gel results are provided in Figure 3, which show significantly high expression levels of the Lirapeptide by the host cells of the present disclosure.
[0185] Out of the 15 combinations tested, it was found that four combinations were secreting the Lirapeptide in E.coli at commercially significant levels (Figure-3). They were identified as:
• gIII(+A) signal peptide with LP2 expression enhancer
• gIII(+A) signal peptide with YebFN13
• gIII(+A) signal peptide with CelCDN20
• OmpT signal peptide with YebFN20
[0186] The above, therefore, shows that the expression cassettes of the present disclosure show enhanced efficacy over other expression cassettes synthesized based on other alternative signal peptides and expression enhancers.

EXAMPLE 3: Lirapeptide expression using constructs lacking signal peptide
[0187] Expression constructs comprising a nucleotide sequence encoding Lirapeptide and expression enhancers (YebFN13, CelCDN20, LP2, and YebFN20 – each incorporated in separate constructs) but lacking signal peptides, were synthesized using PCR from their respective high copy plasmid templates. The plasmids pD861 (high copy), pD431 (low copy), and Lirapeptide cassettes without signal peptides were digested with XbaI and NotI and then ligated to form eight separate vector constructs comprising YebFN13-LP, CelCDN20-LP, LP2-LP, and YebFN20-LP in high copy plasmid and low copy plasmid. The resultant vector constructs containing Lirapeptide expression cassettes were confirmed by nucleotide sequencing.
[0188] These vectors were transformed into E. coli JW1667?Kan?LPP by calcium chloride heat-shock transformation method and the transformed cells were tested for lira-peptide secretion using the same protocol mentioned in the Example 1.
[0189] The tris-tricine gel results are shown in Figure 5a and 5b indicate comparative data of the Lirapeptide expression in the whole cell fractions and culture supernatant. The presence of Lirapeptide in the whole cell fraction and its absence in the culture supernatant indicates that the Lirapeptide transformed host cells failed to secrete Lirapeptide into the culture medium in the absence of signal peptide.

EXAMPLE 4: Lirapeptide expression using constructs lacking expression enhancers
[0190] The nucleotide sequence encoding Lirapeptide was PCR amplified without the expression enhancers (YebFN13, CelCDN20, LP2, and YebFN20) but containing the signal peptide (gIII(+A) or OmpT), 6X his tag and a TEV cleavage recognition site, using suitable primers, and then digested the PCR products with SapI restriction enzyme. Restriction-digested fragments were ligated with the pD861-OmpT and pD861-gIII(+A) linear plasmids. Resultant plasmids containing Lirapeptide expression cassettes were confirmed by nucleotide sequencing.
[0191] These plasmids were transformed into E. coli JW1667?Kan?LPP by calcium chloride heat-shock transformation method and the transformed cells were tested for Lirapeptide secretion using the same protocol mentioned in the example-1.
[0192] The comparative data on the Lirapeptide expression in the culture supernatant and the whole cell fractions, as shown in figure 6, indicates that the transformed cells fail to secrete Lirapeptide into the culture medium in the absence of an expression enhancer.
[0193] Additional embodiments and features of the present disclosure will be apparent to one of ordinary skill in art based on the description provided herein. The embodiments herein provide various features and advantageous details thereof in the description. Descriptions of well-known/conventional methods and techniques are omitted so as to not unnecessarily obscure the embodiments herein.
[0194] The foregoing description of the specific embodiments fully reveals the general nature of the embodiments herein that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. Therefore, while the embodiments in this disclosure have been described in terms of preferred embodiments, those skilled in the art will recognize that the embodiments herein can be practiced with modification within the spirit and scope of the embodiments as described herein.
[0195] While considerable emphasis has been placed herein on the particular features of this disclosure, it will be appreciated that various modifications can be made, and that many changes can be made in the preferred embodiments without departing from the principles of the disclosure. These and other modifications in the nature of the disclosure or the preferred embodiments will be apparent to those skilled in the art from the disclosure herein, whereby it is to be distinctly understood that the foregoing descriptive matter is to be interpreted merely as illustrative of the disclosure and not as a limitation.
All references, articles, publications, general disclosures etc. cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, general methodologies etc. cited herein is not, and should not be taken as, an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world. ,CLAIMS:WE CLAIM:

1. An expression cassette for enhanced expression and secretory production of a protein of interest comprising:
a. a polynucleotide encoding a signal peptide wherein the signal peptide is selected from a group comprising sequence represented by SEQ ID No. 1 and SEQ ID No. 21 or a sequence bearing at least 90% similarity thereto;
b. a polynucleotide encoding an expression enhancer wherein the expression enhancer is selected from a group comprising sequence represented by SEQ ID No. 3, SEQ ID No. 4, SEQ ID No. 5 and SEQ ID No. 23 or a sequence bearing at least 90% similarity thereto; and
c. a polynucleotide encoding the protein(s) of interest.
2. The expression cassette as claimed in claim 1, wherein the expression cassette comprises nucleotides encoding signal peptide and expression enhancer sequences represented by SEQ ID No. 1 and SEQ ID No. 3, SEQ ID No. 1 and SEQ ID No. 4, SEQ ID No. 1 and SEQ ID No. 5 or SEQ ID No. 21 and SEQ ID No.23, respectively.
3. The expression cassette as claimed in claim 1 or claim 2, wherein the expression cassette further comprises a polynucleotide encoding a stretch of amino acids to facilitate affinity purification.
4. The expression cassette as claimed in claim 3, wherein the stretch of amino acids to facilitate the affinity purification is a polyhistidine tag or a polyarginine tag.
5. The expression cassette as claimed in any one of claims 1-4, wherein the expression cassette comprises the polynucleotide encoding the signal peptide, followed by the polynucleotide encoding the expression enhancer and the polynucleotide encoding the stretch of amino acids to facilitate affinity purification in any order, followed by the polynucleotide encoding the protein(s) of interest.
6. The expression cassette as claimed in any one of claims 1-5, wherein the expression cassette further comprises a polynucleotide encoding protease cleavage site, preferably preceding the protein of interest.
7. The expression cassette as claimed in claim 6, wherein the protease cleavage site is selected from a group comprising TEV cleavage site, Enterokinase cleavage site and Factor Xa_cleavage site.
8. The expression cassette as claimed in any one of claims 1-7, wherein the protein of interest is a peptide hormone that is less than 200 amino acids long.
9. The expression cassette as claimed in claim 8, wherein the protein of interest is selected from a group comprising Lirapeptide, Teriparatide, Exenatide, Lixisenatide, Teduglutide, Insulin and its analogues, and Semaglutide.
10. The expression cassette as claimed in any one of claims 1-9, wherein the said polynucleotide sequence of the expression cassette is operably linked to a promoter.
11. An expression vector comprising the expression cassette as claimed in any one of claims 1-10.
12. A bacterial host cell comprising the expression cassette as claimed in any one of claims 1-10 or the expression vector as claimed in claim 11.
13. The bacterial host cell as claimed in claim 12, wherein the said bacterial host is a strain of E.coli.
14. The bacterial host cell as claimed in claim 13, wherein the strain of E.coli comprises one or mutations in the lipoprotein (lpp) gene.
15. A method for producing a protein of interest in a bacterial host cell, the said method comprising steps of:
i. culturing the bacterial host cell as claimed in any one of claims 12-14 under suitable conditions to secrete the protein of interest into culture medium; and
ii. purifying the protein of interest from the culture medium.
16. A protein encoded by the expression cassette as claimed in any one of claims 1-10 or the expression vector as claimed in claim 11.
17. Use of the expression cassette as claimed in any one of claims 1-10 or the expression vector as claimed in claim 11 for the expression of bioactive peptides.
18. A kit comprising the expression cassette as claimed in any one of claims 1-10 or the expression vector as claimed in claim 11 and instructions for their use.