DESC:PROCESS FOR PREPARATION OF LIRAGLUTIDE

FIELD OF THE INVENTION
The present invention relates to a process for the preparation of liraglutide by expression of synthetic oligonucleotide encoding lirapeptide which is operably connected to a oligonucleotide sequence of signal peptide in eukaryotic cell, preferably an yeast cell, an expression construct comprising synthetic oligonucleotide encoding lirapeptide which is operably connected to a oligonucleotide sequence of signal peptide, a vector carrying said expression construct including yeast cells transformed with the vectors.

BACKGROUND OF THE INVENTION AND DISCLOSURE OF PRIOR ART
Liraglutide, marketed under the brand name Victoza, is a long-acting glucagon like peptide agonist developed by Novo Nordisk for the treatment of type 2 diabetes.
Liraglutide is an injectable drug that reduces the level of sugar (glucose) in the blood. It is used for treating type 2 diabetes and is similar to exenatide (Byetta). Liraglutide belongs to a class of drugs called incretin mimetics because these drugs mimic the effects of incretins. Incretins, such as human-glucagon-like peptide-1 (GLP-1), are hormones that are produced and released into the blood by the intestine in response to food. GLP-1 increases the secretion of insulin from the pancreas, slows absorption of glucose from the gut, and reduces the action of glucagon. (Glucagon is a hormone that increases glucose production by the liver.) All three of these actions reduce levels of glucose in the blood. In addition, GLP-1 reduces appetite. Liraglutide is a synthetic (man-made) hormone that resembles and acts like GLP-1. In studies, Liraglutide treated patients achieved lower blood glucose levels and experienced weight loss.
Liraglutide is an acylated human glucagon-like peptide-1 (GLP-1) agonist, with a 97% amino acid sequence identity to endogenous human GLP-1 (7-37). GLP-1 (7-37) represents less than 20% of total circulating endogenous GLP-1. Like GLP-1 (7-37), liraglutide activates the GLP-1 receptor, a membrane-bound cell-surface receptor coupled to adenylyl cyclase by the stimulatory G-protein, Gs, in pancreatic beta cells. Liraglutide increases intracellular cyclic AMP (cAMP), leading to insulin release in the presence of elevated glucose concentrations. This insulin secretion subsides as blood glucose concentrations decrease and approach euglycemia. Liraglutide also decreases glucagon secretion in a glucose-dependent manner. The mechanism of blood glucose lowering also involves a delay in gastric emptying. GLP-1 (7-37) has a half-life of 1.5-2 minutes due to degradation by the ubiquitous endogenous enzymes, dipeptidyl peptidase IV (DPP-IV) and neutral endopeptidases (NEP). Unlike native GLP-1, liraglutide is stable against metabolic degradation by both peptidases and has a plasma half-life of 13 hours after subcutaneous administration. The pharmacokinetic profile of liraglutide, which makes it suitable for once daily administration, is a result of self-association that delays absorption, plasma protein binding and stability against metabolic degradation by DPP-IV and NEP. It reduces meal-related hyperglycemia (for 24 hours after administration) by increasing insulin secretion, delaying gastric emptying, and suppressing prandial glucagon secretion. It is represented by the structure of Formula (I):

U.S. Patent No. 7572884 discloses a process for preparing Liraglutide by recombinant technology followed by acylation and removal of N-terminal extension.
U.S. Patent No. 7273921 and 6451974 discloses a process for acylation of Arg-34GLP-1 to obtain Liraglutide.
WO98/08871 discloses acylation of GLP-1 and analogues.
WO98/08872 discloses acylation of GLP-2 analogues.
WO99/43708 discloses acylation of exendin and analogues.
Yeast is a widely used organism for production of desired peptide or proteins by expression of foreign peptide or proteins in yeast cell which is disclosed in e.g. US4916212, US4870008 and US5618676, US6861237, US6183989, US6861237B2 etc. For commercial application high yields of the expressed peptide or protein is important.
Yeast organisms produce a number of peptide or proteins synthesized intracellularly, but having a function outside the cell. Such extracellular proteins are referred to as secreted peptide or proteins. These secreted proteins are expressed initially inside the cell in a precursor or a pre-form containing a pre-sequence ensuring effective direction of the expressed product across the membrane of the endoplasmic reticulum (ER). The pre-sequence, normally named a signal peptide, is generally cleaved off from the desired product during translocation. Once entered in the secretory pathway, the peptide or protein is transported to the Golgi apparatus and from the Golgi the peptide or protein can follow different routes that lead to compartments such as the cell vacuole or the cell membrane, or it can be routed out of the cell to be secreted to the external medium. (Pfeffer, S.R. and Rothman, J.E. Ann.Rev.Biochem. 56 (1987), 829-852.
A problem encountered with the use of signal peptides heterologous to yeast might be that the heterologous signal peptide does not ensure efficient translocation and/or cleavage of the precursor polypeptide after the signal peptide.
The objective of the present application to provide a process for the preparation of liraglutide by expression of synthetic oligonucleotide encoding lirapeptide which is operably connected to a oligonucleotide sequence of signal peptide in eukaryotic cell, preferably an yeast cell, an expression construct comprising synthetic oligonucleotide encoding lirapeptide which is operably connected to a oligonucleotide sequence of signal peptide, a vector carrying said expression construct including yeast cells transformed with the vectors.

Summary of Invention
In first embodiment, present invention provides a process for producing liraglutide comprising:
a) transforming a yeast organism with an expression construct comprising synthetic oligonucleotide encoding lirapeptide which is operably connected to a oligonucleotide sequence of signal peptide;
b) culturing the transformed yeast; and
c) recovering the lirapeptide from the culture;
d) converting lirapeptide to liraglutide.
wherein, said signal peptide can be selected from:
S. No Signal Peptide Name Amino acid sequence
1 a-mating factor pre-sequence MRFPSIFTAVLFAASSALA SVINYKR
2 a-mating factor
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR
3 a-amylase signal sequence MVAWWSLFLYGLQVAAPALA SVINYKR

4 Glucoamylase signal sequence MSFRSLLALSGLVCSGLA SVINYKR

5 Serum albumin signal sequence MKWVTFISLLFLFSSAYS RGVFRR

6 Inulinase presequence MKLAYSLLLPLAGVSA SVINYKR
7 Invertase signal Sequence (Yeast) MLLQAFLFLLAGFAAKISA SLDRK

8 Killer Protein signal sequence (Yeast) MTKPTQVLVRSVSILFFITLLHLVVA SLDRK
9 Lysozyme signal sequence MLGKNDPMCLVLVLLGLTALLGICQG SLDRK
10 Killer leader sequence (K.lactis) MNIFYIFLFLLSFVQGLEHTHRRG SLDRK

In second embodiment, the present invention provides an expression construct comprising of synthetic oligonucleotide encoding lirapeptide which is operably connected to an oligonucleotide sequence of signal peptide, wherein signal peptide is selected from:
S. No Signal Peptide Name Amino acid sequence
1 a-mating factor pre-sequence MRFPSIFTAVLFAASSALA SVINYKR
2 a-mating factor
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR
3 a-amylase signal sequence MVAWWSLFLYGLQVAAPALA SVINYKR
4 Glucoamylase signal sequence MSFRSLLALSGLVCSGLA SVINYKR

5 Serum albumin signal sequence MKWVTFISLLFLFSSAYS RGVFRR

6 Inulinase presequence MKLAYSLLLPLAGVSA SVINYKR
7 Invertase signal Sequence (Yeast) MLLQAFLFLLAGFAAKISA SLDRK

8 Killer Protein signal sequence (Yeast) MTKPTQVLVRSVSILFFITLLHLVVA SLDRK
9 Lysozyme signal sequence MLGKNDPMCLVLVLLGLTALLGICQG SLDRK
10 Killer leader sequence (K.lactis) MNIFYIFLFLLSFVQGLEHTHRRG SLDRK

In first aspect of the second embodiment, the present invention provide an expression vector fused with expression construct comprising of synthetic oligonucleotide encoding lirapeptide which is operably connected to an oligonucleotide sequence of signal peptide.
In third embodiment, the present invention provides a yeast strain has a non-functional protease gene such as YPS1, PEP 4 or both.
In one aspect of the third embodiment, the process for preparing yeast strain having non-functional protease gene such as YPS1, PEP 4 comprises removing/inactivation of said protease gene from the yeast strain before using it for the expression of the peptide or protein.
In second aspect of the third embodiment, yeast stain of Saccharomyces cerevisiae strain having non-functional protease gene such as YPS1, PEP4 or both.

DETAILED DESCRIPTION
The present invention relates to a process for the preparation of liraglutide by expression of synthetic oligonucleotide encoding lirapeptide which is operably connected to a oligonucleotide sequence of signal peptide in eukaryotic cell, preferably an yeast cell, an expression construct comprising synthetic oligonucleotide encoding lirapeptide which is operably connected to a oligonucleotide sequence of signal peptide, a vector carrying said expression construct including yeast cells transformed with the vectors.
According to the present invention, amino acid sequence of liraglutide herein represent by Formula (I):

According to the present invention, amino acid sequence of Lirapeptide herein represent by Formula (II):

Amino acids may be referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes.
In the first embodiment, present invention provides a process for producing liraglutide comprising:
a) transforming a yeast organism with an expression construct comprising synthetic oligonucleotide encoding lirapeptide which is operably connected to a oligonucleotide sequence of signal peptide;
b) culturing the transformed yeast; and
c) recovering the lirapeptide from the culture;
d) converting lirapeptide to liraglutide.
wherein, said signal peptide can be selected from:
S. No Signal Peptide Name Amino acid sequence
1 a-mating factor pre-sequence MRFPSIFTAVLFAASSALA SVINYKR
2 a-mating factor
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR
3 a-amylase signal sequence MVAWWSLFLYGLQVAAPALA SVINYKR

4 Glucoamylase signal sequence MSFRSLLALSGLVCSGLA SVINYKR

5 Serum albumin signal sequence MKWVTFISLLFLFSSAYS RGVFRR

6 Inulinase presequence MKLAYSLLLPLAGVSA SVINYKR
7 Invertase signal Sequence (Yeast) MLLQAFLFLLAGFAAKISA SLDRK

8 Killer Protein signal sequence (Yeast) MTKPTQVLVRSVSILFFITLLHLVVA SLDRK
9 Lysozyme signal sequence MLGKNDPMCLVLVLLGLTALLGICQG SLDRK
10 Killer leader sequence (K.lactis) MNIFYIFLFLLSFVQGLEHTHRRG SLDRK

In an aspect of first embodiment, expression construct can be prepared by fusion of synthetic oligonucleotide encoding desired peptide or protein at 5’ with oligonucleotide sequence of signal peptide. According to the present invention, expression construct is transformed into yeast cell by transformation method.
In another aspect of first embodiment, different expression construct can be prepared by fusion of synthetic oligonucleotide encoding desired peptide or protein at 5’ with oligonucleotide sequence of signal peptide, wherein signal peptide have amino acid sequence selected from one of above said signal peptide.
In one aspect of the first embodiment, process for producing a protein or peptide or its analogs comprising:
a) ligating expression construct comprising of synthetic oligonucleotide encoding desired peptide or protein which is operably connected to a oligonucleotide sequence of signal peptide in expression vector;
b) transforming said expression vector into yeast and inducing the expression to obtain protein or peptide.
According to the step a), the expression vector can be ligated with expression construct comprising of synthetic oligonucleotide encoding desired peptide or protein which is operably connected to an oligonucleotide sequence of signal peptide. The expression vector may already have desired restriction sites for ligation or can be introduced into expression vector by using restriction enzyme.
In another aspect of first embodiment, lirapeptide can be converted into liraglutide by any known method reported in art or disclosed in US6268343B1, US6844321, US6451974B1, US7273921B2, WO2016059609A1, WO2017021819A1 and the like.
In an aspect, expression vector that is used in the process of the present invention can be commercially available expression vectors or custom designed vectors selected from pYES2, YEp51, YEp351 or YEp352 but are not limited thereto.
The expression vector according to present invention comprises of promoters, selection marker, multiple cloning region, marker gene & origin of replication.
The suitable promoter can be selected from GAL1, TEF1, ADH1, GPD, TEF or derivatives thereof.
The suitable selection marker may be selected from kanamycin, ampicillin, chloramphenicol or tetracycline or their combinations in their wild or mutated forms.
The suitable origin of replication can be selected from pUC Ori, F1 Ori and 2µ (2-micron) and the like in their wild type or mutated form.
The suitable marker gene may be selected from HIS3, TRP1, LEU2, URA3, LYS2, Tn903 kanr, Comr, Hygr, CUP1 or DHFR though they are not limited thereto.
In another aspect, transformed yeast cell can be selected from Saccharomyces cerevisiae in its wild strain or in its mutated strain form.
The suitable Saccharomyces cerevisiae strain can be selected from ABGT40 (genotype: MATa ura3-52 leu2-3,112 ?trp1::hisG pep4-3 prb1-1122 reg1-501 gal1); ABGT50 (genotype: MATa ura3-52 leu2-3,112 ?trp1::hisG pep4-3 prb1-1122 reg1-501 gal1 GAL1-P-KEX2); ABGT60 (genotype: MATa ura3-52 leu2-3,112 ?trp1::hisG pep4-3 prb1-1122 reg1-501 gal1 GAL1-P-STE13), ?YPS1, ?PEP4 or ?YPS1?PEP4 and the like.
The transformation method can be selected from heat shock method, electroporation and the like.
In one variant, heat shock method involves heat shock to cells at about 42oC for about 30 sec to 15 minutes and subsequently keep on ice for about 2-10 minutes.
In second embodiment, the present invention provides an expression construct comprising of synthetic oligonucleotide encoding lirapeptide which is operably connected to an oligonucleotide sequence of signal peptide, wherein signal peptide is selected from:
S. No Signal Peptide Name Amino acid sequence
1 a-mating factor pre-sequence MRFPSIFTAVLFAASSALA SVINYKR
2 a-mating factor
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR
3 a-amylase signal sequence MVAWWSLFLYGLQVAAPALA SVINYKR

4 Glucoamylase signal sequence MSFRSLLALSGLVCSGLA SVINYKR

5 Serum albumin signal sequence MKWVTFISLLFLFSSAYS RGVFRR

6 Inulinase presequence MKLAYSLLLPLAGVSA SVINYKR
7 Invertase signal Sequence (Yeast) MLLQAFLFLLAGFAAKISA SLDRK

8 Killer Protein signal sequence (Yeast) MTKPTQVLVRSVSILFFITLLHLVVA SLDRK
9 Lysozyme signal sequence MLGKNDPMCLVLVLLGLTALLGICQG SLDRK
10 Killer leader sequence (K.lactis) MNIFYIFLFLLSFVQGLEHTHRRG SLDRK

In one aspect of the second embodiment, the expression construct sequence can be as follows:
Lira 00:
AACACA Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Ile Asn Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Glu Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

Lira 01:
CCCGCCGCCACC Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Ile Asn Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Glu Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

Lira 02:
CCCGCCGCCACC Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Glu Lys Arg His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

Lira 01A:
CCCGCCGCCACC Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu Phe Ile Asn Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Ala His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly

Lira 02A:
CCCGCCGCCACC Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln Ile Pro Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe Asp Val Ala Val Leu Pro Phe Ser Ala Ser Ile Ala Ala Lys Glu Glu Gly Val Ser Leu Glu Lys Arg Glu Ala Glu Ala Glu Ala His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly
wherein, AAC… or CCC… represents DNA sequence,
Arg Phe….. (in italics and prior to His which is underline and bold) represents suitable signal peptide and
His Ala…. (bold and underlined) represents the Lirapeptide amino acid sequence.
In first aspect of the second embodiment, the present invention provide an expression vector fused with expression construct expression construct comprising of synthetic oligonucleotide encoding lirapeptide which is operably connected to an oligonucleotide sequence of signal peptide.
In second aspect of the second embodiment, the present invention provides transformant yeast which is transformed by the expression vector having expression construct.
In third embodiment, the present invention provides a yeast strain has a non-functional protease gene such as YPS1, PEP 4 or both.
In one aspect of the third embodiment, the process for preparing yeast strain having non-functional protease gene such as YPS1, PEP 4 comprises removing/inactivation of said protease gene from the yeast strain before using it for the expression of the peptide or protein.
In second aspect of the third embodiment, yeast stain of Saccharomyces cerevisiae strain having non-functional protease gene such as YPS1, PEP4 or both.
In third aspect of the third embodiment, yeast stain of Saccharomyces cerevisiae strains such as ABGT40, ABGT 50, ABGT 60 having non-functional protease gene such as YPS1, PEP4 or both.
According to present invention, PEP4 in S. cerevisiae 2731 YPS1? MAT-A and MAT-a strains can be removed/inactivated/knocked-out by PCR method, which is well known in the art. The Yapsin1 (YPS1) and Proteinase A (PEP4) belongs to Vacuolar Aspartyl protease, known for aberrant proteolytic degradation of recombinant proteins and peptides in Yeast.
In the fourth embodiment, the present invention involves increase in accumulation of resulting protein or peptide by fermentation process comprises:
a) inducing transformant yeast, in fermentation culture medium;
b) culturing the transformant yeast under suitable condition;
c) recovering the expressed protein or peptide;
d) optionally, purifying the expressed protein or peptide;
In an aspect of fourth embodiment, present invention involves fermentation process for increase in accumulation of resulting protein or peptide. Peptide or protein expression of transformant yeast cells having expression construct comprising of synthetic oligonucleotide lirapeptide which is operably connected to an oligonucleotide sequence of signal peptide cloned in expression vector in culture medium is either through constitutive expression or with inducing agent such as galactose. Maintaining cells in glucose gives the most complete repression and the lowest basal transcription of the GAL1 promoter. Transferring cells from glucose- to galactose-containing medium causes the GAL1 promoter to become de-repressed and allows transcription to be induced. Alternatively, cells may be maintained in medium containing raffinose as a carbon source. The presence of raffinose does not repress or induce transcription from the GAL1 promoter. Addition of galactose to the medium induces transcription from the GAL1 promoter even in the presence of raffinose. Induction of the GAL1 promoter by galactose is more rapid in cells maintained in raffinose when compared to those maintained in glucose.
The fermentation may be carried out in fed-batch or batch-mode to produce protein or peptide. Improved expression of the proteins or peptide of the present invention depends on various parameters of the fermentation process. Some of the suitable parameters are fermentation media, concentration of the inducer, feed media and nutrient feed rate.
The fermentation medium is the medium required for the growth and expression of transformant prokaryotic cells at fermenter scale. Typically the fermentation medium comprises of suitable salts, vitamins, carbon source and nitrogen source. The suitable salts can be selected from ammonium chloride, potassium dihydrogen phosphate, disodium hydrogen phosphate, sodium chloride, calcium chloride, magnesium chloride, EDTA sodium salt, sodium molybdate, zinc sulphate, ferrous sulphate, copper sulphate, monopotassium phosphate, dipotassium phosphate, magnesium sulphate and the like or combination thereof. The carbon source may comprise glucose, glycerol, maltose, sucrose, dextrose, fructose or mannitol and the like or combination thereof. The nitrogen source may comprise ammonia, nitrate, peptone, soya peptone, yeast extract, tryptone and the like or combination thereof. The suitable vitamin can be selected from Thiamine (vitamin B or its related compounds) and the like or combination thereof. The fermentation medium further comprises acids selected from citric acid, boric acid and the like or combination thereof.
The feed medium comprises of salt, carbon source, nitrogen source antibiotics and trace elements. The suitable salt, carbon source and nitrogen source are the same as defined herein above. The feed medium may comprise of antibiotics selected from kanamycin, ampicillin, chloramphenicol, tetracycline and the like and will depend upon the antibiotic marker gene embedded in the vector.
In an aspect of present invention protein or peptide may be purified by the purification techniques selected from affinity chromatography, ion exchange chromatography (cation or anion), reverse phase chromatography or any other technique well known in the art.

DEFINITIONS
The following definitions can be used in connection with the words or phrases used in the present application unless the context indicates otherwise.
The term "amino acid" as used herein refers to an organic compound comprising at least one amino group and at least one acidic group. The amino acid may be a naturally occurring amino acid or be of synthetic origin, or an amino acid derivative or amino acid analog.
The term “amplification” as used herein refers to the production of additional copies of a nucleic acid sequence and is generally carried out using polymerase chain reaction (PCR) technologies well known in the art (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer, a Laboratory Manual, Cold 25 Spring Harbor Press, Plainview, N.Y.).
The term “Lirapeptide” is Arg34-GLP-1(7-37) which is liraglutide before acylation.
The term “fermentation”, as used herein, is intended to refer to processes involving the production of recombinant protein products.
The term “Pal” is palmitoyl.
The term "signal peptide" is understood to mean a pre-sequence which is present as an N-terminal sequence on the precursor form of an extracellular protein expressed in yeast. The function of the signal peptide is to allow the heterologous protein to be secreted to enter the endoplasmatic reticulum. The signal peptide is normally cleaved off in the course of this process.
The term S. cerevisiae or Saccharomyces cerevisiae are the same and used in present specification with same meaning.
The reactions of the processes described herein can be carried out in air or under an inert atmosphere. Typically, reactions containing reagents or products that are substantially reactive with air can be carried out using air sensitive synthetic techniques that are well known to the person skilled in art.
The different pLIR plasmids mentioned in the present application are the plasmids resulting from ligation of the corresponding LIR gene in to pYES2 vector.
Analysis
HPLC
An ACE 5 C18 250x4.6mm, 300Å (Part No: ACE-221-2546) was used as a stationary phase. A mobile phase consists of two components of mobile phase A which contains 0.1% Trifluoro acetic acid in Mill-Q water and Acetonitrile and the mobile phase B which contains 0.1% Trifluoro acetic acid in Mill-Q water and Acetonitrile in a gradient mode. The detection was measured at wavelength 220 nm. The injection volume was 10.0 µL. 6M Guanidine HCl is used as a diluent.

LC-MS
A Waters Xevo TQD equipped with AQUITY UPLC H-Class system and Mass Lynx 4.1 Software was used for the identification of peptide. A CORTECS C18+ 100x3 mm, 2.7µ (Part No: 186007402) was used as a stationary phase. Mobile phase A contains 0.1% of formic acid in Milli-Q water and mobile phase B contains 0.1% of formic acid in Acetonitrile. The detection was measured at wavelength 220 nm. The injection volume was 2 µL. The analysis was performed on electrospray ionization positive mode.
Although the exemplified procedures herein illustrate the practice of the present invention in some of its embodiments, the procedures should not be construed as limiting the scope of the invention. Modifications from consideration of the specification and examples within the ambit of current scientific knowledge will be apparent to one skilled in the art.
Example 1:
Oligonucleotide sequence of LIR-01, LIR-01A, LIR-02, LIR-02A were cloned into pYES2 vector and transformed into ABGT40 strain and for each construct at least 5 transformants were tested for expression in a shake flask. Further transformants were inoculated in 30 mL of SD (Synthetic defined) media without Uracil (–Ura), 3% Glucose in 250 mL flask and incubated for16h at 30°C. Then the culture broth was centrifuged at 4000 rpm, 5 min and collected biomass. The collected biomass was transferred into 30 mL of SD–Ura + 3% Glucose + 3% Galactose in 250 mL flask and incubated for 6h at 30°C. Lirapeptide expression was quantified by HPLC and results are tabulated herein below in table 1:
Table 1
Yeast Strain Plasmid Lirapeptide average expression (mg/L)
ABGT 40 pLIR-01

7.0
pLIR-01A
1.4
pLIR-02 1.6
pLIR-02A 1.1

Example 2:
Oligonucleotide sequence of LIR-00, LIR-01, LIR-01A, LIR-02, LIR-02A were cloned into pYES2 vector and transformed into ABGT40 strain and for each construct at least 5 transformants were tested for expression in a shake flask. Further transformants were inoculated in 30 mL of SD (Synthetic defined) media without Uracil (–Ura) 3% Glucose in 250 mL flask and incubated for16h at 30°C. Then the culture broth was centrifuged at 4000 rpm, 5 min and collected biomass. The collected biomass was transferred into 30 mL of YPD + 5% Glucose + 3% Galactose in 250 mL flask (pH regulation with MOPS/TrisHCl), incubated for 24h at 30°C. Lirapeptide expression was quantified by HPLC and results are tabulated herein below in table 2:
Table 2
Strain Plasmid pH (24h) Lira (mg/L)

ABGT40 pYES2 5.05 0.0 YPD 5% glucose, 3% galactose, pH 5.8 (non-adjusted)
pLIR-00 4.93 2.0
pLIR-01 4.93 2.5
pLIR-01A 4.96 0.0
pLIR-02 4.96 0.0
pLIR-02A 4.98 0.0

ABGT40 pYES2 6 0.0 YPD 5% glucose, 3% galactose, MOPS 20g/L, pH 7.0 (adjusted)
pLIR-00 5.96 18.3
pLIR-01 5.85 9.6
pLIR-01A 5.89 0.7
pLIR-02 5.92 8.9
pLIR-02A 5.98 2.2

ABGT40 pYES2 5.02 0.0 YPD 5% glucose, 3% galactose, Tris-Cl 100mM, pH 7.0 (adjusted)
pLIR-00 5.01 4.0
pLIR-01 4.99 2.4
pLIR-01A 5.0 0.0
pLIR-02 4.99 0.0
pLIR-02A 5.0 0.0
Wherein pYES2 means vector without having signal and lirapeptide which is studied as negative control.

Example 3:
Oligonucleotide sequence of LIR-00, LIR-01, LIR-01A, were cloned into pYES2 vector and transformed into ABGT40, ABGT50, ABGT60 strains and for each expression construct at least 5 transformants were tested for expression in a shake flask. Transformants were inoculated in 30 mL of YPD + 3% Glucose + 3% Galactose in 250 mL flask (pH 7.0 regulated with MOPS), incubated for 72h at 30°C. Lirapeptide expression was quantified by HPLC and results are tabulated herein below in table 3:
Table 3
Strain Plasmid Lira (mg/L) 72h

ABGT40 pYES2 0.0
pLIR-00 124.3
pLIR-01 135.7
pLIR-01A 24.7

ABGT60 pYES2 0.0
pLIR-00 13.0
pLIR-01 2.5
pLIR-01A 5.1

ABGT50 pYES2 0.0
pLIR-00 58.7
pLIR-01 63.5
pLIR-01A 15.8

Example 4:
Oligonucleotide sequence of LIR-00, LIR-01, were cloned into pYES2 vector and transformed into ABGT40, ABGT50, ABGT60 strains and for each expression construct at least 5 transformants were tested for expression in a shake flask. Further, transformants were inoculated in 30 mL of YPD + 3% Glucose + 3% Galactose in 250 mL flask (pH 7.0 regulated with MOPS), incubated for 48h at 20oC, 25oC and 30°C. Lirapeptide expression was quantified by HPLC and results are tabulated herein below in table 4:
Table 4
Strain Plasmid Lira (mg/L) 20oC for 48h Lira (mg/L) 25oC for 48h Lira (mg/L) 30oC for 48h
ABGT40 pYES2 0.0 0.0 0.0
pLIR-00 36.3 101.1 134.1
pLIR-01 26.1 83.9 135.2
ABGT60 pYES2 0.0 0.0 0.0
pLIR-00 13.2 42.0 30.1
pLIR-01 10.7 26.8 24.3
ABGT50 pYES2 0.0 0.0 0.0
pLIR-00 37.2 98.3 92.6
pLIR-01 29.5 76.1 84.5

Example 5:
Oligonucleotide sequence of LIR-00, LIR-01, were cloned into pYES2 vector and transformed into ABGT40 strain and for each expression construct at least 5 transformants were tested for expression in a shake flask. Transformants were inoculated in 30 mL of YPD + 3% Glucose + 3% Galactose in 250 mL shake flask (pH 7.0 regulated with MOPS), incubated at 30°C and Lirapeptide yields were measured at 4h, 24h, 32h, 48h, 56h, 72h and 76h by HPLC and tabulated in Table 5:
Table 5
ABGT40/plasmid Fermentation time (h) Lira (mg/L)

pLIR00 4 2.1
24 32.5
32 49.5
48 91.1
56 114.3
72 117.6
76 112.4

pLIR01 4 2.9
24 19.3
32 47.7
48 83.0
56 101.7
72 104.4
76 98.4

Example 6: Transformation of recombinant plasmid, pYES2-MF(a)1-Lira00 into haploid S. cerevisiae 2731 YPS1? PEP4? MAT-a & MAT-A strains and diploid S. cerevisiae 2731 YPS1? PEP4? strain using a Frozen-EZ Yeast Transformation II Kit (Zymo Research).
The recombinant plasmid, pYES2-MF(a)1-Lira00 (wherein MF is Multiple a Mating factor signal sequence) was transformed into chemically competent cells of haploid S. cerevisiae 2731 YPS1? PEP4? MAT-a & MAT-A strains and diploid S. cerevisiae 2731 YPS1? PEP4? strain prepared using Frozen-EZ Yeast Transformation II Kit (Zymo Research).
The transformed cells were selected on SC-U (Synthetic media drop out Uracil) + 2% Glucose selective plates at 30oC for 2-4 days. The transformants will exhibit Uracil prototrophy.
Example 7: Expression of Lirapeptide precursor in S. cerevisiae 2731 YPS1? PEP4? MAT-a strain-pYES2-ML-Lira in Fermenter (10L)
A. Pre-seed culture
1 ml of S. cerevisiae 2731 YPS1? PEP4? MAT-a strain-pYES2-ML-Lira glycerol stock was inoculated into 30ml SC-U (Synthetic media drop out Uracil) + 2% Glucose in a 250ml conical flask and grown at 30oC.
B. Seed culture
30ml pre-seed culture was transferred to 270ml SC-U (Synthetic media drop out Uracil) + 2% Glucose in a 1L conical flask and grown at 30oC.
C. Fermentation
300ml seed culture was used to seed a fermenter containing 2.7L semi synthetic tank media in a 10L fermenter, and is grown at pH 5.0, temperature 30°C and dissolved oxygen (DO) control 30%. Upon glucose exhaustion, an organic feed (20% Yeast extract + 20% Peptone + 50% glucose) was added to the fermenter. The culture is induced with Galactose (20% Yeast extract + 20% Peptone + 40% galactose) after the culture OD600 reached to 40-60 and the target Liraglutide peptide precursor is produced extracellular.
D. Harvest
The pH of the fermentation broth was adjusted to 9.0 with 4M Tris Base and incubated for 1hr at room temperature. The cells were pelleted down and supernatant was collected and analyzed by HPLC and LC-MS to get lirapeptide of 100mg/liter.
,CLAIMS:CLAIMS
1. A process for producing a liraglutide comprising:
a) transforming a yeast organism with an expression construct comprising synthetic oligonucleotide encoding lirapeptide which is operably connected to a oligonucleotide sequence of signal peptide;
b) culturing the transformed yeast; and
c) recovering the lirapeptide from the culture;
d) converting lirapeptide to liraglutide.
wherein, said signal peptide can be selected from:
S. No Signal Peptide Name Amino acid sequence
1 a-mating factor pre-sequence MRFPSIFTAVLFAASSALA SVINYKR
2 a-mating factor
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR
3 a-amylase signal sequence MVAWWSLFLYGLQVAAPALA SVINYKR

4 Glucoamylase signal sequence MSFRSLLALSGLVCSGLA SVINYKR

5 Serum albumin signal sequence MKWVTFISLLFLFSSAYS RGVFRR

6 Inulinase presequence MKLAYSLLLPLAGVSA SVINYKR
7 Invertase signal Sequence (Yeast) MLLQAFLFLLAGFAAKISA SLDRK

8 Killer Protein signal sequence (Yeast) MTKPTQVLVRSVSILFFITLLHLVVA SLDRK
9 Lysozyme signal sequence MLGKNDPMCLVLVLLGLTALLGICQG SLDRK
10 Killer leader sequence (K.lactis) MNIFYIFLFLLSFVQGLEHTHRRG SLDRK

2. The process according to claim 1, further comprises:
a) ligating expression construct comprising of synthetic oligonucleotide encoding lirapeptide which is operably connected to a oligonucleotide sequence of signal peptide in expression vector;
b) transforming said expression vector into yeast and inducing the expression to obtain lirapeptide.
3. A yeast expression construct comprising synthetic oligonucleotide encoding lirapeptide which is operably connected to a oligonucleotide sequence of signal peptide, wherein signal peptide is selected from:
S. No Signal Peptide Name Amino acid sequence
1 a-mating factor pre-sequence MRFPSIFTAVLFAASSALA SVINYKR
2 a-mating factor
MRFPSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR
3 a-amylase signal sequence MVAWWSLFLYGLQVAAPALA SVINYKR

4 Glucoamylase signal sequence MSFRSLLALSGLVCSGLA SVINYKR

5 Serum albumin signal sequence MKWVTFISLLFLFSSAYS RGVFRR

6 Inulinase presequence MKLAYSLLLPLAGVSA SVINYKR
7 Invertase signal Sequence (Yeast) MLLQAFLFLLAGFAAKISA SLDRK

8 Killer Protein signal sequence (Yeast) MTKPTQVLVRSVSILFFITLLHLVVA SLDRK
9 Lysozyme signal sequence MLGKNDPMCLVLVLLGLTALLGICQG SLDRK
10 Killer leader sequence (K.lactis) MNIFYIFLFLLSFVQGLEHTHRRG SLDRK

4. The transformant yeast which is transformed by the expression construct of claim 5.
5. The transformant yeast according to claim 1 & 6 is selected from Saccharomyces cerevisiae in its wild strain or in its mutated strain form, wherein Saccharomyces cerevisiae strain can be selected from ABGT40, ABGT50 or ABGT6.
6. The process according to claim 1, wherein when the yeast strain has a non-functional protease gene selected from YPS1, PEP 4 or both.
7. The process for preparing a yeast strain having non-functional protease gene according to claim 7, comprises removing/inactivation of said protease gene from the yeast strain before using it for the expression of the peptide or protein.
8. The process according to claim 2, wherein the expression vector is selected from commercially available expression vectors or custom designed vectors comprising one or more promoters selected from GAL1 GAL1, TEF1 or derivatives thereof and an antibiotic marker selected from Kanamycin, Ampicillin, Chloramphenicol and Tetracycline.