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WO2025163592A1 - Production de peptides et de protéines à haut niveau - Google Patents

Production de peptides et de protéines à haut niveau

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Publication number
WO2025163592A1
WO2025163592A1 PCT/IB2025/051092 IB2025051092W WO2025163592A1 WO 2025163592 A1 WO2025163592 A1 WO 2025163592A1 IB 2025051092 W IB2025051092 W IB 2025051092W WO 2025163592 A1 WO2025163592 A1 WO 2025163592A1
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WO
WIPO (PCT)
Prior art keywords
seq
multimer
tag
sequence
amino acid
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
PCT/IB2025/051092
Other languages
English (en)
Inventor
Rustom Mody
Sandip Bose
Vaibhav KARNWAL
Shivika Gupta
Ajoy Roy
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sun Pharmaceutical Industries Ltd
Original Assignee
Sun Pharmaceutical Industries Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sun Pharmaceutical Industries Ltd filed Critical Sun Pharmaceutical Industries Ltd
Publication of WO2025163592A1 publication Critical patent/WO2025163592A1/fr
Pending legal-status Critical Current
Anticipated expiration legal-status Critical

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/62—DNA sequences coding for fusion proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/50—Fusion polypeptide containing protease site
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli

Definitions

  • This invention relates to multimers containing a plurality of target peptide sequences and optionally Tag amino acid sequences to modulate pl and hydrophobicity which permit high levels of expression and desired solubility of the multimer expressed in E. coli for ease of downstream processing and purification.
  • Peptides are involved in various biological processes and thousands of different natural peptides have been identified (Barman et al., Int J Peptide Res. and Ther., 29:61, 2023), (BarmanP. etal.,IntJPeptRes Ther. 2023;29(4):61. doi: 10.1007/sl0989-023-10524-3). Peptides are involved in important functions such as hormonal functions, neurotransmission and antimicrobial activity (Barman, supra). Due to this, peptides have gained immense commercial attention in the field of medicine with more than one hundred peptides having been approved for diagnostic and therapeutic use (Barman, supra). Recombinant bioactive peptides have been largely used as biotherapeutic agents.
  • Bioactive peptides can serve as antioxidants, anti-aging agents and anti-inflammatory agents with the potential to enhance skin health and beauty (Le Thi Nhu Ngoc, Feature Papers in Cosmetics, 2023), (Le Thi Nhu Ngoc et. al, Cosmetics 2023,70(4). doi: 10.3390/10040111) Nutraceutical peptides can provide a daily dose of healthy nutrients offering preventive and treatment benefits (A Giriraj, Science direct, 2018;157-181. doi:10.1016/'B978-0-08-100736-5.00006-5).
  • Recombinant peptides are often expressed in a suitable host such as E.coli or yeast.
  • a suitable host such as E.coli or yeast.
  • challenges faced in the production of peptides One of the major problems of expressing peptides is that due to their small size they are highly prone to cleavage via the proteases of host organisms (Ajingi YS, Current Pharmaceutical Biotechnology, 23:645-663, 2022). This cleavage leads to lower yields and truncated versions of the peptide of interest (recombinant peptide of interest).
  • fusion partners have been attached to peptides to increase their expression level and protect them from protease cleavage.
  • TRX thioredoxine
  • SUMO small ubiquitin-like modifier
  • GST glutathione-S-transferase
  • the recombinant peptide of interest is obtained by cleaving off the fusion partner. These fusion partners, however, decrease the efficiency of peptide production by reducing the overall amount of the recombinant peptide of interest produced.
  • the major challenge with expression of a recombinant peptide of interest as a multimer is that multimers often form highly insoluble compact inclusion bodies. Solubilization of these inclusion bodies is extremely challenging as it involves the use of strong chaotropic agent such as guanidium hydrochloride (GuCl). GuCl is an expensive raw material, and its usage increases the cost of production, impacts enzymatic digestion and scalability of the process.
  • U.S. Patent Publication No. 2022/0195004 discloses certain fusion proteins comprising a plurality of target protein sequences, which are connected in series, wherein every two adjacent target protein sequences are connected by means of a linker sequence.
  • Chinese Patent Publication No. 101172996 discloses a connection peptide and poly-fusion expression method.
  • the present invention provides a method for enhanced expression of a multimer (MU) containing a plurality of Target Peptide (TP) sequences. Furthermore, the solubility of the multimer (MU) can be adjusted by using amino acid Tag (Tags) to either express the multimer (MU) as insoluble inclusion bodies or in the soluble form, to allows for convenient downstream processing, conversion of multimer (MU) to target peptide (TP) followed by the purification of the target peptide (TP), to achieve a final purity level of at least 95%.
  • the present invention is directed to the efficient production of a target peptide (TP) using a multimer (MU) comprising a plurality of target peptide sequences connected in series, wherein
  • Every two adjacent target peptide (TP) sequences are connected by a separator sequence, which is referred as a Cleavage Site (CS) is capable of being cleaved by proteolytic enzymes (e.g., Kex2, EK, Carboxypeptidase B) or chemicals to form the target peptide upon complete digestion,
  • proteolytic enzymes e.g., Kex2, EK, Carboxypeptidase B
  • Tag optionally additional separator sequence referred to as Tag, may also be present between cleavage sites (CS), to modify the hydrophobicity and/or pl of the multimer (MU),
  • the N- and/or C-terminus of the multimer comprises of amino acid sequence which is capable of being cleaved by one or more of the proteolytic enzymes (Kex2, EK, Carboxypeptidase B) or chemicals to give target peptide (TP).
  • Kex2, EK, Carboxypeptidase B the proteolytic enzymes
  • TP target peptide
  • each separator sequence independently comprises a cleavage site (also referred to as CS) and optionally a Tag amino acid sequence that alters the hydrophobicity, pl, or both of the multimer (MU).
  • CS cleavage site
  • MU Tag amino acid sequence
  • the affinity tag is added either at the N- or C-terminus of the multimer (MU) separated by the cleavage site (CS).
  • the amino acid at the N-terminus of the multimer is methionine.
  • one or more of the cleavage sites comprise the amino acid sequence KR.
  • the R of the KR amino acid sequence is directly bound to the N-terminus of the target peptide (TP) sequences, and the K of the KR amino acid sequence is directly bound to the C-terminus of the target peptide sequences.
  • the K of the DDDDK sequence (SEQ ID NO: 53) is directly bound to the N-terminus of one of the target peptide (TP) sequences.
  • the multimer includes one or more Tag amino acid sequences to alter the hydrophobicity, pl, or both of the multimer (MU).
  • each Tag amino acid sequence comprises one or more amino acids selected from aspartic acid (D), glutamic acid (E), serine (S), threonine (T), arginine (R), glutamine (Q), lysine (K), histidine (H), leucine (L) and isoleucine (I), alanine (A), valine (V), proline (P), glycine (G), tryptophan (W), phenylalanine (F), tyrosine (Y), cysteine (C), and methionine (M).
  • the multimer has a pl of from about 3 to about 12.
  • the multimer has a hydrophobicity of from 20% to 50%.
  • the multimer (MU) has a hydrophobicity of 25% to about 40% and/or pl from 4 to 10.
  • each Tag amino acid sequence between two target peptide (TP) sequences in the multimer is the same.
  • each Tag amino acid sequence in the target peptide (TP) is the different.
  • the multimer (MU) comprises at least two different Tag amino acid sequences.
  • the multimer (MU) includes 2 to 20 copies of a target peptide (TP) sequence.
  • the multimer (MU) includes 10 copies of a target peptide (TP) sequence.
  • the multimer (MU) has a molecular weight of from about 5 to about 200 kilodaltons. [36] In some embodiments, the multimer (MU) has a molecular weight of from about 30 to about 60 kilodaltons.
  • one or more of the Tag amino acid sequences comprise at least one solubility enhancing domain.
  • the solubility enhancing domain is a naturally occurring solubility enhancing domain such as thyrodoxin, SUMO, Fh8, GB1, CaBP, MsyB, or Skp, with or without any modifications.
  • the solubility enhancing domain is selected from
  • NPGIDAEDANLQDGDADEGEE (SEQ ID NO: 19), IDDQVEDKCLPQ (SEQ ID NO: 20), IDDQVEDKADEGE (SEQ ID NO: 21), LQDGDADEGEEGELSGDGDYD (SEQ ID NO: 23), LEPPLDTD (SEQ ID NO: 25), EGECLPDL(SEQ ID NO: 22), EGELSGDGDYD (SEQ ID NO: 24), LQDGDAD (SEQ ID NO: 26), QDGDADEGEE (SEQ ID NO: 27), NPGIDAEDANL (SEQ ID NO: 28), NPGIDAEDANLQDGDAD (SEQ ID NO: 29),
  • SDKIIHLTDDSFDTDVLKADGAILVDF (SEQ ID NO: 32), MSDKIIHLTDDSFDTDVLKAD (SEQ ID NO: 33), IHLTDDSFDTDVLKADGAILV (SEQ ID NO: 34, II ⁇ AFII ⁇ EHDI ⁇ NI ⁇ DGI ⁇ LDLI ⁇ EL (SEQ ID NO: 35), KEHDKNKDGKLDLKELVSILS (SEQ ID NO: 36), IKAFIKEHDKNKDGKLDLKELVSILSS (SEQ ID NO: 37), and any combination of any of the foregoing.
  • the present invention is directed towards a DNA construct comprising a nucleic acid sequence encoding a multimer.
  • a host cell is transfected with the DNA construct.
  • the present invention uses an expression vector, wherein the DNA sequence coding for the multimer (MU) is introduced under the T7 promoter.
  • a host cell comprises is transformed with the expression vector.
  • the method further comprises digesting the multimer (MU) with one or more proteolytic enzymes or chemicals to obtain a plurality of the target peptide (TP).
  • the present invention is directed a method of preparing a peptide comprising:
  • step (a) of the method comprises expressing the multimer in E. coli, and optionally isolating the multimer.
  • the N-terminus includes methionine and a histidine tag
  • the multimer is isolated using Nickel-NTA resin via the polyhistidine affinity tag.
  • the E. coli is BL21, BL21(DE3), ToplO, or DH5a cells.
  • the multimer (MU) is digested using one or more of the proteases selected from Kex2, carboxypeptidase B, and enterokinase.
  • the cleavage is carried out sequentially or simultaneously using Kex2 and/or enterokinase followed by carboxypeptidase.
  • the cleavage is carried out sequentially or simultaneously using Kex2 followed by carboxypeptidase.
  • the final peptide after complete digestion is target peptide (TP) which is further purified using one or more chromatography or precipitation techniques.
  • the isolating step comprises one or more chromatography steps.
  • the method further comprises conjugating one or more chemical moieties to the target peptide sequence.
  • [55] In some embodiments directed towards a method, wherein the method further comprising conjugating a second peptide to the target peptide sequence to form a third peptide and, optionally, conjugating one or more moieties to the third peptide.
  • a protein of interest is generated through multimers (MU) comprising a plurality of the target peptide sequence.
  • Tag amino acid sequences are interspaced between the target peptide (TP) sequences for convenient downstream processing which includes proteolytic or chemical cleavage to result in a recombinant protein of interest wherein purity of the target protein is at least 95 % after purification.
  • the target peptide (TP) sequences are interspaced with Tag amino amino acid tags having cleavage sites for proteolytic enzymes and/or chemicals.
  • the Tag amino acid sequences alter the intrinsic characteristics of the multimer such as pl and/or hydrophobicity.
  • the building blocks of peptides, i.e., amino acids fall under certain categories such as acidic, basic, hydrophilic, and hydrophobic.
  • Tag amino acid sequences which can modulate overall pl and/or hydrophobicity of the multimer comprises a target peptide (TP) sequence enabling high expression, improved solubility and ease of downstream processing.
  • TP target peptide
  • the Tag amino acid sequences enhance expression levels and improve solubility of the multimer (MU).
  • the Tag amino acid sequences can be designed using a combination of amino acids which can alter the intrinsic properties of the multimer such as pl and/or hydrophobicity.
  • the Tag amino acid sequence comprises aspartic acid (D), glutamic acid (E), histidine (H), lysine (K), or arginine (R) which can alter the pl of the multimer.
  • the Tag amino acid sequences comprise of aspartic acid and/or glutamic acid to reduce the pl of the multimer (MU).
  • the Tag amino acid sequences comprise lysine and/or arginine to can increase the pl of the multimer (MU).
  • the Tag amino acid sequences comprise serine (S).
  • the Tag amino acid sequences comprise leucine (L), isoleucine (I), alanine (A), valine (V), proline (P), glycine (G), tryptophan (W), phenylalanine (F) and other hydrophobic amino acids to increase the hydrophobicity of the multimer (MU).
  • the multimers comprise a target peptide sequence to achieve high level expression of the peptide of interest.
  • the C-terminus of the multimer (after the last target peptide sequence in the multimer) includes a Tag amino acid sequences which is capable of being cleaved from the target peptide (TP) sequence by a protease and/or chemical cleavage site (CS).
  • TP target peptide
  • CS chemical cleavage site
  • the amino acid at the N-terminus of the multimer is methionine and optionally a histidine tag.
  • the amino acid sequence at the N- terminus of the multimer is M(H) P , where p is 1 to 8 (such as MHHHHHH).
  • one or more of the Tag amino acid sequences comprises one or more amino acids to the pl of the multimer, the hydrophobicity of the multimer, or both.
  • one or more Tag amino acid sequences comprise one or more amino acids selected from aspartic acid (D), glutamic acid (E), and any combination of any of the foregoing to reduce the pl of the multimer.
  • one or more Tag amino acid sequences comprise one or more amino acids selected from Histidine (H), lysine (K), arginine (R), and any combination of any of the foregoing to increase the pl of the multimer.
  • one or more Tag amino acid sequences comprise one or more amino acids selected from serine (S), threonine (T), asparagine (N), glutamine (Q) and any other hydrophilic amino acids and any combination of any of the foregoing to increase the hydrophilicity of the multimer.
  • one or more Tag amino acid sequences comprise one or more amino acids selected from leucine (E), isoleucine (I) alanine (A), valine (V), proline (P), Glycine (G), Tryptophan (W), Phenylalanine (F) and any other hydrophobic amino acids and any combination of any of the foregoing to increase the hydrophobicity of the multimer.
  • one or more of the Tag amino acid sequences comprise an amino acid group with a proteolytic and/or chemical cleavage site at both the N and C terminus of the recombinant peptide of interest for cleaving the multimer to generate the recombinant peptide of interest.
  • each of the Tag amino acid sequences comprise an amino acid group with a proteolytic and/or chemical cleavage site at any site of attachment to the N or C-terminus of the target peptide sequence ; this is to permit cleaving of the multimer to generate the target peptide sequence.
  • one or more of the separator sequences includes only proteolytic and/or chemical cleavage sites.
  • one or more of the cleavage sites comprise the amino acid sequence KR. In some instances, each cleavage site sequence is KR.
  • one or more of the Tag amino acid sequences comprise DSSTTDSSTTDDDDDK (SEQ ID NO: 6), DSSDTTDDDDDK (SEQ ID NO: 5), SSTTDDDDK (SEQ ID NO: 8), DSSTTDSSTTSSTT (SEQ ID NO: 7), DSSTTDSSTTDDDDK (SEQ ID NO: 4), SSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 49), SSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 50), SSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 51), SSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 52), SDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 11) , SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 12) , SDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 12)
  • one or more of the Tag amino acid sequences further comprise one or more additional amino acids.
  • each of the Tag amino acid sequences are selected from DSSTTDSSTTDDDDDK (SEQ ID NO: 6), DSSDTTDDDDDK(SEQ ID NO: 5) exactly SSTTDDDDK(SEQ ID NO: 8), DSSTTDSSTTSSTT(SEQ ID NO: 7), DSSTTDSSTTDDDDK(SEQ ID NO: 4), SSTTRSSTTSSTTRSSTTSSTTRDDDDK(SEQ ID NO: 49), SSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK(SEQ ID NO: 50), SSTTSSTTRSSTTSSTTRSSTTRDDDDK(SEQ ID NO: 51), and SSTTSSTTSSTTSSTTSSTTDDDDK(SEQ ID NO: 52), SDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 11) , SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR(SEQ ID NO: 11)
  • one or more of Tag amino acid sequences comprise KRDSSTTDSSTTDDDDDK (SEQ ID NO: 54), KRDSSDTTDDDDDK (SEQ ID NO: 55), KRSSTTDDDDK (SEQ ID NO: 56), KRDSSTTDSSTTSSTT (SEQ ID NO: 57), KRDSSTTDSSTTDDDDK (SEQ ID NO: 58), KRSDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 59), KRSDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 60), KRSDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 61), KRSDSTTSSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 62), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 63), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 64), or any combination of any of the foregoing
  • each of the Tag amino acid sequences are selected from KRDSSTTDSSTTDDDDDK (SEQ ID NO: 54), KRDSSDTTDDDDDK (SEQ ID NO: 55), KRSSTTDDDDK (SEQ ID NO: 56), KRDSSTTDSSTTSSTT (SEQ ID NO: 57), KRDSSTTDSSTTDDDDK (SEQ ID NO: 58), KRSDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 59), KRSDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 60), KRSDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 61), KRSDSTTSSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 62), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 63), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 64).
  • one or more of the aforementioned Tag amino acids further comprise one or more additional amino acids.
  • one or more of Tag amino acid sequences comprise KRSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 65), KRSSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 66), KRSSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 67), KRSSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 68), KRSSTTSSTTRSSTTSSTTSSTTR (SEQ ID NO: 69), KRSDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 70), KRSDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 71), KRSDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 72), KRSDSTTSSTTSSTTSSTTSSTT (SEQ ID NO:
  • one or more of Tag amino acid sequences are selected from KRSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 65), KRSSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 66), KRSSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 67), KRSSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 68), KRSSTTSSTTRSSTTSSTTSSTTR (SEQ ID NO: 69), KRSDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 70), KRSDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 71), KRSDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 72), KRSDSTTSSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 73), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 74
  • one or more of the Tag amino acids comprise one or more additional amino acids in combination with any of the foregoing Tag amino acid sequences (such as to change the pl and/or hydrophobicity of the multimer).
  • the Tag amino acid sequences can be any combination of naturally occurring amino acids (Tag) along with or without a CS targeted to alter intrinsic characteristics of the multimer, such as pl and/or hydrophobicity.
  • the Tag amino acid sequence can lower the pl of the multimer.
  • the Tag amino acid sequence can lower the hydrophobicity of the multimer.
  • the Tag amino acid sequence can include one or more of SEQ ID NOs. 4-8, 11-16, and 19-29.
  • one or more of the Tag amino acid sequence include one or more naturally occurring solubility enhancing partner tags.
  • the naturally occurring solubility enhancing partner tags have one or more domains which are responsible for enhancing the solubility of the multimer. These domains can also be used to modulate the pl of the multimer.
  • the naturally occurring solubility enhancing partner tag has one or more domains from Thyrodoxin, SUMO, Fh8, GB1, CaBP, MsyB, and Skp.
  • the naturally occurring solubility enhancing domain has one or more domains selected from NPGIDAEDANLQDGDADEGEE (SEQ ID NOL: 19), IDDQVEDKCLPQ (SEQ ID NO: 20), IDDQVEDKADEGE (SEQ ID NO: 21), EGECLPDL (SEQ ID NO: 22), LQDGDADEGEEGELSGDGDYD (SEQ ID NO: 23), EGELSGDGDYD (SEQ ID NO: 24), LEPPLDTD (SEQ ID NO: 25), LQDGDAD (SEQ ID NO: 26), QDGDADEGEE (SEQ ID NO: 27), NPGIDAEDANL (SEQ ID NO: 28), NPGIDAEDANLQDGDAD (SEQ ID NO: 29) (a domain from MsyB), SDKIIHLTDDSFDTDVLKADGAILVDF (SEQ ID NO: 32), MSDKIIHLTDDSFDTDVLKAD (SEQ ID NO: 33), IHLTDDSFDTDV
  • one or more of Tag amino acid sequences comprise NPGIDAEDANLQDGDADEGEEKR (SEQ ID NO: 38), KRIDDQVEDKCLPQ(SEQ ID NO: 39), KRIDDQVEDKADEGE (SEQ ID NO: 40), KREGECLPDL(SEQ ID NO: 41), KRLQDGDADEGEEGELSGDGDYD (SEQ ID NO: 42), KREGELSGDGDYD (SEQ ID NO: 43), KRLEPPLDTD (SEQ ID NO: 44), KRLQDGDAD (SEQ ID NO: 45), KRQDGDADEGEE (SEQ ID NO: 46), KRNPGIDAEDANL(SEQ ID NO: 47), KRNPGIDAEDANLQDGDAD (SEQ ID NO: 48) or any combination of any of the foregoing.
  • one or more of Tag amino acid sequences further comprise one or more additional amino acids.
  • one or more of Tag amino acid sequences are selected from NPGIDAEDANLQDGDADEGEEKR (SEQ ID NO: 38), KRIDDQVEDKCLPQ (SEQ ID NO: 39), KRIDDQVEDKADEGE (SEQ ID NO: 40), KREGECLPDL (SEQ ID NO: 41), KRLQDGDADEGEEGELSGDGDYD (SEQ ID NO: 42), KREGELSGDGDYD (SEQ ID NO: 43), KRLEPPLDTD (SEQ ID NO: 44), KRLQDGDAD (SEQ ID NO: 45), KRQDGDADEGEE(SEQ ID NO: 46), KRNPGIDAEDANL(SEQ ID NO: 47), KRNPGIDAEDANLQDGDAD (SEQ ID NO: 48) or any combination of any of the foregoing.
  • one or more of Tag amino acid sequences further comprise one or more additional amino acids.
  • every Tag amino acid sequence in the multimer is the same.
  • the multimer comprises two or more different Tag amino acid sequence.
  • the N-terminus of the multimer includes a methionine and histidine tag.
  • the N-terminus of the multimer include but not limited to methioninepoly histidine (such as -MHHHHHH (SEQ ID NO: 76)).
  • the C-terminus of the multimer includes a histidine tag followed by a stop codon. In some instances, the C-terminus of the multimer include a poly histidine.
  • the poly histidine tag can be within the multimer, i.e. not at the N or C terminus.
  • the multimer comprises greater than 2 copies of the target peptide sequence. In some embodiments, the multimer has 5 to 40 copies of the target peptide sequence. In some embodiments, the multimer has 10 copies of the target peptide sequence. In some embodiments, the multimer has 20 copies of the target peptide sequence.
  • one or more occurrences of MO comprise one or more naturally occurring solubility enhancing partner tags with or without CS as described herein.
  • the affinity tag or variable A comprises a tag to facilitate purification of the multimer .
  • the variable A affinity tag comprise a histidine tag (e.g., poly histidine).
  • each occurrence of CS is independently KR or any other proteolytic/chemical cleavage site. In some embodiments, each occurrence of CS is KR .
  • each occurrence of CS is independently DDDDK (SEQ ID NO: 53) or any other proteolytic/chemical cleavage site. In some embodiments, each occurrence of CS is DDDDK (SEQ ID NO: 53).
  • the multimer has a pl from about 4 to about 12.
  • the multimer has a hydrophobicity of from about 20 to about 50%.
  • the multimer has a hydrophobicity range of from about 24 to about 42%.
  • the multimer has a molecular weight more than 2 kilodalton.
  • the target peptide (TP) sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 1).
  • the target peptide sequence can be but not limited to 10- 29 amino acids.
  • the target peptide sequence is but not limited to EGTFTSDVSSYLEGQ AAKEFIAWLVRGRG , GTFTSDVSSYLEGQAAKEFIAWLVRGRG, FTSD VS SYLEGQ AAKEFIAWLVRGRG, TSD VS S YLEGQ AAKEFIAWLVRGRG, SDVSSYLEGQ AAKEFIAWLVRGRG, DVSSYLEGQ AAKEFIAWLVRGRG, DVSSYLEGQ AAKEFIAWLVRGRG, DVSSYLEGQ AAKEFIAWLVRGRG, DVSSYLEGQ AAKEFIAWLVRGRG, DVSSYLEGQ AAKEFIAWLVRGRG,
  • LEGQ AAKEFIAWLVRGRG LEGQ AAKEFIAWLVRGRG, EGQ AAKEFIAWLVRGRG, GQ AAKEFIAWLVRGRG, Q AAKEFIAWLVRGRG, AAKEFIAWLVRGRG, AKEFIAWLVRGRG, KEFIAWLVRGRG, EFIAWLVRGRG, and FIAWLVRGRG
  • the target peptide sequence is HAEGTFTSD VS SYLEGQ AAKEFIAWLVRGRG In some instances, the target peptide sequence is SEQ ID NO. 1.
  • the sequence of the multimer of a target peptide sequence is SEQ ID NO. 2. In some instances, the sequence of the multimer of a target peptide sequence is SEQ ID NO. 9. In some instances, the sequence of the multimer of a target peptide sequence is SEQ ID NO. 17. In some instances, the sequence of the multimer of a target peptide sequence is SEQ ID NO. 30.
  • the multimer nucleotide sequence of a target peptide sequence is SEQ ID NO. 3. In some instances, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO. 10. In some instances, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO. 18. In some instances, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO. 31.
  • DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.
  • host cell transfected with a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.
  • Yet another aspect is an expression vector comprising a DNA sequence encoding the multimer as described herein.
  • Yet another aspect is a host cell comprising the expression vector described herein.
  • the expression vector has any promoter.
  • the promoter is T7.
  • Yet another aspect is a method of preparing a multimer comprising expressing a DNA construct comprising a nucleic acid sequence encoding the multimer described herein in a host cell.
  • the method further comprises digesting the multimer with one or more enzymes to obtain a plurality of the target peptide sequences in free form.
  • yet another aspect is a method of preparing a peptide comprising:
  • Step (b) Digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form.
  • Step (a) comprise expressing the multimer in the host and isolating the multimer.
  • the host Prior to performing step (a), the host be transfected with an expression vector for expressing the multimer (such as that described herein).
  • the N-terminus of the multimer includes a methionine and; with or without not limited to affinity tag (prefrably poly-histidine) tag.
  • affinity tag prefrably poly-histidine
  • step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide.
  • the method further comprises step (c) isolating the peptide.
  • multimer can be digested with single protease.
  • the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and/or chemicals in a single step.
  • the target peptide sequence comprises of one or more of the target peptide sequences.
  • a target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG is obtained by
  • a multimer described herein where the peptide in the multimer (MU) has the sequence of the target peptide (TP) sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleaving site (CS).
  • TP target peptide
  • CS chemical cleaving site
  • Step (b) Digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form.
  • Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein).
  • the N-terminus of the multimer includes a methionine and poly -histidine tag. The multimer be isolated in a Nickel column through use of the histidine tag.
  • step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide.
  • the method further comprises step
  • multimer can be digested with single protease.
  • the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and/or chemicals in a single step.
  • the target peptide sequence is not limited to TFTSD VS S YLEGQ AAKEFIAWLVRGRG.
  • the target peptide sequence can be but not limited to 10- 29 amino acids.
  • the target peptide sequence is selected from EGTFTSDVSSYLEGQ AAKEFIAWLVRGRG, GTFTSDVSSYLEGQ AAKEFIAWLVRGRG, FTSD VS S YLEGQAAKEFIAWLVRGRG, TSD VS S YLEGQ AAKEFIAWLVRGRG, SDVSSYLEGQ AAKEFIAWLVRGRG, DVSSYLEGQ AAKEFIAWLVRGRG, DVSSYLEGQ AAKEFIAWLVRGRG, DVSSYLEGQ AAKEFIAWLVRGRG, DVSSYLEGQ AAKEFIAWLVRGRG, DVSSYLEGQ AAKEFIAWLVRGRG,
  • LEGQ AAKEFIAWLVRGRG LEGQ AAKEFIAWLVRGRG, EGQ AAKEFIAWLVRGRG, GQ AAKEFIAWLVRGRG, Q AAKEFIAWLVRGRG, AAKEFIAWLVRGRG, AKEFIAWLVRGRG, KEFIAWLVRGRG, EFIAWLVRGRG, and FIAWLVRGRG
  • Figure 1 is an SDS-PAGE analysis for expression confirmation of the multimer of the target peptide (SEQ ID NO: 2).
  • Lane 1 Molecular weight marker; and
  • Lane 2 multimer of target peptide (SEQ ID 2) (33.8 KDa).
  • Figure 2 is a Ni-NTA purification sample run on SDS PAGE of the multimeric target peptide (SEQ ID No. 2).
  • Lane 1 Molecular weight marker
  • Lane 2 Load sample of multimeric target peptide
  • Lane 3 Flow through sample of multimeric target peptide
  • Lane 4 Wash of multimeric target peptide
  • Lane 5 Eluate of multimeric target peptide.
  • Figure 3 is an RPHPLC analysis of the multimer and after enzymatic digestion with Kex2 and CpB protease to generate the monomer of the target peptide.
  • the black peak indicates the multimer (SEQ ID NO: 2)
  • the blue peak indicates the intermediate after Kex2 digestion
  • the red peak indicates the monomer of target peptide (SEQ ID NO:1) after CpB digestion.
  • Figure 4 is an SDS-PAGE analysis for expression confirmation of the multimer of a target peptide (SEQ ID NO: 9) having improved solubility.
  • Lane 1 Molecular weight marker
  • Lane 2 multimer of target peptide (SEQ ID NO: 9) (48.5 kDa)
  • Figure 5 shows the solubilization of the multimer of the target peptide in different solubilization buffers.
  • the multimer of the target peptide (SEQ ID NO:2) is insoluble in 8M Urea, whereas the multimer of the target peptide with improved solubility (SEQ ID NO:9) is readily soluble in a mild solubilizing agent such as 6M Urea.
  • Figure 6 is an SDS-PAGE analysis for expression confirmation of the multimer of the target peptide (SEQ ID NO: 17).
  • Lane 1 Molecular weight, marker;
  • Lane 2 multimer of target peptide (SEQ ID 17) (51.3 kDa)
  • Figure 7 is a comparative SDS-PAGE analysis of the multimers (SEQ ID NO: 17) and (SEQ ID NO: 2).
  • Lane 1 Molecular weight, marker
  • Lane 2 multimer of target peptide (SEQ ID NO:17 (51.3 kDa).
  • Lane 3 multimer of target peptide (SEQ ID NO: 2)
  • Figure 8 is a Ni-NTA purification sample run on SDS PAGE of the multimer (SEQ ID NO: 17).
  • Lane 1 Load sample of multimeric target peptide
  • Lane 2 Molecular weight marker
  • Lane 3 Flow through sample of multimeric target peptide
  • Lane 4 Wash 1 of multimeric target peptide
  • Lane 5 Wash 2 of multimeric target peptide
  • Lane 6 Eluate 1 of multimeric target peptide
  • Lane 7 Eluate 2 of multimeric target peptide
  • Figure 9 is an elute sample of the multimer (SEQ ID NO: 17) which was buffer exchanged in different concentrations of urea solution.
  • Tube 1 Sample dissolved in 2M Urea.
  • Lane 2 Sample dissolved in 3M Urea.
  • Lane 3 Sample dissolved in 4MUrea.
  • Figure 10 is an SDS-PAGE analysis for expression confirmation of the multimer of target peptide (SEQ ID NO: 30).
  • Lane 1 Molecular weight, marker;
  • Lane 2 multimer of target peptide (SEQ ID 30) (50.9 kDa)
  • Figure 11 is a Ni-NTA purification sample run on SDS PAGE of the multimer of target peptide (SEQ ID NO. 30); Lane 1 : Molecular weight marker, Lane 2: Load sample of multimeric target peptide, Lane 3: Flow through sample of multimeric target peptide, Lane 4: Wash 1 of multimeric target peptide, Lane 5: Wash 2 of multimeric target peptide, Lane 6: Eluate of multimeric target peptide. DETAILED DESCRIPTION OF THE INVENTION
  • polypeptide In the present context the terms "polypeptide”, “protein” and “peptide” be used interchangeably to designate a polypeptide. It is to be understood that the particular term used has no limitation as to the size of the molecule (unless directly stated in the particular context). Amino acid residues are generally designated according to single letter abbreviation according to IUPAC nomenclature, e.g., D meaning aspartic acid (Asp) and G meaning glycine. In one embodiment, the peptide of interest has between 2 and 50 amino acids. In another embodiment, the peptide of interest has more than 50 amino acids, such as 51 to 200 amino acids, 51 to 100 amino acids, or 101 to 200 amino acids.
  • target peptide sequence and “peptide of interest” are interchangeable.
  • the term “promoter” generally refers to a regulatory region of DNA usually located upstream of the inserted gene of interest providing a control point for regulated gene transcription.
  • the term "host cell” as used herein is intended to mean a microorganism which is used for the expression of a peptide of interest.
  • a host cell encompasses any progeny of a parent cell that is not identical to the parent cell due to mutations that occur during replication.
  • Host cell can be but not limited to bacteria, Yeast, CHO.
  • the hydrophobicity of a peptide may be calculated by methods known in the art, such as by Peptide 2.0 (www.peptide2.com/N_peptide_hydrophobicity_hydrophilicity.php) or the use of the Monera scale (Monera et al., 1995, J. Protein Sci., 1 :319-329).
  • each amino acid in the peptide is assigned a hydrophobicity value based on established scales, such as the Monera scale.
  • the scale quantifies the hydrophobic nature of each amino acid.
  • the hydrophobicity values of all amino acids in the peptide are summed up. This total may then be divided by the number of amino acids in the peptide to get an average hydrophobicity value.
  • the average hydrophobicity value can be normalized to a percentage scale. This involves comparing the peptide's hydrophobicity to a reference value, which could be the maximum hydrophobicity value on the scale used.
  • digestion of the multimer can be performed by proteases (for example, the Kex2 protease and carboxypeptidase B) and chemical reactions.
  • the Kex2 protease specifically hydrolyzes a carboxyl terminal peptide bond in an alpha factor precursor, in particular a carboxyl terminal peptide bond of two consecutive basic amino acids, such as Lys- Arg, Lys-Lys, or Arg-Arg.
  • Recombinant carboxypeptidase B (CPB) can selectively hydrolyze arginine or lysine at the carboxyl terminus of a peptide.
  • the present invention provides a process for the development of a recombinant peptide of interest in a suitable host resulting in high expression in the form of 2 or more monomers of a target peptide sequences wherein each monomer is interspaced with a Tag amino acid sequence, one or more protease and /or chemical cleavage sites, and with or without additional amino acids, to modulate pl and/or hydrophobicity so as to enhance the solubility of the resulting target peptide of interest.
  • the multimeric target peptide sequence will be larger in size as compared to the monomeric target peptide sequence, thereby resulting in higher expression.
  • the expressed multimeric target peptide sequence will be in the form of insoluble (inclusion body) or soluble or in a combination of both forms that will require to be fully solubilized and digested with one or more specific proteases, whose sites have been created within the tags to generate the final monomeric target peptide sequence.
  • the process according to the present invention is not limited to target peptide sequences and can be used for any proteins.
  • the Tag amino acid sequences can be designed using a combination of amino acids which can alter the intrinsic properties of the multimer such as pl and/or hydrophobicity.
  • the Tag amino acid sequence comprises aspartic acid (D), glutamic acid (E), histidine (H), lysine (K), or arginine (R) which can alter the pl of the multimer.
  • the Tag amino acid sequences comprises aspartic acid and/or glutamic acid to reduce the pl of the multimer.
  • the Tag amino acid sequences comprise lysine and/or arginine to can increase the pl of the multimer.
  • the Tag amino acid sequences comprise serine (S).
  • the Tag amino acid sequences comprise leucine (L), isoleucine (I), alanine (A), valine (V), proline (P), glycine (G), tryptophan (W), phenylalanine (F) and other hydrophobic amino acids to increase the hydrophobicity of the amino acid.
  • the multimers comprise a target peptide sequence to achieve high level expression of the peptide of interest.
  • the C-terminus of the multimer (after the last target peptide sequence in the multimer) includes a Tag amino acid sequences which is capable of being cleaved from the target peptide (TP) sequence by a protease and/or chemical cleavage site (CS).
  • TP target peptide
  • CS chemical cleavage site
  • the amino acid at the N-terminus of the multimer is methionine and optionally a histidine tag.
  • the amino acid sequence at the N- terminus of the multimer is M(H) P , where p is 1 to 8 (such as MHHHHHH).
  • one or more of the Tag amino acid sequences comprises one or more amino acids to the pl of the multimer, the hydrophobicity of the multimer, or both.
  • one or more Tag amino acid sequences comprise one or more amino acids selected from aspartic acid (D), glutamic acid (E), and any combination of any of the foregoing to reduce the pl of the multimer.
  • one or more Tag amino acid sequences comprise one or more amino acids selected from Histidine (H), lysine (K), arginine (R), and any combination of any of the foregoing to increase the pl of the multimer.
  • one or more Tag amino acid sequences comprise one or more amino acids selected from serine (S), threonine (T), asparagine (N), glutamine (Q) and any other hydrophilic amino acids and any combination of any of the foregoing to increase the hydrophilicity of the multimer.
  • one or more Tag amino acid sequences comprise one or more amino acids selected from leucine (L), isoleucine (I) alanine (A), valine (V), proline (P), Glycine (G), Tryptophan (W), Phenylalanine (F) and any other hydrophobic amino acids and any combination of any of the foregoing to increase the hydrophobicity of the multimer.
  • one or more of the Tag amino acid sequences comprise an amino acid group with a proteolytic and/or chemical cleavage site at both the N and C terminus of the recombinant peptide of interest for cleaving the multimer to generate the recombinant peptide of interest.
  • each of the Tag amino acid sequences comprise an amino acid group with a proteolytic and/or chemical cleavage site at any site of attachment to the N or C-terminus of the target peptide sequence ; this is to permit cleaving of the multimer to generate the target peptide sequence.
  • one or more of the separator sequences includes only proteolytic and/or chemical cleavage sites.
  • one or more of the cleavage sites comprise the amino acid sequence KR. In some instances, each cleavage site sequence is KR.
  • one or more of the Tag amino acid sequences comprise DSSTTDSSTTDDDDDK (SEQ ID NO: 6), DSSDTTDDDDDK (SEQ ID NO: 5), SSTTDDDDK (SEQ ID NO: 8), DSSTTDSSTTSSTT (SEQ ID NO: 7), DSSTTDSSTTDDDDK (SEQ ID NO: 4), SSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 49), SSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 50), SSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 51), SSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 52), SDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 11) , SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 12) , SDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 12)
  • one or more of the Tag amino acid sequences further comprise one or more additional amino acids.
  • each of the Tag amino acid sequences are selected from DSSTTDSSTTDDDDDK (SEQ ID NO: 6), DSSDTTDDDDDK(SEQ ID NO: 5) exactly SSTTDDDDK(SEQ ID NO: 8), DSSTTDSSTTSSTT(SEQ ID NO: 7), DSSTTDSSTTDDDDK(SEQ ID NO: 4), SSTTRSSTTSSTTRSSTTSSTTRDDDDK(SEQ ID NO: 49), SSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK(SEQ ID NO: 50), SSTTSSTTRSSTTSSTTRSSTTRDDDDK(SEQ ID NO: 51), and SSTTSSTTSSTTSSTTSSTTDDDDK(SEQ ID NO: 52), SDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 11) , SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR(SEQ ID NO: 11)
  • one or more of Tag amino acid sequences comprise KRDSSTTDSSTTDDDDDK (SEQ ID NO: 54), KRDSSDTTDDDDDK (SEQ ID NO: 55), KRSSTTDDDDK (SEQ ID NO: 56), KRDSSTTDSSTTSSTT (SEQ ID NO: 57), KRDSSTTDSSTTDDDDK (SEQ ID NO: 58), KRSDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 59), KRSDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 60), KRSDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 61), KRSDSTTSSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 62), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 63), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 64), or any combination of any of the foregoing
  • each of the Tag amino acid sequences are selected from KRDSSTTDSSTTDDDDDK (SEQ ID NO: 54), KRDSSDTTDDDDDK (SEQ ID NO: 55), KRSSTTDDDDK (SEQ ID NO: 56), KRDSSTTDSSTTSSTT (SEQ ID NO: 57), KRDSSTTDSSTTDDDDK (SEQ ID NO: 58), KRSDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 59), KRSDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 60), KRSDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 61), KRSDSTTSSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 62), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 63), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 64).
  • one or more of Tag amino acid sequences comprise KRSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 65), KRSSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 66), KRSSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 67), KRSSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 68), KRSSTTSSTTRSSTTSSTTSSTTR (SEQ ID NO: 69), KRSDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 70), KRSDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 71), KRSDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 72), KRSDSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 73), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 66), KRSSTTSST
  • one or more of Tag amino acid sequences are selected from KRSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 65), KRSSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK (SEQ ID NO: 66), KRSSTTSSTTRSSTTSSTTRSSTTRDDDDK (SEQ ID NO: 67), KRSSTTSSTTSSTTSSTTSSTTDDDDK (SEQ ID NO: 68), KRSSTTSSTTRSSTTSSTTSSTTR (SEQ ID NO: 69), KRSDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 70), KRSDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 71), KRSDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 72), KRSDSTTSSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 73), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 74
  • one or more of the Tag amino acids comprise one or more additional amino acids in combination with any of the foregoing Tag amino acid sequences (such as to change the pl and/or hydrophobicity of the multimer).
  • the Tag amino acid sequences can be any combination of naturally occurring amino acids (Tag) along with or without a CS targeted to alter intrinsic characteristics of the multimer, such as pl and/or hydrophobicity.
  • the Tag amino acid sequence can lower the pl of the multimer. In yet another embodiment of the multimer described herein, the Tag amino acid sequence can lower the hydrophobicity of the multimer. In some instances, the Tag amino acid sequence can include one or more of SEQ ID NOs. 4-8, 11-16, and 19-29.
  • one or more of the Tag amino acid sequence include one or more naturally occurring solubility enhancing partner tags.
  • the naturally occurring solubility enhancing partner tags have one or more domains which are responsible for enhancing the solubility of the multimer. These domains can also be used to modulate the pl of the multimer.
  • the naturally occurring solubility enhancing partner tag has one or more domains from Thyrodoxin, SUMO, Fh8, GB1, CaBP, MsyB, and Skp.
  • the naturally occurring solubility enhancing domain has one or more domains selected from NPGIDAEDANLQDGDADEGEE (SEQ ID NO: 19), IDDQVEDKCLPQ (SEQ ID NO: 20), IDDQVEDKADEGE (SEQ ID NO: 21), EGECLPDL (SEQ ID NO: 22), LQDGDADEGEEGELSGDGDYD (SEQ ID NO: 23), EGELSGDGDYD (SEQ ID NO: 24), LEPPLDTD (SEQ ID NO: 25), LQDGDAD (SEQ ID NO: 26), QDGDADEGEE (SEQ ID NO: 27), NPGIDAEDANL (SEQ ID NO: 28), NPGIDAEDANLQDGDAD (SEQ ID NO: 29) (a domain from MsyB), SDKIIHLTDDSFDTDVLKADGAILVDF (SEQ ID NO: 32), MSDKIIHLTDDSFDTDVLKAD (SEQ ID NO: 33), IHLTDDSFDTDVL
  • one or more of Tag amino acid sequences comprise NPGIDAEDANLQDGDADEGEEKR (SEQ ID NO: 38), KRIDDQVEDKCLPQ(SEQ ID NO: 39), KRIDDQVEDKADEGE (SEQ ID NO: 40), KREGECLPDL(SEQ ID NO: 41), KRLQDGDADEGEEGELSGDGDYD (SEQ ID NO: 42), KREGELSGDGDYD (SEQ ID NO: 43), KRLEPPLDTD (SEQ ID NO: 44), KRLQDGDAD (SEQ ID NO: 45), KRQDGDADEGEE (SEQ ID NO: 46), KRNPGIDAEDANL(SEQ ID NO: 47), KRNPGIDAEDANLQDGDAD (SEQ ID NO: 48) or any combination of any of the foregoing.
  • one or more of Tag amino acid sequences further comprise one or more additional amino acids.
  • one or more of Tag amino acid sequences are selected from NPGIDAEDANLQDGDADEGEEKR (SEQ ID NO: 38), KRIDDQVEDKCLPQ (SEQ ID NO: 39), KRIDDQVEDKADEGE (SEQ ID NO: 40), KREGECLPDL (SEQ ID NO: 41), KRLQDGDADEGEEGELSGDGDYD (SEQ ID NO: 42), KREGELSGDGDYD (SEQ ID NO: 43), KRLEPPLDTD (SEQ ID NO: 44), KRLQDGDAD (SEQ ID NO: 45), KRQDGDADEGEE(SEQ ID NO: 46), KRNPGIDAEDANL(SEQ ID NO: 47), KRNPGIDAEDANLQDGDAD (SEQ ID NO: 48) or any combination of any of the foregoing.
  • one or more of Tag amino acid sequences further comprise one or more additional amino acids.
  • every Tag amino acid sequence in the multimer is the same.
  • the multimer comprises two or more different Tag amino acid sequence.
  • the N-terminus of the multimer includes a methionine and histidine tag.
  • the N-terminus of the multimer include but not limited to methioninepoly histidine (such as -MHHHHHH (SEQ ID NO: 76)).
  • the C-terminus of the multimer includes a histidine tag followed by a stop codon. In some instances, the C-terminus of the multimer include a poly histidine.
  • the poly histidine tag can be within the multimer, i.e. not at the N or C terminus.
  • the multimer comprises greater than 2 copies of the target peptide sequence. In some embodiments, the multimer has 5 to 40 copies of the target peptide sequence. In some embodiments, the multimer has 10 copies of the target peptide sequence. In some embodiments, the multimer has 20 copies of the target peptide sequence.
  • One aspect of the invention is a multimer comprising a plurality of target peptide sequences connected in series, wherein (i)
  • every two adjacent target peptide sequences are connected by an amino acid tag sequence (Tag) capable of being cleaved by a protease and/or chemical cleavage (CS) to form the target peptide sequence (TP) sequences in free form, wherein the Tag amino acid sequence (Tag) between each two adjacent target peptide sequences are the same or different,
  • the C-terminus of the multimer (after the last target peptide in the multimer) includes a Tag amino acid sequence which is capable of being cleaved from the target peptide sequence by a protease and/or chemical cleavage.
  • one or more of the Tag amino acid sequences comprise an amino acid group with a proteolytic and/or chemical cleavage (CS) site at both the N and C terminus of the target peptide sequence for cleaving the multimer.
  • CS chemical cleavage
  • each of the Tag amino acid sequences comprise an amino acid group with a proteolytic and/or chemical cleavage site (CS) at any site of attachment to the N or C-terminus of the target peptide sequence; this is to permit cleaving of the multimer.
  • CS proteolytic and/or chemical cleavage site
  • Amino acids in the Tag amino acid sequence act as both a modulator of pl and/or hydrophobicity as well as a proteolytic and/or chemical cleavage site.
  • one or more of the separator sequences includes only proteolytic and/or chemical cleavage sites.
  • one or more of the Tag amino acid sequences are KR. In some embodiments, each amino acid tag sequence is KR.
  • one or more of the Tag amino acid sequences comprise one or more naturally occurring solubility enhancing fusion partner tags as described herein.
  • variable A comprises a tag to facilitate purification of the multimer.
  • variable A comprise a histidine tag (e.g., poly histidine).
  • Tag is absent and r is 0, in which case MO will be TP-CS.
  • each occurrence of Tag is absent and each occurrence of r is 0.
  • the multimer has a pl from about 4 to about 12.
  • the multimer has a molecular weight more than 2 kilodalton.
  • the target peptide sequence is TFTSD VS S YLEGQ AAKEFIAWLVRGRG.
  • the target peptide sequence can be but not limited to 10- 29 amino acids.
  • the target peptide sequence is HAEGTFTSDVSSYLEGQ AAKEFIAWLVRGRG.
  • Yet another aspect is a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.
  • Yet another aspect is a host cell transfected with a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.
  • a host cell transfected with a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.
  • an expression vector comprising a DNA sequence encoding the multimer as described herein.
  • Yet another aspect is a host cell comprising the expression vector described herein.
  • the expression vector has any promoter.
  • the promoter is T7.
  • Yet another aspect is a method of preparing a multimer comprising expressing a DNA construct comprising a nucleic acid sequence encoding the multimer described herein in a host cell.
  • the method further comprises digesting the multimer with one or more enzymes to obtain a plurality of the target peptide sequences in free form.
  • each occurrence of CS is independently KR or any other proteolytic/chemical cleavage site. In some embodiments, each occurrence of CS is KR .
  • each occurrence of CS is independently DDDDK or any other proteolytic/chemical cleavage site.
  • each occurrence of CS is DDDDK (SEQ ID NO: 53).
  • the multimer has a hydrophobicity range of from about 24 to about 42%.
  • the multimer has a molecular weight more than 2 kilodalton.
  • the target peptide (TP) sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 1).
  • the target peptide sequence can be but not limited to 10- 29 amino acids.
  • the target peptide sequence is but not limited to EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 78), GTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 79), FTSD VS SYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 80), TSDVS SYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 81), SDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 82), D VS SYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 83), VS SYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 84), S SYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 85), SYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 86), YLEGQAAKEFIAWLVRGRG (SEQ ID NO: 87), LEGQAAKEFIAWLVRGRG (SEQ ID NO: 78), GTFT
  • the target peptide sequence is HAEGTFTSD VS SYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 97). In some instances, the target peptide sequence is SEQ ID NO. 1.
  • the sequence of the multimer of a target peptide sequence is SEQ ID NO. 2. In some instances, the sequence of the multimer of a target peptide sequence is SEQ ID NO. 9. In some instances, the sequence of the multimer of a target peptide sequence is SEQ ID NO. 17. In some instances, the sequence of the multimer of a target peptide sequence is SEQ ID NO. 30.
  • the multimer nucleotide sequence of a target peptide sequence is SEQ ID NO. 3. In some instances, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO. 10. In some instances, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO. 18. In some instances, the multimer nucleic acid sequence of a target peptide sequence is SEQ ID NO. 31.
  • DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.
  • host cell transfected with a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.
  • Yet another aspect is an expression vector comprising a DNA sequence encoding the multimer as described herein.
  • Yet another aspect is a host cell comprising the expression vector described herein.
  • the expression vector has any promoter.
  • the promoter is T7.
  • Yet another aspect is a method of preparing a multimer comprising expressing a DNA construct comprising a nucleic acid sequence encoding the multimer described herein in a host cell.
  • the method further comprises digesting the multimer with one or more enzymes to obtain a plurality of the target peptide sequences in free form.
  • Yet another aspect is a method of preparing a peptide comprising:
  • Step (b) Digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form.
  • Step (a) comprise expressing the multimer in the host and isolating the multimer.
  • the host Prior to performing step (a), the host be transfected with an expression vector for expressing the multimer (such as that described herein).
  • the N-terminus of the multimer includes a methionine and; with or without not limited to poly-histidine tag.
  • the multimer be isolated in a Nickel column through use of the histidine tag.
  • step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide.
  • the method further comprises step (c) isolating the peptide.
  • multimer can be digested with single protease.
  • the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and/or chemicals in a single step.
  • the recombinant peptide of interest described herein could comprise of one or more of the peptides.
  • the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 1) obtained by
  • Step (b) digesting the multimer with one or more enzymes or chemicals to obtain a plurality of the target peptide sequences in free form.
  • Step (a) comprise expressing the multimer in the host and isolating the multimer. Prior to performing step (a), the host has to be transfected with an expression vector for expressing the multimer (such as that described herein).
  • the N-terminus of the multimer includes a methionine and poly-histidine tag. The multimer be isolated in a Nickel NTA resin through use of the histidine tag.
  • step (b) comprises (i) subjecting the multimer to digestion with any protease enzyme or chemical to produce a first intermediate peptide mixture, (ii) subjecting the first intermediate peptide mixture to digestion with second protease or chemical to produce a second intermediate peptide mixture, and (iii) subjecting the second intermediate peptide mixture with third protease enzyme or chemical to form the target peptide.
  • the method further comprises step
  • multimer can be digested with single protease.
  • the multimer can be digested with two different proteases. Wherein the multimers can be digested using multiple protease and/or chemicals in a single step.
  • the target peptide sequence is not limited to TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 1).
  • the target peptide sequence can be but not limited to 10- 29 amino acids.
  • the target peptide sequence is but not limited to EGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 78), GTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 79), FTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 80), TSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 81), SDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 82), DVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 83), VSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 84), SSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 85), SYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 86), YLEGQAAKEFIAWLVRGRG
  • TP sequence is conjugated to His-Aib-Glu-Gly.
  • target peptide sequence is conjugated to the fatty acid moiety N-(17- carboxy-l-oxoheptadecyl)-L-y-glutamyl-2-[2-(2-aminoethoxy)ethoxyl]acetyl-2-[2-(2- aminoethoxy)ethoxy]acetyl at the lysine at position 26 to form semaglutide.
  • the target peptide sequence is conjugated to His- Aib-Glu-Gly and the fatty acid moiety N-(17-carboxy-l-oxoheptadecyl)-L-y-glutamyl-2-[2-(2- aminoethoxy)ethoxyl]acetyl-2-[2-(2-aminoethoxy)ethoxy]acetyl at the lysine at position 26 to form semaglutide
  • a multimer of the target peptide (SEQ ID NO:1) was constructed (SEQ ID NO: 2).
  • the multimer included a methionine and a histidine tag (at the N-terminus followed by repeating units of target peptide conjugated to a KR tag.
  • the N-terminus of the first target peptide (SEQ ID NO: 1) in the multimer includes a KR tag.
  • the monomeric target peptide (SEQ ID NO:1) is obtained by cleavage of the multimer of target peptide using Kex2 and CpB proteases.
  • a multimer of the target peptide (SEQ ID NO:1) was constructed (SEQ ID NO: 2) where monomeric peptide units were connected by specific amino acid residues.
  • these amino acid residues could be lysine (K) and arginine (R) which are cleavage site for the proteases Kex2 and CpB.
  • K lysine
  • R arginine
  • This approach significantly increases the efficiency of peptide expression as it eliminates usage of fusion partner tags.
  • the monomeric target peptide is obtained by cleavage of multimer of target peptide using Kex2 and CpB protease.
  • the cloned expression vector was transformed into E. coli BL21 DE3 for expression studies. A flask study was performed where cells were seeded at 0.1 optical density (OD), induced with IPTG and harvested 9 hours post induction. Expression of the multimer SEQ ID NO: 2 (molecular weight 33.8 kDa) was confirmed by SDS PAGE as shown in Figure 1.
  • the harvested pellet was weighed and resuspended in 20X 8M GuCl solubilization buffer. The sample was sonicated for 15 minutes and incubated overnight under shaking conditions till the solution became clear. The sample was filtered and processed through a Ni-NTA column for capturing the multimer (SEQ ID NO: 2). The captured multimer was eluted and further analyzed for protein expression by SDS PAGE as shown in Figure 2.
  • the sample was subjected to cleavage by Kex2 and CpB protease to obtain the target peptide monomer (SEQ ID NO: 1).
  • Kex2 protease specifically recognizes the amino acid sequence KR and cleaves at the C-terminus of R.
  • CpB protease is an exopeptidase which subsequently removes R and K leaving behind the target peptide (SEQ ID NO: 1).
  • Reverse phase HPLC (RP-HPLC) analysis was performed for confirmation of target peptide monomer as shown in Figure 3.
  • the multimer (SEQ ID NO: 2) had high expression level but inclusion bodies formed that were difficult to solubilize, requiring 8M GuCl which increases cost of production and is un-favorable for downstream enzymatic processing.
  • the multimer (SEQ ID NO: 2) had a pl of 10.28 and hydrophobicity of 37.1%. Since these parameters were unfavorable with respect to solubility, specific amino acid tag sequences (SEQ ID NOs: 4-8) were incorporated into the multimer (SEQ ID NO:2). The resulting additions led to the pl decreasing from 10.28 to 4.49 and hydrophobicity decreasing from 37.1% to 25.4%.
  • the multimer has been designed in such a way that monomers of target peptide are interspaced with the specific amino acid tag sequences (SEQ ID Nos:4-8). Protease cleavage sites have been added at the N and C terminus of the target peptide to ensure generation of the monomer of the target peptide (SEQ ID NO. 1) after enzymatic digestion with one or more proteases.
  • the resulting multimer is SEQ ID NO: 9, which had improved solubility.
  • the amino acid tag sequences include aspartic acid (D), serine (S) and threonine (T).
  • Aspartic acid (D) helps in lowering the pl of the multimer.
  • Serine (S) and threonine (T) help in lowering the overall hydrophobicity of the multimer.
  • the sequence DDDDK SEQ ID NO: 53 was incorporated, which also provides a cleavage site as it can be digested with an enterokinase enzyme (EK).
  • the cloned expression vector was transformed into E. coli BL21 DE3 for expression studies. A flask study was performed where cells were seeded at 0.1 OD, induced with IPTGand harvested 9 hours post induction. Expression of the multimer (SEQ ID NO: 9, molecular weight 48.5 kDa) was confirmed by SDS PAGE analysis as shown in Figure 4.
  • Inclusion bodies or cell pellet formed by the multimer is soluble in mild solubilizing agents such as 6M urea as compared to the multimer of Example 1 (SEQ ID NO: 2) which is insoluble in 8M Urea and requires a strong chaotropic agent for solubilization as shown in Figure 5.
  • This modulation of pl and hydrophobicity enables improved solubility and ease of downstream processing, however multimer expression level is on lower side as shown in Figure 4.
  • SEQ ID NO: 17 For producing the target peptide (SEQ ID NO: 1) a multimer of target peptide was constructed (SEQ ID NO: 17). As shown in example 2, multimer of the target peptide with improved solubility (but low expression) SEQ ID NO: 9 had low pl 4.49 and low hydrophobicity of 25.4%. To enhance expression level, SEQ ID NO: 17 was designed where the hydrophobicity was kept low (23.77%), however the pl was increased from 4.49 to 10.3. The hydrophobicity was reduced by incorporating S (Serine) and T (Threonine). To increase the pl to 10.3, R (Arginine) was added to the tag sequence. Lower hydrophobicity is required to enhance solubility of multimer, however, pl modulation is required for enhanced multimer expression.
  • SEQ ID NO: 17 the corresponding nucleotide sequence was codon optimized and synthesized yielding SEQ ID NO: 18.
  • the nucleotide sequence (SEQ ID NO: 18) was cloned in a suitable expression vector with a T7 promoter and transformed in E. coli ToplO cells to generate expression vector.
  • the cloned expression vector was transformed into E. coli BL21 DE3 for expression studies. A flask study was performed where cells were seeded at 0.1 OD, induced with IPTG and harvested 9 hours post induction. Expression of the multimer SEQ ID NO: 17 (molecular weight 51.3 kDa) was confirmed by SDS PAGE analysis as shown in Figure 6. The expression level was improved compared to the expression level of multimer (SEQ ID NO:2) as shown in Figure 7.
  • the inclusion bodies or cell pellet obtained after the flask study of multimer (SEQ ID NO: 17) are easily soluble in urea buffer.
  • the samples were purified using Ni-NTA column where eluates were in 4M urea buffer (mild solubilizing agent) as shown in Figure 8.
  • the eluate containing multimer of the target peptide is soluble in mild urea concentrations of 2-4 M ( Figure 9). This improved solubility of the high expressing multimer enable ease of enzymatic digestion and further downstream processing.
  • SEQ ID NO: 1 For producing a target peptide (SEQ ID NO: 1), a multimer of the target peptide was constructed (SEQ ID NO: 30). As shown in Example 3, the multimer of target peptide (SEQ ID NO: 1) was designed with improved solubility and expression (SEQ ID NO: 17). The inclusion bodies obtained from the multimer of SEQ ID NO: 17 were soluble in urea when eluted into 4M urea.
  • a multimer (SEQ ID NO: 30) was design incorporating domains from the naturally occurring solubility enhancing molecule MsyB.
  • the pl was reduced from 10.3 (SEQ ID NO: 17) to 5.09 (SEQ ID NO: 30) and hydrophobicity was changed from 23.77% (SEQ ID NO: 17) to 32.68 % (SEQ ID NO: 30).
  • the cloned expression vector was transformed into E. coli BL21 DE3 for expression studies. A flask study was performed where cells were seeded at 0.1 OD, induced with IPTG and harvested 9 hours post induction. Expression of the multimer (SEQ ID NO: 30) (molecular weight 50.9 kDa) was confirmed by SDS-PAGE analysis ( Figure 10).

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Abstract

La présente invention concerne des multimères contenant une pluralité de séquences peptidiques cibles qui permettent des niveaux élevés d'expression et une solubilité améliorée pour une facilité de traitement et de purification en aval.
PCT/IB2025/051092 2024-01-31 2025-01-31 Production de peptides et de protéines à haut niveau Pending WO2025163592A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101172996A (zh) 2006-09-29 2008-05-07 上海新生源医药研究有限公司 用于多肽融合表达的连接肽及多肽融合表达方法
WO2014097323A1 (fr) * 2012-12-21 2014-06-26 Enzene Biosciences Ltd. Nouvelles étiquettes de fusion et système de vecteur d'expression pour l'expression de la parathormone humaine (rhpth)
WO2018172921A1 (fr) 2017-03-20 2018-09-27 Lupin Limited Expression et production à grande échelle de peptides
US20220195004A1 (en) 2019-06-26 2022-06-23 Peg-Bio Biopharm Co., Ltd. (Chongqing) Method for preparing target polypeptide by means of recombination and series connection of fused proteins
CN115975047A (zh) * 2022-10-24 2023-04-18 扬州奥锐特药业有限公司 一种重组融合蛋白生产多肽的方法及其应用
CN116948013A (zh) * 2023-04-28 2023-10-27 江苏创健医疗科技股份有限公司 重组小分子胶原蛋白及其表达系统与制备方法

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101172996A (zh) 2006-09-29 2008-05-07 上海新生源医药研究有限公司 用于多肽融合表达的连接肽及多肽融合表达方法
WO2014097323A1 (fr) * 2012-12-21 2014-06-26 Enzene Biosciences Ltd. Nouvelles étiquettes de fusion et système de vecteur d'expression pour l'expression de la parathormone humaine (rhpth)
WO2018172921A1 (fr) 2017-03-20 2018-09-27 Lupin Limited Expression et production à grande échelle de peptides
US20220195004A1 (en) 2019-06-26 2022-06-23 Peg-Bio Biopharm Co., Ltd. (Chongqing) Method for preparing target polypeptide by means of recombination and series connection of fused proteins
CN115975047A (zh) * 2022-10-24 2023-04-18 扬州奥锐特药业有限公司 一种重组融合蛋白生产多肽的方法及其应用
CN116948013A (zh) * 2023-04-28 2023-10-27 江苏创健医疗科技股份有限公司 重组小分子胶原蛋白及其表达系统与制备方法

Non-Patent Citations (13)

* Cited by examiner, † Cited by third party
Title
A GIRIRAJ, SCIENCE DIRECT, 2018, pages 157 - 181
AJINGY YS, CURRENT PHARMACEUTICAL BIOTECHNOLOGY, vol. 23, 2022, pages 645 - 663
BARMAN ET AL., INT J PEPTIDE RES. AND THER., vol. 29, no. 61, 2023
BARMAN P. ET AL., INT J PEPT RES THER., vol. 29, no. 4, 2023, pages 61
HOU ET AL., BIOSCI BIOTECHNOL BIOCHEM., vol. 71, no. 6, 2007, pages 1462 - 1469
LAVALLIE ET AL., BIOTECHNOLOGY, vol. 11, no. 2, 1993, pages 187 - 93
LE THI NHU NGOC, COSMETICS, vol. 10, no. 4, 2023
LE THI NHU NGOC, FEATURE PAPERS IN COSMETICS, 2023
MONERA ET AL., J. PROTEIN SCI., vol. 1, 1995, pages 319 - 329
RYAN M. KRAMER ET AL., BIOPHYS J, vol. 102, no. 8, 2012, pages 1907 - 15
S C BERNIER, PROTEIN EXPR PURIF, vol. 152, 2018, pages 92 - 106
S. COASTA, FRONT MICROBIOL, vol. 19, no. 5, 2014, pages 63
SHEN SH, PROC NATL ACAD SCI U S A, vol. 81, no. 15, 1984, pages 4627 - 31

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