High level production of peptides and proteins

Abstract

This invention relates to multimers containing a plurality of target peptide sequences which permit high levels of expression and improved solubility for ease of downstream processing and purification.

Description

[1] This application claims the benefit of the Indian provisional application 202421006467, filed January 31, 2024, which is hereby incorporated by reference. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING [2] The instant application contains a Sequence Listing that has been submitted electronically and is hereby incorporated by reference in its entirety. The Sequence Listing was created on January 30, 2024, is named “159121-06201_SL.xml” and is 101,792 bytes in size. FIELD OF THE INVENTION [3] 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. BACKGROUND OF THE INVENTION [4] 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. However, there is an increasing demand in the fields of cosmetics and nutraceuticals. 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, / 0(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:10.10I6 / B978-0-08-I00736-5.00006-5). [5] Recombinant peptides are often expressed in a suitable host such as E.coli or yeast. However, there are 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). To increase yield, fusion partners have been attached to peptides to increase their expression level and protect them from protease cleavage. For example, thioredoxine (TRX), small ubiquitin-like modifier (SUMO) and glutathione-S-transferase (GST) can be fused with the recombinant peptide of interest, and later separated by a protease cleavage site. The fusion partner protects it from intrinsic proteolytic enzymes of the host (S. Coasta 2014, SC Bernier 2018, LaVallie et al.; S. Coasta etal., Front microbiol, 2014; 19:5:63; S C Bernier et.al, Protein Expr Purif, 2018; 152:92-106; LaVallie et al. BioTechnology, 1993; 11(2):187-93). 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. [6] There is a need to not only overcome the problem of cytosolic cleavage of peptides, but improve the efficiency of peptide production that is lost from using fusion proteins tags. One method includes adding repeats of the recombinant peptide of interest upon itself (Shen SH 1984 (Shen SH, Proc Natl Acad Set U SA, 1984; 81(15).4627--31). The intrinsic properties of a peptide such as its isoelectric point (pl) and hydrophobicity play a major role in its solubility (Ryan M. Kramer 2012 (RyanM. Kramer etal., Biophys J, 2012; 18; 102(8): 1907-15). [7] 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. [8] Hou et aL, Biosci Biotechnol Biochem., 71(6):1462-1469, 2007, discloses expression of a fusion protein containing 10 tandem repeated GLP-1 analogs in yeast Pichia pastoris. [9] International Publication No. WO 2018 / 172921 discloses a method for preparation of a biologically active recombinant peptide by overexpressing it as a concatemer having specific intervening Kex2 protease and Carboxypeptidase B cleavage sites separating each monomer.

[10] Chinese Patent Publication No. 101172996 discloses a connection peptide and poly-fusion expression method.

[11] The prior arts clearly reflects the continuing need for improved processes for producing polypeptides in high yield. SUMMARY OF THE INVENTION

[12] 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%.

[13] 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 (i) 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, (ii) 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), (iii) the choice of cleavage sites (CS) shall have no identical sequence within the target peptide (TP), (iv) the N- and / or C-terminus of the multimer (MU) 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).

[14] In one embodiment, 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).

[15] In another embodiment a multimer (MU) having the formula MU = Met - (M0)n or MU = (M0)n, where n is an integer ranging from 2-50, preferably 2-20 and more precisely 10. Each MO has the formula - MO = (CS-TP-CS) and / or MO = (Tag-CS-TP-CS) and / or MO = (CS-TP-CS-Tag), where Tag is a sequence of amino acids to modulate pl and / or hydrophobicity of MO and MU. i. The Tag is not positioned between TP and CS,

[16] The number of Tag moieties to be included will depend on the degree of change in the pl and / or hydrophobicity desired for the MO and / or MU, ii.   The choice of amino acids used in the Tags shall be based on whether the expression of the MU is desired in the soluble form or as insoluble inclusion bodies, iii. The choice of the cleavage site (CS) should be such that upon cleavage, by chemicals or one or more proteases, shall leave no additional amino acids at either the N or the C terminus of the TP. [

[17] In some embodiments, the affinity tag is added either at the N- or C-terminus of the multimer (MU) separated by the cleavage site (CS).

[18] In some embodiments, the amino acid at the N-terminus of the multimer is methionine.

[19] In some embodiment and optionally poly-histidine is added either at N- or C-terminus of the multimer (MU), represented by the formula M(H)P, where p is 1 to 8 (SEQ ID NO: 76) (such as MHHHHHH (SEQ ID NO: 77)).

[20] In some embodiments, one or more of the cleavage sites comprise the amino acid sequence KR. Pl] In some embodiments, 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.

[22] In some embodiments, one or more of the cleavage site (CS) comprises of amino acid sequences DDDDK (SEQ ID NO: 53).

[23] In some embodiments, 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.

[24] In some embodiments, the multimer includes one or more Tag amino acid sequences to alter the hydrophobicity, pl, or both of the multimer (MU).

[25] In some embodiments, 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).

[26] In some embodiments, the multimer has a pl of from about 3 to about 12.

[27] In some embodiments, the multimer has a hydrophobicity of from 20% to 50%.

[28] In some embodiments, the multimer (MU) has a hydrophobicity of 25% to about 40% and / or pl from 4 to 10.

[29] In some embodiments, each Tag amino acid sequence between two target peptide (TP) sequences in the multimer is the same.

[30] In some embodiments, each Tag amino acid sequence in the target peptide (TP) is the different.

[31] In some embodiments, the multimer (MU) comprises at least two different Tag amino acid sequences.

[32] In some embodiments, the multimer (MU) includes 2 to 50 copies of a target peptide (TP) sequence.

[33] In some embodiments, the multimer (MU) includes 2 to 20 copies of a target peptide (TP) sequence.

[34] In some embodiments, the multimer (MU) includes 10 copies of a target peptide (TP) sequence.

[35] In some embodiments, 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.

[37] In some embodiments, one or more of the Tag amino acid sequences comprise at least one solubility enhancing domain.

[38] In some embodiments, 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.

[39] In some embodiments, 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, IKAFIKEHDKNKDGKLDLKEL (SEQ ID NO: 35), KEHDKNKDGKLDLKELVSILS (SEQ ID NO: 36), IKAFIKEHDKNKDGKLDLKELVSILSS (SEQ ID NO: 37), and any combination of any of the foregoing.

[40] In some embodiments, the present invention is directed towards a DNA construct comprising a nucleic acid sequence encoding a multimer.

[41] In some instances, a host cell is transfected with the DNA construct.

[42] In some embodiments, the present invention uses an expression vector, wherein the DNA sequence coding for the multimer (MU) is introduced under the T7 promoter.

[43] In some instances, a host cell comprises is transformed with the expression vector.

[44] In some embodiments, 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).

[45] In some embodiments, the present invention is directed a method of preparing a peptide comprising: (a) obtaining a multimer; and (b) digesting the multimer with one or more digestive enzymes or chemicals to obtain a plurality of the target peptide sequence in free form.

[46] In some embodiments, step (a) of the method comprises expressing the multimer in E. coli, and optionally isolating the multimer.

[47] In some embodiments directed towards a method, the N-terminus includes methionine and a histidine tag, and the multimer is isolated using Nickel-NTA resin via the polyhistidine affinity tag.

[48] T In some embodiments directed towards a method, the E. coli is BL21, BL21(DE3), ToplO, orDH5a cells.

[49] In some embodiments directed towards a method, the multimer (MU) is digested using one or more of the proteases selected from Kex2, carboxypeptidase B, and enterokinase.

[50] In some embodiments when multiple proteases are used the cleavage is carried out sequentially or simultaneously using Kex2 and / or enterokinase followed by carboxypeptidase.

[51] In some embodiments when multiple proteases are used the cleavage is carried out sequentially or simultaneously using Kex2 followed by carboxypeptidase.

[52] In another embodiment the final peptide after complete digestion is target peptide (TP) which is further purified using one or more chromatography or precipitation techniques.

[53] In some embodiments directed towards a method, the isolating step comprises one or more chromatography steps.

[54] In some embodiments directed towards a method, wherein 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.

[56] In some embodiments, a protein of interest is generated through multimers (MU) comprising a plurality of the target peptide sequence. In some instances, 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. In some embodiments, within the multimer (MU), the target peptide (TP) sequences are interspaced with Tag amino amino acid tags having cleavage sites for proteolytic enzymes and / or chemicals.

[57] In some embodiments, 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. Disclosed are 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. In some embodiments, the Tag amino acid sequences enhance expression levels and improve solubility of the multimer (MU).

[58] In some embodiments, 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. In some instances, 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. In some instances, the Tag amino acid sequences comprise of aspartic acid and / or glutamic acid to reduce the pl of the multimer (MU). In some instances, the Tag amino acid sequences comprise lysine and / or arginine to can increase the pl of the multimer (MU). In some instances, the Tag amino acid sequences comprise serine (S). threonine (T), asparagine (N), glutamine (Q) and other hydrophilic amino acids to render the multimer (MU) more hydrophilic. In some instances, 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). In some embodiments, the multimers comprise a target peptide sequence to achieve high level expression of the peptide of interest.

[59] In one embodiment, 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).

[60] In some embodiments, the amino acid at the N-terminus of the multimer is methionine and optionally a histidine tag. In some instances, the amino acid sequence at the N-terminus of the multimer is M(H)P, where p is 1 to 8 (such as MHHHHHH).

[61] In some embodiments, 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. In some instances, 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. In some instances, 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. In some instances, 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. In some instances, 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.

[62] In one embodiment of the multimer described herein, 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.

[63] In one embodiment of the multimer described herein, 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.

[64] In some embodiments, one or more of the separator sequences includes only proteolytic and / or chemical cleavage sites.

[65] In some embodiments, one or more of the cleavage sites comprise the amino acid sequence KR. In some instances, each cleavage site sequence is KR.

[66] In some embodiments, 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(SEQIDNO: 11) , SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 12) , SDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 13), SDSTTSSTTSSTTSSTTSSTT(SEQ ID NO: 14), SSTTSDSTTRSDSTTSSTTRSDSTTR(SEQ ID NO: 15), SDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 16) and any combination of any of the foregoing. In some embodiments, one or more of the Tag amino acid sequences further comprise one or more additional amino acids. In some embodiments, each of the Tag amino acid sequences are selected from 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), and SSTTSSTTSSTTSSTTSSTTDDDDK(SEQ ID NO: 52), SDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 11), SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR(SEQ ID NO: 12) , SDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 13), SDSTTSSTTSSTTSSTTSSTT(SEQ ID NO: 14), SSTTSDSTTRSDSTTSSTTRSDSTTR(SEQ ID NO: 15), and SDSTTSDSTTRSDSTTSDSTTSDSTTR(SEQ ID NO: 16). In one embodiment, one or more of the aforementioned Tag amino acid sequences further comprise one or more additional amino acids.

[67] In some embodiments, 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), KRSDSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 62), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 63), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 64), or any combination of any of the foregoing. In some instances, 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), KRSDSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 62), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 63), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 64). In some embodiments, one or more of the aforementioned Tag amino acids further comprise one or more additional amino acids.

[68] In some embodiments, 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: 74), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 75), KR, or any combination of any of the foregoing. In some embodiments, 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), KRSDSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 73), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 74), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 75), KR, and any combination of any of the foregoing. In some embodiments, one or more of the aforementioned Tag amino acids further comprise one or more additional amino acids.

[69] In some embodiments, 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).

[70] In some embodiments, 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.

[71] In some instances, 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.

[72] In some embodimetns, one or more of the Tag amino acid sequence include one or more naturally occurring solubility enhancing partner tags. In some embodiments, 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.

[73] In one embodiment, the naturally occurring solubility enhancing partner tag has one or more domains from Thyrodoxin, SUMO, Fh8, GB1, CaBP, MsyB, and Skp.

[74] In another embodiment, 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), IHLTDDSFDTDVLKADGAILV (SEQ ID NO: 34) (a domain from Thyrodoxin), IKAFIKEHDKNKDGKLDLKEL(SEQ ID NO: 35), KEHDKNKDGKLDLKELVSILS (SEQ ID NO: 36), IKAFIKEHDKNKDGKLDLKELVSILSS(SEQ ID NO: 37) (a domain from Fh8), and any combination of any of the foregoing. These domains observed to improve solubility of the multimer.

[75] In some embodiments, 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 In some embodiments, one or more of Tag amino acid sequences further comprise one or more additional amino acids. In some embodiments, 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. In some embodiments, one or more of Tag amino acid sequences further comprise one or more additional amino acids.

[76] In some embodiments, every Tag amino acid sequence in the multimer is the same.

[77] In some embodiments, the multimer comprises two or more different Tag amino acid sequence.

[78] In some embodiments, the N-terminus of the multimer includes a methionine and histidine tag. For instance, the N-terminus of the multimer include but not limited to methioninepoly histidine (such as -MHHHHHH (SEQ ID NO: 76)).

[79] In some embodiments, 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.

[80] In some embodiments, the poly histidine tag can be within the multimer, i.e. not at the N or C terminus.

[81] In some embodiments, 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.

[82] In some embodiments, one or more occurrences of MO comprise one or more naturally occurring solubility enhancing partner tags with or without CS as described herein.

[83] In some embodiments, the affinity tag or variable A comprises a tag to facilitate purification of the multimer . In some embodiments, the variable A affinity tag comprise a histidine tag (e.g., poly histidine).

[84] In some embodiments, each occurrence of CS is independently KR or any other proteolytic / chemical cleavage site. In In some embodiments, each occurrence of CS is KR.

[85] In some embodiments, 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).

[86] In some embodiments, the multimer has a pl from about 4 to about 12.

[87] In some embodiments, the multimer has a hydrophobicity of from about 20 to about 50%.

[88] In In some embodiments, the multimer has a hydrophobicity range of from about 24 to about 42%.

[89] In some embodiments, the multimer has a molecular weight more than 2 kilodalton.

[90] In some embodiments, the target peptide (TP) sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 1).

[91] In some embodiments, the target peptide sequence can be but not limited to 1029 amino acids.

[92] In some embodiments, the target peptide sequence is but not limited to EGTFTSDVSSYLEGQ AAKEFIAWLVRGRG, GTFTSDVSSYLEGQAAKEFIAWLVRGRG, FTSD VS SYLEGQ AAKEFIAWLVRGRG, TSD VS SYLEGQ AAKEFIAWLVRGRG, SDVSSYLEGQAAKEFIAWLVRGRG, DVSSYLEGQAAKEFIAWLVRGRG, VS SYLEGQAAKEFIAWLVRGRG, S SYLEGQAAKEFIAWLVRGRG, SYLEGQAAKEFIAWLVRGRG, YLEGQAAKEFIAWLVRGRG, LEGQAAKEFIAWLVRGRG, EGQAAKEFIAWLVRGRG, GQAAKEFIAWLVRGRG, QAAKEFIAWLVRGRG, AAKEFIAWLVRGRG, AKEFIAWLVRGRG, KEFIAWLVRGRG, EFIAWLVRGRG, and FIAWLVRGRG.

[93] In some embodiments, the target peptide sequence is HAEGTFTSDVS SYLEGQAAKEFIAWLVRGRG In some instances, the target peptide sequence is SEQ ID NO. 1.

[94] In some embodiments, 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.

[95] In some embodiments, 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.

[96] Yet another aspect is a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.

[97] Yet another aspect is a host cell transfected with a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.

[98] Yet another aspect is an expression vector comprising a DNA sequence encoding the multimer as described herein.

[99] Yet another aspect is a host cell comprising the expression vector described herein. The expression vector has any promoter. In one preferred embodiment, the promoter is T7.

[100] 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. In some embodiments, the method further comprises digesting the multimer with one or more enzymes to obtain a plurality of the target peptide sequences in free form.

[101] In some embodiments , yet another aspect is a method of preparing a peptide comprising: (a) obtaining a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without Tag amino acid sequences along with protease or chemical cleavage sites (CS). (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 be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, the N-terminus of the multimer includes a methionine and; with or without not limited to affinity tag (prefrably poly-histidine) tag. The multimer be isolated in a Nickel column through use of the histidine tag. In another embodiment, 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. In another embodiment, multimer can be digested with single protease. In another embodiment, 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.

[102] In one embodiment of any of the methods, the target peptide sequence comprises of one or more of the target peptide sequences.

[103] In some embodiments, a target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG is obtained by (a) 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). (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). In one embodiment, 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. In another embodiment, 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. In another embodiment, multimer can be digested with single protease. In another embodiment, 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.

[104] In some embodiments, the target peptide sequence is not limited to TFTSD VS S YLEGQ AAKEFIAWLVRGRG.

[105] In some embodiments, the target peptide sequence can be but not limited to 10- 29 amino acids.

[106] In some embodiments, the target peptide sequence is selected from EGTFTSDVSSYLEGQ AAKEFIAWLVRGRG, GTFTSDVSSYLEGQ AAKEFIAWLVRGRG, FTSDVS SYLEGQAAKEFIAWLVRGRG, TSD VS S YLEGQ AAKEFIAWLVRGRG, SDVSSYLEGQAAKEFIAWLVRGRG, DVSSYLEGQAAKEFIAWLVRGRG, VS SYLEGQAAKEFIAWLVRGRG, S SYLEGQAAKEFIAWLVRGRG, SYLEGQAAKEFIAWLVRGRG, YLEGQAAKEFIAWLVRGRG, LEGQAAKEFIAWLVRGRG, EGQAAKEFIAWLVRGRG, GQAAKEFIAWLVRGRG, QAAKEFIAWLVRGRG, AAKEFIAWLVRGRG, AKEFIAWLVRGRG, KEFIAWLVRGRG, EFIAWLVRGRG, and FIAWLVRGRG. BRIEF DESCRIPTION OF THE FIGURES

[107] 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).

[108] 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; and Lane 5: Eluate of multimeric target peptide.

[109] 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 and the red peak indicates the monomer of target peptide (SEQ ID NO:1) after CpB digestion.

[110] 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)

[111] 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.

[112] 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)

[113] 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)

[114] 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

[115] 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 3MUrea. Lane 3: Sample dissolved in 4MUrea.

[116] 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)

[117] 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 DEFINITIONS

[118] The term "about" as used herein indicates values that deviate up to 1%, more specifically 5%, more specifically 10%, more specifically 15%, and in some cases up to 20% higher or lower than the value referred to, the deviation range including integer values, and, if applicable, non-integer values as well, constituting a continuous range. Disclosed and described, it is to be understood that this invention is not limited to the specific examples, methods steps, and compositions disclosed herein as such methods steps and compositions vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing specific embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

[119] It must be noted that, as used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise.

[120] 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.

[121] The term “target peptide sequence” and “peptide of interest” are interchangeable.

[122] 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.

[123] 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.

[124] 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). In one embodiment, 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.

[125] Throughout this specification and the examples and claims which follow, unless the context requires otherwise, the word “comprise”, and variations such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. The term comprising will also be understood to include “consisting of’ and “consisting essentially of.”

[126] In some embodiments, 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.

[127] 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.

[128] One of the major challenges for improving the efficiency of production of recombinant peptide or protein is the solubility of the inclusion body which requires strong chaotropic agents such as guanidine HC1. To mitigate this insolubility issue some of the characteristics of the multimeric target peptide sequence such as pl and / or hydrophobicity are / is modified using intervening specific amino acid tags which aides in higher solubility and expression of the multimeric peptide. This invention provides a technology platform that enables small target peptide sequences having more than 3 amino acids in length to be produced in the form of multimers in microbial cells such as E. coli or yeast.

[129] In some embodiments, 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. In some instances, 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. In some instances, the Tag amino acid sequences comprises aspartic acid and / or glutamic acid to reduce the pl of the multimer. In some instances, the Tag amino acid sequences comprise lysine and / or arginine to can increase the pl of the multimer. In some instances, the Tag amino acid sequences comprise serine (S). threonine (T), asparagine (N), glutamine (Q) and other hydrophilic amino acids to render the multimer more hydrophilic. In some instances, 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. In some embodiments, the multimers comprise a target peptide sequence to achieve high level expression of the peptide of interest.

[130] In one embodiment, 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).

[131] In some embodiments, the amino acid at the N-terminus of the multimer is methionine and optionally a histidine tag. In some instances, the amino acid sequence at the N-terminus of the multimer is M(H)P, where p is 1 to 8 (such as MHHHHHH).

[132] In some embodiments, 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. In some instances, 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. In some instances, 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. In some instances, 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. In some instances, 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.

[133] In one embodiment of the multimer described herein, 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.

[134] In one embodiment of the multimer described herein, 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.

[135] In some embodiments, one or more of the separator sequences includes only proteolytic and / or chemical cleavage sites.

[136] In some embodiments, one or more of the cleavage sites comprise the amino acid sequence KR. In some instances, each cleavage site sequence is KR.

[137] In some embodiments, 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(SEQIDNO: 11) , SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR (SEQ ID NO: 12) , SDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 13), SDSTTSSTTSSTTSSTTSSTT(SEQ ID NO: 14), SSTTSDSTTRSDSTTSSTTRSDSTTR(SEQ ID NO: 15), SDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 16) and any combination of any of the foregoing. In some embodiments, one or more of the Tag amino acid sequences further comprise one or more additional amino acids. In some embodiments, each of the Tag amino acid sequences are selected from 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), and SSTTSSTTSSTTSSTTSSTTDDDDK(SEQ ID NO: 52), SDSTTRSDSTTSDSTTRSDSTTSDSTTR (SEQ ID NO: 11), SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR(SEQ ID NO: 12) , SDSTTSDSTTRSSTTSSTTRSSTTR (SEQ ID NO: 13), SDSTTSSTTSSTTSSTTSSTT(SEQ ID NO: 14), SSTTSDSTTRSDSTTSSTTRSDSTTR(SEQ ID NO: 15), and SDSTTSDSTTRSDSTTSDSTTSDSTTR(SEQ ID NO: 16). In one embodiment, one or more of the aforementioned Tag amino acid sequences further comprise one or more additional amino acids.

[138] In some embodiments, 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), KRSDSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 62), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 63), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 64), or any combination of any of the foregoing. In some instances, 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), KRSDSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 62), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 63), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 64). In some embodiments, one or more of the aforementioned Tag amino acids further comprise one or more additional amino acids.

[139] In some embodiments, 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: 74), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 75), KR, or any combination of any of the foregoing. In some embodiments, 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), KRSDSTTSSTTSSTTSSTTSSTT (SEQ ID NO: 73), KRSSTTSDSTTRSDSTTSSTTRSDSTTR (SEQ ID NO: 74), KRSDSTTSDSTTRSDSTTSDSTTSDSTTR (SEQ ID NO: 75), KR, and any combination of any of the foregoing. In some embodiments, one or more of the aforementioned Tag amino acids further comprise one or more additional amino acids.

[140] In some embodiments, 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).

[141] In some embodiments, 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.

[142] In some instances, 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.

[143] In some embodimetns, one or more of the Tag amino acid sequence include one or more naturally occurring solubility enhancing partner tags. In some embodiments, 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.

[144] In one embodiment, the naturally occurring solubility enhancing partner tag has one or more domains from Thyrodoxin, SUMO, Fh8, GB1, CaBP, MsyB, and Skp.

[145] In another embodiment, 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), IHLTDDSFDTDVLKADGAILV (SEQ ID NO: 34) (a domain from Thyrodoxin), IKAFIKEHDKNKDGKLDLKEL(SEQ ID NO: 35), KEHDKNKDGKLDLKELVSILS (SEQ ID NO: 36), IKAFIKEHDKNKDGKLDLKELVSILSS(SEQ ID NO: 37) (a domain from Fh8), and any combination of any of the foregoing. These domains observed to improve solubility of the multimer.

[146] In some embodiments, 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 In some embodiments, one or more of Tag amino acid sequences further comprise one or more additional amino acids. In some embodiments, 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. In some embodiments, one or more of Tag amino acid sequences further comprise one or more additional amino acids.

[147] In some embodiments, every Tag amino acid sequence in the multimer is the same.

[148] In some embodiments, the multimer comprises two or more different Tag amino acid sequence.

[149] In some embodiments, the N-terminus of the multimer includes a methionine and histidine tag. For instance, the N-terminus of the multimer include but not limited to methioninepoly histidine (such as -MHHHHHH (SEQ ID NO: 76)).

[150] In some embodiments, 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.

[151] In some embodiments, the poly histidine tag can be within the multimer, i.e. not at the N or C terminus.

[152] In some embodiments, 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.

[153] In some embodiments One aspect of the invention is a multimer comprising a plurality of target peptide sequences connected in series, wherein (i) (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, (ii) The choice of the CS should be such that upon cleavage by one or more proteases or chemicals shall leave no additional amino acids at either the N or the C terminus of the TP. (iii) The choice of the CS between the TP sequences shall be selected such that similar sites are not present within the TP

[154] In some embodiments, 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.

[155] In some embodiments, 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.

[156] In some embodiments, 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. 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.

[157] In some embodiments, one or more of the separator sequences includes only proteolytic and / or chemical cleavage sites.

[158] In some embodiments, one or more of the Tag amino acid sequences are KR. In some embodiments, each amino acid tag sequence is KR.

[159] In some embodiments, one or more of the Tag amino acid sequences comprise one or more naturally occurring solubility enhancing fusion partner tags as described herein.

[160] In some instances, the variable A comprises a tag to facilitate purification of the multimer. In some instances, the variable A comprise a histidine tag (e.g., poly histidine).

[161] In some embodiments, at least for one monomer Mn or MO, Tag is absent and r is 0, in which case MO will be TP-CS. In some embodiments, each occurrence of Tag is absent and each occurrence of r is 0.

[162] In some embodiments, the multimer has a pl from about 4 to about 12.

[163] In some embodiments, the multimer has a molecular weight more than 2 kilodalton.

[164] In some embodiments, the target peptide sequence is TFTSD VS SYLEGQ AAKEFIAWLVRGRG.

[165] In one embodiment of the multimer described herein, the target peptide sequence can be but not limited to 10- 29 amino acids.

[166] In one embodiment of the multimer described herein, the target peptide sequence is HAEGTFTSD VS SYLEGQ AAKEFIAWLVRGRG.

[167] Yet another aspect is a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.

[168] Yet another aspect is a host cell transfected with a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.

[169] Yet another aspect is an expression vector comprising a DNA sequence encoding the multimer as described herein.

[170] Yet another aspect is a host cell comprising the expression vector described herein. The expression vector has any promoter. In one preferred embodiment, the promoter is T7.

[171] 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.

[172] In some embodiments, each occurrence of CS is independently KR or any other proteolytic / chemical cleavage site. In In some embodiments, each occurrence of CS is KR.

[173] In some embodiments, each occurrence of CS is independently DDDDK or any other proteolytic / chemical cleavage site. In some embodiments, each occurrence of CS is DDDDK (SEQ ID NO: 53).

[174] In In some embodiments, the multimer has a hydrophobicity range of from about 24 to about 42%.

[175] In some embodiments, the multimer has a molecular weight more than 2 kilodalton.

[176] In some embodiments, the target peptide (TP) sequence is TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 1).

[177] In some embodiments, the target peptide sequence can be but not limited to 1029 amino acids.

[178] In some embodiments, 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: 88), EGQAAKEFIAWLVRGRG (SEQ ID NO: 89), GQAAKEFIAWLVRGRG (SEQ ID NO: 90), QAAKEFIAWLVRGRG (SEQ ID NO: 91), AAKEFIAWLVRGRG (SEQ ID NO: 92), AKEFIAWLVRGRG (SEQ ID NO: 93), KEFIAWLVRGRG (SEQ ID NO: 94), EFIAWLVRGRG (SEQ ID NO: 95), and FIAWLVRGRG (SEQ ID NO: 96).

[179] In some embodiments, the target peptide sequence is HAEGTFTSD VS SYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 97). In some instances, the target peptide sequence is SEQ ID NO. 1.

[180] In some embodiments, 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.

[181] In some embodiments, 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.

[182] Yet another aspect is a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.

[183] Yet another aspect is a host cell transfected with a DNA construct comprising a nucleic acid sequence encoding the multimer as described herein.

[184] Yet another aspect is an expression vector comprising a DNA sequence encoding the multimer as described herein.

[185] Yet another aspect is a host cell comprising the expression vector described herein. The expression vector has any promoter. In one preferred embodiment, the promoter is T7.

[186] 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. In some embodiments, the method further comprises digesting the multimer with one or more enzymes to obtain a plurality of the target peptide sequences in free form.

[187] Yet another aspect is a method of preparing a peptide comprising: (a) obtaining a multimer described herein, where the peptide in the multimer has the sequence of the target peptide sequence and preferably with or without amino acid tags along with protease or chemical cleaving site (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 be transfected with an expression vector for expressing the multimer (such as that described herein). In one embodiment, 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. In another embodiment, 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. In another embodiment, multimer can be digested with single protease. In another embodiment, 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.

[188] In one embodiment of any of the methods, the recombinant peptide of interest described herein could comprise of one or more of the peptides.

[189] In some embodiments, the target peptide sequence TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 1) obtained by (a) a multimer described herein, where the target peptide sequence in the multimer and preferably with or without Tag amino acid sequences along with protease or chemical cleaving site (CS). (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). In one embodiment, 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. In another embodiment, 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. In another embodiment, multimer can be digested with single protease. In another embodiment, 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.

[190] In some embodiments, the target peptide sequence is not limited to TFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 1).

[191] In some embodiments, the target peptide sequence can be but not limited to 10- 29 amino acids.

[192] In some embodiments, 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 (SEQ ID NO: 87), LEGQAAKEFIAWLVRGRG (SEQ ID NO: 88), EGQAAKEFIAWLVRGRG (SEQ ID NO: 89), GQAAKEFIAWLVRGRG (SEQ ID NO: 90), QAAKEFIAWLVRGRG (SEQ ID NO: 91), AAKEFIAWLVRGRG (SEQ ID NO: 92), AKEFIAWLVRGRG (SEQ ID NO: 93), KEFIAWLVRGRG (SEQ ID NO: 94), EFIAWLVRGRG (SEQ ID NO: 95), and FIAWLVRGRG (SEQ ID NO: 96).

[193] In some embodiments in any of the methods described herein, wherein the TP sequence is conjugated to His-Aib-Glu-Gly. In some embodiments in the any of the methods described herein, wherein the 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. In some embodiments in any of the methods described herein, wherein 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

[194] The sequences referenced by sequence number herein are provided in Table A at the end of the specification.

[195] In order that the invention may be readily understood and put into practical effect, particular embodiments will now be described by way of the following nonlimiting examples. EXAMPLES EXAMPLE 1 PREPARATION OF A MULTIMER OF TARGET PEPTIDE

[196] To produce the target peptide (SEQ ID NO:1), 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.

[197] 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. For example, these amino acid residues could be lysine (K) and arginine (R) which are cleavage site for the proteases Kex2 and CpB. 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.

[198] To express the multimer (SEQ ID NO:2) of the target peptide (SEQ ID NO: 1) in E. coli, the corresponding nucleotide sequence was codon optimized and synthesized yielding SEQ ID NO: 3. The nucleotide sequence (SEQ ID NO: 3) was cloned into a suitable expression vector with a T7 promoter and transformed in E. coli ToplO cells to generate expression vector.

[199] 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.

[200] 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.

[201] After purification of the multimer, 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. EXAMPLE 2 PREPARATION OF MULTIMER WITH IMPROVED SOLUBILITY

[202] 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 (MU) 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.

[203] 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. In order to increase the number of D (aspartic acid) and decrease the overall pl 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).

[204] To express the multimer (SEQ ID NO: 9) in E. coli, the corresponding nucleotide sequence was codon optimized and synthesized yielding SEQ ID NO: 10. The nucleotide sequence (SEQ ID NO: 10) was cloned into a suitable expression vector with a T7 promoter and transformed in E. coli ToplO cells to generate expression vector.

[205] 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.

[206] Inclusion bodies or cell pellet formed by the multimer (SEQ ID NO: 9) 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. EXAMPLE 3 PREPARATION OF MULTIMER WITH IMPROVED SOLUBILITY AND EXPRESSION

[207] 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.

[208] To express the multimer (SEQ ID NO: 17) of the target peptide in E. coli, 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.

[209] 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. EXAMPLE 4 PREPARATION OF MULTIMER WITH IMPROVED SOLUBILITY AND EXPRESSION USING NATURALLY AVAILABLE SOLUBILITY TAG DOMAINS

[210] Specific sequences (i.e., solubility domains) within naturally occurring solubility partners were explored to modulate the pl and hydrophobicity of the multimer to enhance its expression and solubility. 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. To further improve the solubility of the multimer where it could be eluted in less than 4M urea, a multimer (SEQ ID NO: 30) was design incorporating domains from the naturally occurring solubility enhancing molecule MsyB. Using solubility enhancing domains from 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).

[211] To express the multimer (SEQ ID NO: 30) of the target peptide in E. coli, the corresponding nucleotide sequence was codon optimized and synthesized yielding (SEQ ID NO: 31). The nucleotide sequence (SEQ ID NO: 31) was cloned into a suitable expression vector with a T7 promoter and transformed in E. coli ToplO cells to generate an expression vector.

[212] 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).

[213] The inclusion bodies or cell pellet obtained after the flask study of the multimer (SEQ ID NO: 30) was readily soluble in urea buffer. The samples were purified using Ni-NTA column where eluates were in 2M urea buffer (mild solubilizing agent), as shown in Figure 11. These modulations of the multimer using the solubility domains of naturally occurring fusion tags improved the expression at lower pl. Additionally, they enhanced the solubility of the multimer in 2M urea which would enable ease of enzymatic digestion and further downstream processing. Table A Description SEQ ID NO: Sequence Target Peptide in Example 1 1 TFTSDVS S YLEGQ A A KEFIA WLVRGRG Multimer of Target Peptide in Example 1 2 MHHHHHHKRTFTSD VS S YLEGQ A AI< EFIA WLVRGRGK R TFTSDVSSYLEGQAAKEFIAWLVRGRGKRTFTSDVSSYLE GQAAKEFIAWLVRGRGKRTFTSDVSSYLEGQAAKEFIAW L VRGRGKRTFTSD VS S YLEGQ AAKEFIAWL VRGRGKRTF TSDVS S YLEGQ AAKEFIAWLVRGRGKRTFTSDVS SYLEG QAAKEFI AWLVRGRGKRTFTSD VS S YLEGQ AAKEFIAWL VRGRGKRTFTSDVS S YLEGQ AAKEFIAWLVRGRGKRTFT SDVSS YLEGQ AAKEFIAWL VRGRGKR Nucleotide sequence for Multimer of Example 1 3 ATGCATCATCACCATCATCATAAACGCACCTTTACCTC AGATGTTTCAAGCTATCTGGAAGGTCAGGCAGCCAAA GAATTTATTGCATGGCTGGTTCGTGGTCGTGGTAAACG TACATTTACAAGTGATGTTAGCAGTTACCTGGAAGGCC AAGCCGCAAAAGAGTTTATCGCCTGGTTAGTGCGTGGC CGTGGCAAAAGAACCTTTACCAGCGACGTTAGCAGCT ATTTAGAAGGACAAGCTGCGAAAGAATTTATAGCGTG GTTGGTACGCGGACGCGGTAAGCGTACCTTCACATCTG ATGTTAGTTCATATTTAGAGGGCCAAGCGGCAAAAGA ATTTATTGCGTGGCTTGTGAGAGGCCGTGGAAAACGCA CGTTCACCAGTGATGTGTCATCATATCTTGAGGGTCAA GCAGCGAAAGAGTTCATAGCCTGGCTTGTCCGTGGTAG AGGCAAACGGACGTTTACATCCGATGTGAGTAGTTACT TGGAGGGACAAGCAGCTAAAGAATTTATCGCATGGTT AGTTAGAGGTCGCGGTAAAAGAACATTCACGTCAGAT GTAAGCTCTTATCTTGAAGGCCAGGCTGCAAAAGAATT TATAGCCTGGCTGGTGCGTGGACGGGGAAAGCGCACT TTTACATCAGACGTTAGCTCGTACTTAGAAGGGCAAGC AGCAAAAGAATTTATCGCTTGGCTGGTAAGAGGAAGA GGAAAGCGGACCTTTACGAGTGACGTGAGTTCATACCT TGAAGGACAGGCAGCGAAAGAGTTTATTGCTTGGTTA GTTCGCGGTAGAGGAAAACGAACCTTCACTTCGGATGT GAGCAGCTACTTAGAAGGTCAGGCAGCGAAAGAATTT ATCGCGTGGCTTGTCCGTGGTCGCGGTAAACGTTAATA A Tag + CS in Example 2 to lower pl and / or hydrophobicity of multimer, where Tag is DSSTTDSSTT and CS is DDDDK 4 D S S TTD S STTDDDDK Tag + CS in Example 2 to lower pl and / or hydrophobicity of multimer where Tag is DSSDTTD and CS is DDDDK 5 DSSDTTDDDDDK Tag + CS in Example 2 to lower pl and / or hydrophobicity of multimer where Tag is DSSTTDSSTTD and CS is DDDDK 6 D S S TTD S STTDDDDDK Tag in Example 2 to lower pl and / or 7 DSSTTDSSTTSSTT hydrophobicity of multimer Tag + CS in Example 2 to lower pl and / or hydrophobicity of multimer where Tag is SSTT and CS is DDDDK 8 SSTTDDDDK Multimer of Target Peptide in Example 2 9 MHHHHHHKRDSSTTDSSTTDDDDKTFTSDVSSYLEGQA AKEFIAWLVRGRGKRDS S TTDS STTDDDDDKTFTSD VS S YLEGQ AAKEFIAWLVRGRGKRDSSDTTDDDDDKTFTSD VSSYLEGQAAKEFIAWLVRGRGKRDSSDTTDDDDDKTFT SDVSSYLEGQAAKEFIAWLVRGRGKRDSSDTTDDDDDK TFTSD VS S YLEGQ AAKEFIAWLVRGRGKRSS TTDDDDKT FTSDVSSYLEGQAAKEFIAWLVRGRGKRDSSDTTDDDDD KTFTSD VS S YLEGQ AAKEFIAWLVRGRGKRD SSDTTDDD DDKTFTSDVS S YLEGQ AAKEFIAWLVRGRGKRDS SDTTD DDDDKTFTSD VS S YLEGQ AAKEFIAWLVRGRGKRD S SDT TDDDDDKTFTSDVS SYLEGQ AAKEFIAWLVRGRGKRDS S TTDSSTTSSTT Nucleotide sequence for Multimer of Example 2 10 ATGCATCATCACCATCATCATAAACGTGATAGCAGCAC CACCGATAGCTCAACCACCGATGATGATGATAAAACC TTTACCAGTGATGTGAGCAGCTATCTGGAAGGTCAGGC AGCAAAAGAATTTATTGCATGGCTGGTTCGTGGTCGTG GTAAACGCGATTCAAGTACCACCGATTCTAGCACAAC GGATGATGACGATGACAAAACATTCACCTCAGATGTT AGCTCATACCTGGAAGGCCAAGCTGCCAAAGAGTTTA TCGCCTGGTTAGTGCGTGGCCGTGGCAAACGTGACAGC TCAGATACGACCGATGACGACGACGATAAGACGTTTA CATCTGATGTTTCCAGCTATTTAGAAGGGCAAGCTGCG AAAGAATTTATAGCGTGGCTTGTACGCGGACGCGGAA AACGTGATTCTAGCGATACTACCGACGACGATGATGA CAAGACTTTTACGTCCGATGTTAGTAGTTATCTTGAAG GACAAGCAGCCAAAGAATTTATTGCGTGGTTAGTCAG AGGTCGGGGTAAGCGTGATAGTAGCGATACGACAGAT GATGATGACGACAAGACCTTCACCAGCGACGTTTCAA GTTACTTAGAGGGTCAAGCCGCTAAAGAGTTCATTGCT TGGTTGGTCCGTGGAAGAGGTAAACGTAGCAGTACCA CAGACGATGACGACAAAACATTTACAAGTGACGTTTCT AGTTATTTGGAAGGCCAAGCCGCAAAAGAATTTATCG CTTGGTTAGTTCGCGGAAGAGGCAAGCGTGATTCAAGT GATACCACTGATGATGACGACGATAAAACTTTTACGA GCGACGTTAGTTCATATTTGGAGGGTCAAGCAGCTAAA GAATTTATCGCCTGGCTGGTAAGAGGCAGAGGTAAGC GCGACTCATCAGACACCACAGATGACGATGATGATAA GACATTCACATCAGACGTGTCCTCATACTTAGAAGGCC AGGCAGCCAAAGAATTTATCGCGTGGCTTGTGCGTGGT AGAGGCAAACGCGATAGTTCTGACACTACCGACGATG ACGACGACAAAACCTTCACAAGTGATGTTAGTTCTTAC TTGGAAGGACAGGCTGCTAAAGAGTTCATAGCCTGGC TGGTTCGCGGACGTGGGAAGCGTGATAGTTCTGATACC ACGGACGACGATGACGATAAAACCTTCACTTCTGATGT GTCAAGTTACCTTGAAGGTCAAGCGGCAAAAGAGTTC ATTGCCTGGCTTGTGCGTGGTCGCGGTAAGCGTGATTC ATCAACCACCGATAGCAGCACAACCAGCAGCACCACA TAA Tag in Example 3 to lower hydrophobicity and to maintain pl of multimer 11 SDSTTRSDSTTSDSTTRSDSTTSDSTTR Tag in Example 3 to lower hydrophobicity and to maintain pl of multimer 12 SDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR Tag in Example 3 to lower hydrophobicity and to maintain pl of multimer 13 SDSTTSDSTTRSSTTSSTTRSSTTR Tag in Example 3 to lower hydrophobicity and to maintain pl of multimer 14 SDSTTSSTTSSTTSSTTSSTT Tag in Example 3 to lower hydrophobicity and to maintain pl of multimer 15 SSTTSDSTTRSDSTTSSTTRSDSTTR Tag in Example 3 to lower hydrophobicity and to maintain pl of multimer 16 SDSTTSDSTTRSDSTTSDSTTSDSTTR Multimer of Target Peptide in Example 3 17 MHHHHHHKRSD S TTRSD S TTSD S TTRSD S TTSD STTRKRT FTSDVSSYLEGQAAKEFIAWLVRGRGKRSDSTTSSTTRSD STTSDSTTRSDSTTSSTTRKRTFTSDVSSYLEGQAAKEFIA WLVRGRGKRSD S TTSD STTRS STTS S TTRS STTRKRTFTSD VS SYLEGQ AAKEFIAWLVRGRGKRTFTSDVS SYLEGQAA KEFIAWLVRGRGKRTFTSD VS SYLEGQ AAKEFIAWLVRG RGKRTFTSD VS SYLEGQ AAKEFIAWLVRGRGKRTFTSDV SSYLEGQAAKEFIAWLVRGRGKRTFTSDVSSYLEGQAAK EFIAWLVRGRGKRSDSTTSSTTSSTTSSTTSSTTKRTFTSD VSSYLEGQAAKEFIAWLVRGRGKRSSTTSDSTTRSDSTTS STTRSDSTTRKRTFTSDVSSYLEGQAAKEFIAWLVRGRGK RSDSTTSDSTTRSDSTTSDSTTSDSTTR Nucleotide sequence for Multimer of Example 3 18 ATGCATCATCACCATCATCATAAACGTAGCGATAGCAC AACCCGTAGCGATTCAACCACCAGTGATAGTACCACA CGTTCAGATAGCACCACCTCAGATAGTACGACCCGTAA ACGTACCTTTACCAGTGATGTTAGCAGCTATCTGGAAG GTCAGGCAGCAAAAGAATTTATTGCATGGCTGGTTCGT GGTCGTGGTAAACGCAGTGATTCTACCACCTCAAGCAC CACACGCTCTGATAGTACAACCAGTGATTCAACAACCC GTTCAGATTCTACAACCAGCTCAACCACACGCAAACGC ACATTTACCTCAGATGTTAGTTCATACCTGGAAGGCCA AGCTGCCAAAGAGTTTATCGCCTGGTTAGTGCGTGGCC GTGGCAAACGTTCTGATTCAACGACCTCTGATTCCACC ACACGTAGCAGCACCACCAGTAGCACTACCCGTTCAA GTACAACGCGCAAGCGCACGTTTACAAGCGACGTTTC ATCATATTTAGAAGGGCAAGCAGCGAAAGAGTTCATA GCTTGGCTGGTACGCGGACGCGGAAAAAGAACCTTCA CATCAGATGTTTCATCCTATCTTGAGGGTCAAGCTGCG AAAGAATTTATAGCGTGGCTTGTGAGAGGTCGCGGAA AACGTACATTCACCAGCGACGTGTCAAGTTATCTTGAA GGACAAGCAGCCAAAGAGTTCATTGCCTGGCTTGTCA GAGGCCGTGGGAAACGCACTTTCACAAGTGATGTGTC CAGCTACTTAGAGGGCCAAGCCGCTAAAGAATTTATTG CTTGGTTAGTAAGAGGCCGTGGTAAGCGTACATTTACA TCCGATGTGAGTAGTTACTTGGAGGGACAAGCGGCAA AAGAGTTTATAGCCTGGCTTGTTCGCGGTAGAGGTAAA AGAACATTTACGTCTGATGTCTCAAGCTACCTTGAAGG CCAGGCTGCTAAAGAATTTATCGCGTGGTTGGTTCGCG GAAGAGGCAAACGCTCCGATAGCACGACCAGCAGCAC AACCTCATCAACCACAAGCTCAACAACCAGCAGTACC ACCAAACGGACGTTTACGTCAGACGTGTCCTCTTACTT AGAAGGCCAGGCAGCCAAAGAATTTATAGCCTGGTTA GTACGCGGTCGGGGAAAACGCAGTAGCACCACAAGCG ACAGTACTACCCGCAGCGACAGCACCACGAGCAGTAC AACACGTAGTGACTCTACAACTCGCAAACGGACCTTCA CGAGTGACGTGAGCAGTTATTTAGAAGGTCAAGCCGC AAAAGAATTTATCGCTTGGCTGGTAAGAGGTAGAGGT AAACGTTCTGATAGCACAACAAGCGATAGCACCACTC GCTCAGATTCTACCACATCTGATTCAACCACATCCGAT AGTACAACGCGGTAATAA Tag in Example 4 to lower hydrophobicity and pl of multimer 19 NPGIDAEDANLQDGDADEGEE Tag in Example 4 to lower hydrophobicity and pl of multimer 20 IDDQVEDKCLPQ Tag in Example 4 to lower hydrophobicity and pl of multimer 21 IDDQVEDKADEGE Tag in Example 4 to lower hydrophobicity and pl of multimer 22 EGECLPDL Tag in Example 4 to lower hydrophobicity and pl of multimer 23 LQDGDADEGEEGELSGDGDYD Tag in Example 4 to lower hydrophobicity and pl of multimer 24 EGELSGDGDYD Tag in Example 4 to lower hydrophobicity and pl of multimer 25 LEPPLDTD Tag in Example 4 to lower hydrophobicity and pl of multimer 26 LQDGDAD Tag in Example 4 to lower hydrophobicity and pl of multimer 27 QDGDADEGEE Tag in Example 4 to lower hydrophobicity and pl of multimer 28 NPGIDAEDANL Tag in Example 4 to lower hydrophobicity and pl of multimer 29 NPGIDAEDANLQDGDAD Multimer of Target Peptide in Example 4 30 MHHHHHHNPGIDAEDANLQDGDADEGEEKRTFTSDVSS YLEGQ AAKEFIAWLVRGRGKRIDDQVEDKCLPQKRTFTS DVSSYLEGQAAKEFIAWLVRGRGKRIDDQVEDKADEGE KRTFTSDVSSYLEGQAAKEFIAWLVRGRGKREGECLPDL KRTFTSDVSSYLEGQAAKEFIAWLVRGRGKRLQDGDAD EGEEGELS GDGDYDKRTFTSD VS S YLEGQ AAKEFIAWLV RGRGKREGELSGDGD YDKRTFTSDVS S YLEGQ AAKEFIA WLVRGRGKRLEPPLDTDKRTFTSDVSSYLEGQ AAKEFIA WLVRGRGKRLQDGDADKRTFTSDVSSYLEGQ AAKEFIA WLVRGRGKRQDGDADEGEEKRTFTSDVSSYLEGQAAKE FIAWLVRGRGKRNPGIDAEDANLKRTFTSDVSSYLEGQA AKEFIAWLVRGRGKRNPGIDAEDANLQDGDAD Nucleotide sequence for Multimer of Example 4 31 ATGCACCACCACCACCACCATAACCCGGGTATCGACG CTGAAGACGCTAATCTGCAAGATGGCGACGCAGACGA GGGTGAGGAAAAACGCACGTTTACCAGCGACGTGTCT TCCTATCTGGAAGGCCAGGCAGCGAAAGAGTTCATTG CTTGGCTGGTGCGTGGCCGCGGCAAACGTATTGATGAC CAGGTTGAAGACAAGTGTCTGCCGCAGAAACGTACCT TCACCAGCGATGTTTCTTCCTACCTGGAAGGTCAGGCT GCTAAAGAATTTATTGCATGGCTGGTTCGTGGCCGTGG TAAACGCATTGATGACCAAGTGGAGGATAAAGCAGAC GAAGGTGAAAAGCGTACGTTTACCTCTGACGTGTCTTC TTATCTGGAAGGCCAGGCAGCAAAAGAATTCATCGCTT GGCTGGTGCGTGGTCGTGGTAAGCGTGAGGGCGAGTG TCTGCCGGACCTGAAACGTACCTTCACCTCCGATGTCT CTTCCTACCTGGAAGGTCAAGCCGCGAAAGAGTTTATT GCTTGGCTGGTCCGTGGTCGTGGCAAACGTCTGCAAGA TGGTGACGCTGACGAGGGCGAGGAGGGTGAACTGTCC GGCGATGGCGATTACGACAAGCGTACCTTCACCTCCGA CGTGTCCTCCTACCTGGAGGGTCAGGCTGCAAAGGAGT TCATCGCTTGGCTGGTTCGTGGTCGTGGCAAACGCGAA GGTGAGCTGTCCGGCGACGGCGATTACGATAAACGTA CCTTTACCAGCGACGTGTCTAGCTACCTGGAAGGTCAG GCCGCTAAGGAATTCATCGCGTGGCTGGTTCGCGGCCG CGGTAAACGTCTGGAACCTCCGCTGGATACTGATAAAC GTACGTTCACTAGCGATGTGTCTAGCTATCTGGAAGGT CAGGCCGCAAAAGAATTCATTGCATGGCTGGTTCGCG GTCGTGGCAAGCGTCTGCAGGATGGTGACGCCGACAA ACGCACCTTTACCTCCGACGTTTCTTCTTACCTGGAAG GCCAGGCTGCAAAAGAATTCATCGCATGGCTGGTCCGT GGTCGCGGCAAACGCCAGGATGGTGACGCCGATGAAG GCGAAGAGAAGCGCACCTTCACCAGCGATGTGTCTTCC TACCTGGAAGGTCAGGCGGCGAAAGAATTCATTGCGT GGCTGGTGCGTGGTCGCGGCAAACGTAACCCGGGCAT CGATGCCGAGGACGCGAACCTGAAACGCACTTTTACTT CCGATGTCAGCTCCTATCTGGAAGGTCAGGCTGCGAAA GAATTCATCGCATGGCTGGTGCGTGGCCGTGGTAAACG CAACCCGGGTATCGACGCAGAGGATGCGAACCTGCAG GACGGTGACGCTGATTAATAA Naturally occurring solubility enhancing partner tag 32 SDKIIHLTDDSFDTDVLKADGAILVDF Naturally occurring solubility enhancing partner tag 33 MSDKIIHLTDDSFDTDVLKAD Naturally occurring solubility enhancing partner tag 34 IHLTDDSFDTDVLKADGAILV Naturally occurring solubility enhancing partner tag 35 IKAFIKEHDKNKDGKLDLKEL Naturally occurring solubility enhancing partner tag 36 KEHDKNKDGKLDLKELVSILS Naturally occurring solubility enhancing partner tag 37 IKAFIKEHDKNKDGKLDLKELVSILS S Naturally occurring solubility enhancing partner tag + CS , where Tag NPGIDAEDANLQD GDADEGEE and CS isKR 38 NPGIDAEDANLQDGDADEGEEKR CS + Naturally occurring solubility enhancing partner tag, where Tag 39 KRIDDQVEDKCLPQ IDDQVEDKCLPQ and CS is KR CS + Naturally occurring solubility enhancing partner tag, where Tag IDDQVEDKADEGE and CS is KR 40 KRIDDQVEDKADEGE CS + Naturally occurring solubility enhancing partner tag, where Tag EGECLPDL and CS isKR 41 KREGECLPDL CS + Naturally occurring solubility enhancing partner tag, where Tag LQDGDADEGEEGE LSGDGDYD and CS isKR 42 KRLQDGDADEGEEGELSGDGDYD CS + Naturally occurring solubility enhancing partner tag, where Tag EGELSGDGDYD and CS is KR 43 KREGELSGDGDYD CS + Naturally occurring solubility enhancing partner tag, where Tag EPPLDTD and CS is KR 44 KRLEPPLDTD CS + Naturally occurring solubility enhancing partner tag, where Tag LQDGDAD and CS isKR 45 KRLQDGDAD CS + Naturally occurring solubility enhancing partner tag, where Tag QDGDADEGEE and CS is KR 46 KRQDGDADEGEE CS + Naturally occurring solubility enhancing partner tag, where Tag NPGIDAEDANL and CS is KR 47 KRNPGIDAEDANL CS + Naturally occurring solubility enhancing partner tag, where Tag 48 KRNPGIDAEDANLQDGDAD NPGIDAEDANLQD GDAD and CS is KR Amino Acid Tag Sequence + CS , where tag SSTTRSSTTSSTTRS STTSSTTRandCS is DDDDK 49 S S TTRS STTS S TTRS STTS STTRDDDDK Amino Acid Tag Sequence + CS , where tag SSTTSSTTRSSTTSS TTRSSTTSSTTRand CS is DDDDK 50 S S TTS STTRS S TTS STTRS STTS STTRDDDDK Amino Acid Tag Sequence + CS , where tag SSTTSSTTRSSTTSS TTRSSTTR and CS is DDDDK 51 S S TTS STTRS S TTS STTRS STTRDDDDK Amino Acid Tag Sequence + CS , where tag SSTTSSTTSSTTSST TSSTT and CS is DDDDK 52 SSTTSSTTSSTTSSTTSSTTDDDDK Cleavage Site 53 DDDDK Amino acid tag sequence 54 KRDS STTDS STTDDDDDK Amino acid tag sequence 55 KRDSSDTTDDDDDK Amino acid tag sequence 56 KRSSTTDDDDK Amino acid tag sequence 57 KRDSSTTDSSTTSSTT Amino acid tag sequence 58 KRDSSTTDSSTTDDDDK Amino acid tag sequence 59 KRSDSTTRSDSTTSDSTTRSDSTTSDSTTR Amino acid tag sequence 60 KRSDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR Amino acid tag sequence 61 KRSDSTTSDSTTRSSTTSSTTRSSTTR Amino acid tag sequence 62 KRSDSTTSSTTSSTTSSTTSSTT Amino acid tag sequence 63 KRSSTTSDSTTRSDSTTSSTTRSDSTTR Amino acid tag sequence 64 KRSDSTTSDSTTRSDSTTSDSTTSDSTTR Amino acid tag sequence 65 KRSSTTRSSTTSSTTRSSTTSSTTRDDDDK Amino acid tag sequence 66 KRSSTTSSTTRSSTTSSTTRSSTTSSTTRDDDDK Amino acid tag sequence 67 KRSSTTSSTTRSSTTSSTTRSSTTRDDDDK Amino acid tag sequence 68 KRSSTTSSTTSSTTSSTTSSTTDDDDK Amino acid tag sequence 69 KRSSTTSSTTRSSTTSSTTSSTTR Amino acid tag sequence 70 KRSDSTTRSDSTTSDSTTRSDSTTSDSTTR Amino acid tag sequence 71 KRSDSTTSSTTRSDSTTSDSTTRSDSTTSSTTR Amino acid tag sequence 72 KRSDSTTSDSTTRSSTTSSTTRSSTTR Amino acid tag sequence 73 KRSDSTTSSTTSSTTSSTTSSTT Amino acid tag sequence 74 KRSSTTSDSTTRSDSTTSSTTRSDSTTR Amino acid tag sequence 75 KRSDSTTSDSTTRSDSTTSDSTTSDSTTR Methionine-Poly Histidine tag 76 MHHHHHH

[214] All publications, patents and patent applications cited herein are hereby incorporated by reference as if set forth in their entirety herein. While this invention has been described with reference to illustrative embodiments, this description is not intended to be construed in a limiting sense. Various modifications and combinations of illustrative embodiments, 5 as well as other embodiments of the invention, will be apparent to persons skilled in the art upon reference to the description. It is therefore intended that the appended claims encompass such modifications and enhancements.

Claims

1. High expression of the target peptide (TP) by constructing multiple repeats of the TP along with flanking amino acids that constitute a cleavage site (CS) which can be cleaved by one or more chemicals / proteases, and is referred to as Monomer (MO).i. Each MO has the formula -MO = (CS-TP-CS) and / orMO = (Tag-CS-TP-CS) and / orMO = (CS-TP-CS-Tag),where Tag is a sequence of amino acids to modulate pl and / orhydrophobicity of MO and MU.Multiple units of MO are arranged in tandem to make a Multimer (MU), which is represented as MU = (MO)n, the plurality of MO is defined by n which is an integer ranging from 2-50, preferably 2-20 and more precisely 10.ii. If TP does not have methionine at the N-terminus, the MU will have Met at the N-terminus, represented as MU = Met - (MO)n,iii. To modulate the hydrophobicity and / or pl of the MO and / or the MU, additional amino acids are added, referred as a Tag,iv. The Tag is not positioned between TP and CS,v. The number of Tag moieties to be included will depend on the degree of change in the pl and / or hydrophobicity desired for the MO and / or MU,vi. The choice of amino acids used in the Tags shall be based on whether the expression of the MU is desired in the soluble form or as insoluble inclusion bodies,vii. The choice of the CS between the TP sequences shall be selected such that similar sites are not present within the TP.viii. The choice of the CS should be such that upon cleavage, by chemicals or one or more proteases, shall leave no additional amino acids at either the N or the C terminus of the TP.

2. A Multimer (MU) comprising a plurality of target peptide (TP) sequences connected in series, wherein -i. Every two adjacent TP sequences are flanked on the N and C terminus by a cleavage site (CS), which is capable of being cleaved by one or more chemicals or proteases to form the TP without leaving any additional amino acids at either the N or the C terminus of the TP.ii. The amino acid sequence that make up the CS site shall not be present within the TP,iii. Additional amino acid sequence (referred as Tag) may or may not be present, to modulate the pl and / or hydrophobicity of the MO and / or MU.iv.   The number of Tag moieties to be included will depend on the degree of change desiredin the pl and / or hydrophobicity of the MO and MU,v. The Tag moieties will not be positioned between TP and CS sites,vi. The first TP sequence at the N-terminal of MU is preceded by methionine in the absence of a methionine at the N-terminal of TP,vii. Additional amino acid sequence (such as poly-histidine) to facilitate purification of the MU, may or may not be added at the N and / or C-terminus of the MU.

3. The MU in any of the preceding claims, wherein one or more of the Tag amino acid sequencescomprise one or more aspartic acid (D), lysine (K) and arginine (R) residues.

4. Tag sequence in any of the preceding claims comprises of one or more of aspartic acid (D), lysine (K), arginine (R), glutamic acid (E), and histidine (H) amino acid in any order to modulate the pl of the MO and / or MU.

5. Tag sequence in any of the preceding claims comprises of one or more serine (S), threonine (T), glutamine (Q), leucine (L) and isoleucine (I), alanine (A), valine (V), proline (P), glycine (G), tryptophan (W), phenylalanine (F), tyrosine (Y), cysteine (C), and methionine (M) residues to modulate the hydrophobicity of the MO and / or MU.

6. The MU in any one of the preceding claims, wherein one or more of the CS comprise of the amino acid KR wherein the R of the KR sequence is directly bound to the N-terminus of the TP while the K of the KR sequence is directly bound to the C-terminus of one of the preceding TP. The MU in any of the preceding claims, wherein one or more of the CS comprise of the amino acid DDDDK (SEQ ID NO: 53) where the K of the DDDDK sequence is directly bound to the N-terminus of the TP. The MU in any of the preceding claims, wherein the multimer includes one or more Tag amino acid sequences to alter the hydrophobicity, pl, or both of the multimer or a cleaved product of the multimer.

7. The MU in any of the preceding claims has a pl ranging from about 3 to 12, depending on the desired pl of the MU.

8. The MU in any of the preceding claims has a hydrophobicity ranging from 20% to 50% depending on the desired hydrophobicity of the MU9. The MU in any of the preceding claims, wherein each Tag amino acid sequence between two target peptides is the same or different.

10. The MU in any of the preceding claims, wherein the multimer includes 2 to 50 copies of a target peptide sequence.

11. The MU in any of the preceding claims include 2 to 20 copies of the TP, more specifically 10 copies of the TP sequence.

12. The MU in any of the preceding claims, wherein the MU has a molecular weight of from about 5 to about 200 kilodaltons, more precisely from about 30 to about 60 kilodaltons.

13. The MU in any one of the preceding claims, wherein one or more of the Tag amino acid sequences may comprise of solubility enhancing sequence that are naturally occurring or modified solubility enhancing tags such as thyrodoxin, SUMO, Fh8, GB1, CaBP, MsyB, or Skp.

14. The MU of any one of the preceding claims, wherein the solubility enhancing domain is selected fromNPGIDAEDANLQDGDADEGEE (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), IKAFIKEHDKNKDGKLDLKEL (SEQ ID NO: 35), KEHDKNKDGKLDLKELVSILS (SEQ ID NO: 36), IKAFIKEHDKNKDGKLDLKELVSILSS (SEQ ID NO: 37) and any combination of any of the foregoing.

15. The MU in any one of the preceding claims is designed using a DNA construct comprising a nucleic acid sequence encoding the MU as described below, but not restricted to the following steps -i. The nucleic acid sequence is inserted in to an expression vector / plasmid DNA.ii. The expression vector used contains a T7 promoter.iii. The plasmid DNA is transformed in to a host cell (e.g. E. coli) to express the desired MU, wherein theE. coli strain used is BL21, BL21(DE3), ToplO, or DH5a cells.iv. Optimize the growth conditions to maximize production of the MU either in the soluble or insoluble form.v. The MU is extracted from the cells either in the insoluble form (as inclusion bodies) or in the soluble form,vi. The insoluble form of MU (inclusion bodies) are extracted from cell lysate and then solubilized using Urea and GuCl in to a soluble form,vii. The soluble form of MU bearing the affinity tag as described in claim 2 is extracted by loading the cell lysate on an affinity column via the affinity tag,16. The digestion of the MU as described in claim 15 is done using Kex2 and / or enterokinase followed by carboxypeptidase B.

17. The MU as described in claim 15 can also be digested using Kex2 followed by carboxypeptidase B.

18. The digestion as described in claim 16 and claim 17 when taken to completion will result in TP.

19. The resulting TP from the preceding claim 18, may or may not be further subjected to further purification by any of the standard purification methods, such as one or more chromatography steps or solvent fractionation.

20. The purified TP as described in claim 19, may or may not be conjugated to a second peptide to form a third peptide and, optionally, conjugating one or more moieties to the third peptide.

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