KR102345011B1 - Method for production of glucagon-like peptide-1 or analogues with groes pusion - Google Patents

Abstract

One embodiment of the present invention relates to a fusion tag, a fusion polypeptide comprising the fusion tag and glucagon-like peptide-1 or an analog thereof. When the novel fusion tag according to the present invention is used, the yield of glucagon-like peptide-1 or an analog thereof can be improved compared to the conventional fusion tag. In particular, it is useful when a glucagon-like peptide-1 having a relatively small molecular weight and easily degraded amino terminus or an analog thereof is prepared using genetic recombination technology. In addition, the fusion polypeptide comprising the fusion tag according to an embodiment of the present invention is protected from proteolytic enzymes of the host cell by inducing expression in the form of an inclusion body in the host cell, and stably glucagon-like peptide-1 or its It has the advantage of being able to produce analogues. Therefore, it is possible to provide a method for producing recombinant glucagon-like peptide-1 or an analog thereof with improved stability and yield than when a conventional fusion tag is used.

Description

Method for producing glucagon-like peptide-1 or an analog thereof using GroES fusion {METHOD FOR PRODUCTION OF GLUCAGON-LIKE PEPTIDE-1 OR ANALOGUES WITH GROES PUSION}

The present invention is a novel fusion tag, and relates to a fusion polypeptide comprising the fusion tag and glucagon-like peptide-1 or an analog thereof.

Recently, with the development of genetic engineering and biotechnology, many useful foreign proteins are produced using E. coli, yeast, animal and plant cells, etc., and these proteins are widely used as pharmaceuticals. Specifically, the development and industrialization of production process technology for pharmaceutical and research proteins such as immune modulators, enzyme inhibitors and hormones, or industrial proteins such as reactive additive enzymes are being promoted.

Among them, the genetic recombination technique is a method of producing a target protein or a target polypeptide by cloning the nucleic acids of various target proteins into an expression vector to obtain a recombinant expression vector, transforming it into an appropriate host cell, and culturing it. However, all or part of the target protein or target polypeptide is degraded by a degradation enzyme (eg, protease or peptidase) inherent in the host cell, resulting in a lower yield or use as a fusion tag The size of the peptide to be prepared is too large compared to the size of the target protein or target polypeptide to be prepared, so there is a problem in that the yield is significantly lowered.

Therefore, when a target protein or target polypeptide is to be mass-produced using genetic recombination technology, it is important to develop a fusion tag capable of improving the production yield while stably expressing the target protein or target polypeptide.

An object of the present invention is to provide a fusion tag comprising an amino acid sequence for producing glucagon-like peptide-1 or an analog thereof.

The technical problems to be achieved by the present invention are not limited to the technical problems mentioned above, and other technical problems not mentioned can be clearly understood by those of ordinary skill in the art to which the present invention belongs from the description below. There will be.

In order to achieve the above technical object, the fusion tag for producing glucagon-like peptide-1 or an analog thereof according to an aspect of the present invention may include the amino acid sequence of SEQ ID NO:6.

In an embodiment of the present invention, the fusion tag may be located at the N-terminus of the glucagon-like peptide-1 or an analog thereof.

In an embodiment of the present invention, the fusion tag may be expressed together with the glucagon-like peptide-1 or an analog thereof to form an inclusion body.

In an embodiment of the present invention, the fusion tag may include a linker connecting the fusion tag and the glucagon-like peptide-1 or an analog thereof.

In an embodiment of the present invention, the linker may include an affinity tag.

In an embodiment of the present invention, the linker may include a protease recognition sequence.

In an embodiment of the present invention, the protease recognition sequence may be located at the C-terminus of the linker.

In an embodiment of the present invention, the protease recognition sequence is selected from the group consisting of a tobacco etch virus protease recognition sequence, an enterokinase recognition sequence, a ubiquitin carboxy terminal hydrolase recognition sequence, a factor Xa recognition sequence, a purine recognition sequence, and combinations thereof. can be selected.

In an embodiment of the present invention, the protease recognition sequence may include the amino acid sequence of SEQ ID NO: 9.

When the novel fusion tag according to an embodiment of the present invention is used, the yield of glucagon-like peptide-1 or an analog thereof can be improved compared to the conventional fusion tag. In particular, it is useful when a glucagon-like peptide-1 having a relatively small molecular weight and easily degraded amino terminus or an analog thereof is prepared using genetic recombination technology. In addition, the fusion polypeptide comprising the fusion tag according to an embodiment of the present invention is protected from proteolytic enzymes of the host cell by inducing expression in the form of an inclusion body in the host cell, thereby stably glucagon-like peptide-1 or an analog thereof. has the advantage of being able to produce Therefore, it is possible to provide a method for producing a recombinant peptide with improved stability and yield compared to the case of using a conventional fusion tag.

The effects of the present invention are not limited to the above-described effects, and it should be understood to include all effects that can be inferred from the configuration of the invention described in the detailed description or claims of the present invention.

1 is a result of SDS-PAGE analysis of the total cell fraction of GLP-1K28R fusion polypeptides expressed in recombinant E. coli (lane M: marker protein, lane 1: H6TEV-GLP-1K28R (strain number PG005), lane 2) : PG43-H6TEV-GLP-1K28R (strain number PG011), lane 3: PG57-H6TEV-GLP-1K28R (strain number PG035), lane 4: PG68-H6TEV-GLP-1K28R (strain number PG036), lane 5: PG79 -H6TEV-GLP-1K28R (strain number PG037); lane 6: PG88-H6TEV-GLP-1K28R (strain number PG038); lane 7: PG97-H6TEV-GLP-1K28R (strain number PG039)).
Figure 2 is the result of analyzing the total cell fraction of H6TEV-GLP-1K28R, PG97-H6TEV-GLP-1K28R, SUMO-H6TEV-GLP-1K28R expressed in recombinant E. coli by SDS-PAGE (lane M: marker protein, lane 1: H6TEV-GLP-1K28R (strain number PG005), lane 2: PG97-H6TEV-GLP-1K28R (strain number PG039), lane 3: SUMO-H6TEV-GLP-1K28R (strain number PG049))
3 is a result of SDS-PAGE analysis of the PG97-H6TEV-GLP-1K28R fusion polypeptide purified through chromatography (lane M: marker protein, lane S: sample before purification, lane FT: flow-through fraction, lane E: fraction of eluate). Arrows indicate the G97-H6TEV-GLP-1K28R fusion polypeptide.
4 is a result of SDS-PAGE analysis of fractions after cleavage of purified PG97-H6TEV-GLP-1K28R using TEV protease (lane M: marker protein, lane C: sample not treated with TEV protease, lane T: sample treated with TEV protease).
5 is a result of SDS-PAGE analysis of fractions obtained by separating PG97-H6TEV and GLP-1K28R cleaved from PG97-H6TEV-GLP-1K28R using reverse phase chromatography (lane M: marker protein, lane S: purification Whole sample, lane FT: flow-through fraction, lane W: wash fraction, lane E: eluate fraction).
6 shows the results of measuring the molecular weight of chemically synthesized GLP-1K28R.
7 shows the results of measuring the molecular weight of GLP-1K28R purified according to the present invention.
8 is a graph confirming the retention time and purity of GLP-1K28R chemically synthesized by reverse-phase HPLC method and recombinant GLP-1K28R according to the present invention. 8(a) and 8(b) show reverse-phase HPLC analysis chromatograms of chemically synthesized GLP-1K28R and recombinant GLP-1K28R.

Hereinafter, the present invention will be described with reference to the accompanying drawings. However, the present invention may be embodied in several different forms, and thus is not limited to the embodiments described herein. And in order to clearly explain the present invention in the drawings, parts irrelevant to the description are omitted, and similar reference numerals are attached to similar parts throughout the specification.

Throughout the specification, when a part is “connected (connected, contacted, coupled)” with another part, it is not only “directly connected” but also “indirectly connected” with another member interposed therebetween. "Including cases where In addition, when a part "includes" a certain component, this means that other components may be further provided without excluding other components unless otherwise stated.

The terms used herein are used only to describe specific embodiments, and are not intended to limit the present invention. The singular expression includes the plural expression unless the context clearly dictates otherwise. In this specification, terms such as "comprises" or "have" are intended to designate that the features, numbers, steps, operations, components, parts, or combinations thereof described in the specification exist, but one or more other features It is to be understood that this does not preclude the possibility of addition or existence of numbers, steps, operations, components, parts, or combinations thereof.

Hereinafter, embodiments of the present invention will be described in detail with reference to the accompanying drawings.

The fusion tag for producing glucagon-like peptide-1 (GLP-1) or an analog thereof according to an aspect of the present invention may include the amino acid sequence of SEQ ID NO: 6.

In the case of producing various target peptides or target proteins using a recombinant microbial system, depending on the physical properties of the target material, the yield may be reduced due to degradation by enzymes in the host cell, low expression level, improper protein folding, and/or low mRNA stability. There is a risk of deterioration. For example, maltose binding protein (MBP), glutathione-S-transferase, thioredoxin, SUMO, and ubiquitin used as conventional fusion tags are 397, 216, and 106, respectively. , 101, 76 amino acids, there was a problem in that the yield is not good when producing a target polypeptide of a relatively low molecular weight.

In contrast, the fusion tag according to the present invention is a peptide having a relatively small molecular weight, and when the target polypeptide or target protein such as GLP-1, GLP-2 or hPTH 1-34 is produced by applying the fusion tag, the existing fusion tag It is possible to obtain the target polypeptide or target protein in an improved yield compared to the case where they are utilized. For example, the proportion of GLP-1 in the recombinant fusion polypeptide is schematically shown in Table 1 below.

fusion partner number of amino acids Molecular Weight (kDa) Target peptide ratio (%)* Maltose binding protein (MBP) 397 44.2 8 glutathione S-transferase 216 23.8 12 Fusion tag comprising the amino acid sequence of SEQ ID NO: 6 57 ~ 97 6.3 - 10.4 23 - 33

*Including the linker based on GLP-1 As shown in Calculation Table 1, in the conventional fusion tags, the ratio of GLP-1 in the fusion polypeptide is only 8% to 12%, whereas the fusion tag according to the present invention is a fusion tag. The proportion of GLP-1 in the obtained fusion polypeptide is found to be 23% to 33%. Therefore, since the amount of GLP-1 obtainable from the fusion polypeptide of the same concentration is higher than that of the conventional fusion tag, the final production yield can be improved.

In addition, the fusion tag according to the present invention can induce insoluble expression of the fusion polypeptide so that the fusion polypeptide is accumulated at a high concentration into the host cell as an insoluble inclusion body. Therefore, to obtain glucagon-like peptide-1 (GLP-1) or an analog thereof in high yield, which is likely to be completely or partially degraded or cleaved by proteases and peptidases in host cells such as Escherichia coli It has the advantage of being easy.

Specifically, the fusion tag including the amino acid sequence of SEQ ID NO: 6 may include a peptide having 97 amino acids.

In addition, the fusion tag may include variously modified peptides, ie, variants. The modification may be performed through a method of substituting, deleting, or adding one or more proteins without altering the function of the fusion tag. These various peptides may be at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any one of the amino acid sequences of SEQ ID NO: 6.

Another aspect of the present invention is a fusion polypeptide comprising the fusion tag as described above, glucagon-like peptide-1 or an analog thereof, and a fusion tag comprising a linker for linking the glucagon-like peptide-1 or analog thereof can provide

The linker may include an affinity tag. The term "affinity tag" used in the present invention is a peptide or nucleic acid sequence that can be introduced into a recombinant fusion polypeptide or a nucleic acid encoding the same, and can be used for various purposes, for example, purification of the target polypeptide or protein of interest. This may be to increase efficiency. As the affinity tag that can be used in the present invention, any suitable material known in the art can be used as desired. For example, the affinity tag used in the present invention may be a polyhistidine tag (SEQ ID NO:8).

In addition, the linker may include a protease recognition sequence. A protease is a protease that recognizes a specific amino acid sequence and cleaves a peptide bond within the recognized sequence or a peptide bond with the first amino acid of a fused polypeptide with the last amino acid of the sequence. The fusion polypeptide according to the present invention includes a linker having a protease recognition sequence, so that when the polypeptide is purified in the final step, the amino terminus containing the cleavage enzyme recognition sequence (in the case of using an affinity tag, the affinity tag is also included) and the purpose By separating the N-terminus of the polypeptide or target protein, the target polypeptide or target protein can be recovered.

Specifically, the protease recognition sequence may be a recognition sequence selected from the group consisting of a tobacco etch virus (TEV) protease recognition sequence, an enterokinase recognition sequence, a ubiquitinase recognition sequence, a factor Xa, a purine, and combinations thereof. For example, the protease recognition sequence may include any one of the amino acid sequence of SEQ ID NO:9.

The glucagon-like peptide-1 or an analog thereof may preferably be a liraglutide precursor peptide.

*The liraglutide precursor peptide (GLP-1K28R) is a polypeptide derived from glucagon-like peptide-1 (GLP-1) consisting of 31 amino acids, and the 28th lysine of GLP-1 is substituted with arginine (K28R) ) has been In this regard, liraglutide has a form in which N-Palmitoyl-L-glutamic acid composed of palmitic acid and glutamic acid is bonded to the amino group of the 20th lysine residue of GLP-1K28R. Liraglutide can be used as a treatment for type 2 diabetes or obesity. In general, liraglutide produces a liraglutide precursor peptide (GLP-1K28R) to which N-Palmitoyl-L-glutamic acid is not bound, It can be produced by a process of binding N-Palmitoyl-L-glutamic acid to its 20th lysine residue (Dunweber, Jensen et al. 2007).

Another aspect of the present invention may provide a polynucleotide encoding the above-described fusion polypeptide.

Another aspect of the present invention may provide an expression vector comprising a polynucleotide molecule encoding the above-described fusion polypeptide. The term "vector" used in the present invention can be introduced into a host cell, recombination and insertion into the host cell genome. Such vectors include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogs thereof. Examples of viral vectors can include retroviruses, adenoviruses, and adeno-associated viruses. In addition, the plasmid may contain a selectable marker, such as an antibiotic resistance gene, and host cells maintaining the plasmid may be cultured under selective conditions.

As used herein, the term "gene expression" or "expression" is understood to mean transcription of a DNA sequence, translation of an mRNA transcript, and secretion of a fusion protein product or a fragment thereof.

As used herein, the term "host cell" refers to prokaryotic and eukaryotic cells into which recombinant expression vectors can be introduced. As used herein, the terms "transfected", "transformed" and "transfected" refer to the introduction of a nucleic acid (eg, a vector) into a cell using techniques known in the art.

In addition, an expression vector comprising a nucleotide sequence encoding a fusion polypeptide comprising a glucagon-like peptide-1 or an analog thereof of the present invention may be provided as a part of an inducible expression vector. For example, a host cell transformed with an expression vector is cultured, and expression of the fusion protein is induced from the expression vector. The expression vector may be a plasmid. For example, the expression vector is a plasmid further comprising a gene encoding a recombinant fusion protein and a gene for a selection marker, and the host cell is cultured under selection conditions to maintain the plasmid.

In addition to the protein coding sequence, the expression vector may include regulatory elements related to protein expression, such as a promoter, a ribosome binding site (RBS), a transcription terminator, and a translation initiation and stop signal. For example, RBS that affects the expression level of a useful target polypeptide can be obtained through genes or synthesis of various organisms.

In addition, the promoter used in the present invention is a regulated inducible promoter, and may be, for example, a promoter derived from the T7 promoter, Lac promoter, Tac promoter and Trc promoter. Specifically, the LacI gene may be present in the expression system as an expression regulator in the expression system using the T7 promoter, which is generally a Lac-based promoter. The LacI gene, which is usually a constitutively expressed gene, encodes the Lac repressor protein LacI protein, which binds to the lac operator of the Lac family promoter. Thus, when a Lac family promoter is used, the Lac gene can also be included and expressed in an expression system.

Another aspect of the present invention may provide a host cell comprising the expression vector. A suitable host cell, which has been transfected or transfected with the DNA sequence of the present invention, can be used for expression and/or secretion of glucagon-like peptide-1 or an analogue thereof, and a preferred host cell that can be used in the present invention is an E. coli cell. , immortalized hybridoma cells, NS/0 myeloma cells, 293 cells, Chinese hamster ovary cells (CHO cells), HeLa cells, human amniotic fluid derived cells (CapT cells) or COS cells. For example, the host strain used to express the fusion polypeptide in the present invention is Escherichia coli BL21(DE3), and the gene and methods for using them are known in the art.

In addition, high throughput screening can be performed to determine optimal conditions for expressing the recombinant fusion protein. Conditions that may be changed in screening include the host cell, the genetic background of the host cell (eg, deletion of certain genes such as various proteases), the type of promoter in the expression construct, the type of RBS in the expression construct, the incubation temperature, When an inducible promoter is used, cell turbidity at the time of induction, when a lac-based promoter is used, the concentration of IPTG used for induction, protein induction period, culture temperature after addition of an inducer to the culture, agitation speed of the culture, selection method for plasmid maintenance, the volume of the culture in the vessel, and the method of cell lysis.

In addition, "optimal host strain" or "optimal expression system" is identified based on the quality, expression amount, expression level and/or location of the expressed recombinant fusion protein compared to various host cells of different genotypes. or you can choose Accordingly, an optimal host strain is a strain that produces a recombinant fusion protein suitable for production of a target polypeptide or target protein. High suitability for the production of a target polypeptide or target protein may vary depending on the target polypeptide or target protein to be produced, but the quality, expression amount, expression level of the recombinant fusion protein, for example, intracellularity of the target polypeptide or target protein It includes whether degradation or partial cleavage has occurred, what level of expression or expression level, whether the protein is properly or preferably folded or accumulated in insoluble inclusion bodies, and the like. That is, the optimal host strain or optimal expression system has a production yield, expression amount, expression level, amount of recoverable recombinant fusion protein, and suitably accumulated in the form of insoluble inclusion bodies at a certain level or higher compared to the control host strain or expression system. Any amount of recombinant fusion protein can be produced.

Hereinafter, embodiments of the present invention will be described in detail so that those of ordinary skill in the art can easily carry out the present invention. However, the present invention may be embodied in several different forms and is not limited to the embodiments described herein.

Example 1. Preparation and production of GLP-1K28R fusion polypeptide

Example 1.1 Construction of GLP-1K28R fusion polypeptide expression plasmid

The gene for the GLP-1K28R fusion polypeptide was synthesized using the overlap extension polymerase chain reaction (OE-PCR) method. In this case, the GLP-1K28R fusion tag is an amino-terminal fusion tag, such as PG43 (SEQ ID NO: 1), PG57 (SEQ ID NO: 2), PG68 (SEQ ID NO: 3), PG79 (SEQ ID NO: 4), PG88 (SEQ ID NO: 5), PG97 (SEQ ID NO: 6) and 6 histidine tag (SEQ ID NO: 8) and TEV protease recognition sequence (SEQ ID NO: 9) and amino acid sequence of GLP-1K28R (SEQ ID NO: 17).

The GLP-1K28R fusion polypeptide (H6TEV-GLP-1K28R, SEQ ID NO: 18) used as a negative control did not contain an amino-terminal fusion tag, 6 histidine tag (SEQ ID NO: 7), TEV protease recognition sequence (SEQ ID NO: 8) ) and the amino acid sequence of GLP-1K28R (SEQ ID NO: 17).

The GLP-1K28R fusion polypeptide (SUMO-H6TEV-GLP-1K28R, SEQ ID NO: 25) used as a positive control was an amino-terminal fusion tag SUMO (SEQ ID NO: 7) and 6 histidine tag (SEQ ID NO: 8) and a TEV protease recognition sequence ( SEQ ID NO: 9) and the amino acid sequence of GLP-1K28R (SEQ ID NO: 17). GLP comprising any one of PG43 (SEQ ID NO: 1), PG57 (SEQ ID NO: 2), PG68 (SEQ ID NO: 3), PG79 (SEQ ID NO: 4), PG88 (SEQ ID NO: 5), PG97 (SEQ ID NO: 6)- The amino acid sequence of the 1K28R fusion polypeptide corresponds to SEQ ID NOs: 19 to 24.

The gene of each fusion polypeptide contains recognition sequences of restriction enzymes NdeI, NcoI and XhoI and one stop codon. The nucleotide sequences encoding the GLP-1K28R fusion polypeptide correspond to SEQ ID NOs: 59 to 64, and negative and positive controls correspond to SEQ ID NOs: 58 and SEQ ID NO: 65.

In order to construct the GLP-1K28R fusion polypeptide expression plasmids pSGK530, pSGK497, pSGK699, pSGK700, pSGK701, pSGK702, pSGK490, and pSGK752 shown in Table 2 below, the GLP-1K28R fusion protein gene fragment synthesized by OE-PCR was subjected to NdeI and It was digested using an XhoI restriction enzyme and cloned into an expression vector pET26b capable of expression control by IPTG, including the T7 promoter, Lac operator and LacI gene.

strain host cell plasmid Recombinant Fusion Polypeptides PG005 E. coli BL21 (DE3) pSGK530 H6TEV-GLP-1K28R PG011 E. coli BL21 (DE3) pSGK497 PG43-H6TEV-GLP-1K28R PG035 E. coli BL21 (DE3) pSGK699 PG57-H6TEV-GLP-1K28R PG036 E. coli BL21 (DE3) pSGK700 PG68-H6TEV-GLP-1K28R PG037 E. coli BL21 (DE3) pSGK701 PG79-H6TEV-GLP-1K28R PG038 E. coli BL21 (DE3) pSGK702 PG88-H6TEV-GLP-1K28R PG039 E. coli BL21 (DE3) pSGK490 PG97-H6TEV-GLP-1K28R PG049 E. coli BL21 (DE3) pSGK752 SUMO-H6TEV-GLP-1K28R

The prepared GLP-1K28R fusion polypeptide expression plasmids were confirmed for correct cloning through DNA sequencing. E. coli BL21(DE3) cells were transformed by a chemical method using calcium chloride using the prepared GLP-1K28R fusion polypeptide expression plasmids. E. coli transduced with the expression plasmid of the GLP-1K28R fusion polypeptide formed colonies in LB solid medium containing kanamycin at a concentration of 50 μg/ml. Transformed E. coli are cultured in LB liquid medium in which kanamycin is present at a concentration of 50 μg/ml, respectively, and then 50% glycerol is added in the same volume as the culture solution to make a cell stock, which is heated at -80 ° C. was stored in the freezer of

Example 1.2. Culture of Transformed Cells and Expression of GLP-1K28R

After dissolving the E. coli cell concentrate transformed with the expression plasmid of the GLP-1K28R fusion polypeptide stored at -80°C at room temperature, 50 μl of LB liquid medium containing kanamycin at a concentration of 50 μg/ml is included. It was added to the test tube and cultured for 12 hours in a shaker incubator at a temperature of 37°C. 2 ml of the seed culture E. coli was added to a flask containing 200 ml of LB liquid medium in which kanamycin was present at a concentration of 50 μg/ml, and cultured in a shaker incubator at a temperature of 37°C. When the optical density (OD600) of the cells reached about 1.0 after about 3 hours of culture, IPTG was added to a final concentration of 0.1 mM to induce expression of the GLP-1K28R fusion polypeptide. After 4 hours of induction of expression, the optical density was measured to measure the optical density of the cells.

Example 1.3. Preparation of samples for comparative analysis of expression levels

The cells were concentrated so that the optical density of the expression-induced cells was 10.0, resuspended in 50 mM sodium phosphate, pH 7.2 buffer, and then the cells were disrupted using an ultrasonic processor (Cole-Parmer). The lysed cells were labeled with the total cell fraction. The cell lysate was centrifuged for 15 minutes at 12,000×g and 4°C. The supernatant was recovered and labeled as a soluble fraction. The insoluble fraction was resuspended in 500 µl of 50 mM sodium phosphate, pH 7.2 buffer using an ultrasonic cell disrupter and labeled as the insoluble fraction.

Example 1.4. Identification of the produced GLP-1K28R fusion polypeptide by SDS-PAGE analysis

50 μl of total cells, soluble and insoluble fractions were mixed with 50 μl of each of the two-fold concentrated SDS sample buffer (SDS sample buffer 2× concentrate, Sigma), and then heated at 95° C. for 5 minutes to release the protein of each sample. allowed to be transformed. Denatured proteins in the sample were separated in the gel according to the molecular weight size using a 16% SDS-PAGE gel and TANK buffer. After SDS-PAGE, the gel was stained with a staining buffer containing Coomassie blue R-250, and then bleached with a decolorization buffer so that only the stained protein could be seen. The results are shown in FIG. 1 .

Referring to FIG. 1, the band of H6TEV-GLP-1K28R (molecular weight of 5.1 kDa) that does not contain a fusion tag including the amino acid sequence of SEQ ID NO: 1 to SEQ ID NO: 6 as a negative control was not detected on the SDS-PAGE gel. After expression, it appears to be degraded by intracellular proteolytic enzymes. PG43-H6TEV-GLP-1K28R (molecular weight of 9.8 kDa), PG57-H6TEV-GLP-1K28R (11.3 kDa), which are GLP-1K28R fusion polypeptides fused with amino-terminal fusion tags PG43, PG57, PG68, PG79, PG88 and PG97 of molecular weight), PG68-H6TEV-GLP-1K28R (molecular weight of 12.5 kDa), PG79-H6TEV-GLP-1K28R (molecular weight of 13.7 kDa), PG88-H6TEV-GLP-1K28R (molecular weight of 14.7 kDa) and PG97-H6TEV- It was confirmed that the expression level of GLP-1K28R (molecular weight of 15.3 kDa) was significantly improved compared to the control (H6TEV-GLP-1K28R).

Through densitometric analysis, it was confirmed that the PG97-H6TEV-GLP-1K28R fused with PG97 was significantly higher than the polypeptides fused with PG43, PG57, PG68, PG79 and PG88 (FIG. 1). In addition, the expression level of PG97-H6TEV-GLP-1K28R was significantly higher than that of the positive control, SUMO-H6TEV-GLP-1K28R (molecular weight of 16.2 kDa) (FIG. 2). Therefore, subsequent experiments were performed with PG97-H6TEV-GLP-1K28R.

Example 2. Recovery and purification of PG97-H6TEV-GLP-1K28R

Example 2.1. Cell disruption and recovery of insoluble inclusion bodies

Frozen cell pellets of expressed cells at flask scale were thawed by adding 50 ml of 50 mM sodium phosphate, pH 7.2 buffer. The resuspended cells were disrupted using an ultrasonic processor (Cole-Parmer). The lysed cells were centrifuged at 12,000 rpm (12,000×g) for 30 minutes and the supernatant was removed to recover the insoluble inclusion body fraction containing the recombinant fusion polypeptide.

Example 2.2. Solubilization of insoluble inclusion bodies

20 ml of inclusion body solubilization buffer (8 M urea, 20 mM Tris, 500 mM sodium chloride, 50 mM imidazole, pH 7.4) was added to the recovered insoluble inclusion body fraction and incubated with shaking at 25° C. for 4 hours to obtain an insoluble fraction Recombinant fusion polypeptides in the form of inclusion bodies were allowed to solubilize. The solubilized insoluble fraction sample was centrifuged at 12,000Х for 30 minutes, and the supernatant was filtered through a membrane filter (0.45/0.2 μm).

Example 2.3. Purification of PG97-H6TEV-GLP-1K28R

AKTA pure 25 chromatography system (GE Healthcare) equipped with S9 sample pump S9 and F9-C fraction collector F9-C for purification of PG97-H6TEV-GLP-1K28R in solubilized insoluble fraction was used. The solubilized insoluble fraction sample was injected onto a HisTrap FF 1 ml column (GE Healthcare) pre-equilibrated with inclusion body solubilization buffer (8 M urea, 20 mM Tris, 500 mM sodium chloride, 50 mM imidazole, pH 7.4). After injection is complete, the column is washed with 5 column volumes of equilibration buffer, and 5 column volumes of elution buffer (8 M urea, 20 mM Tris, 500 mM sodium chloride, 500 mM imidazole, pH 7.4) in steps of 100% PG97-H6TEV-GLP-1K28R bound to the resin of the column was eluted. The results of the fractions obtained after elution are shown in FIG. 3 , respectively.

Example 3. Linker sequence cleavage through protease treatment

After combining about 5 ml of purified PG97-H6TEV-GLP-1K28R fusion polypeptide fractions, 140 ml of dilution buffer (20 mM Tris, pH 7.4) was added to dilute the urea concentration to 1 M. The diluted recombinant fusion polypeptide was added to a final concentration of TEV protease of 500 nM, and the cleavage reaction was allowed to proceed at room temperature for 12 hours.

To confirm whether cleavage by TEV protease was performed, SDS-PAGE analysis was performed after cleavage, and the results are shown in FIG. 4 . On the other hand, referring to FIG. 4 , the 15.4 kDa PG97-H6TEV-GLP-1K28R fusion with the N-terminal fusion tag, 6 histidine tag, and TEV protease recognition sequence, the PG97-H6TEV fragment (12.0 kDa) in a yield of almost 100% It was confirmed that the target polypeptide is separated into a GLP-1K28R (3.4 kDa) fragment.

Example 4. Purification of GLP-1K28R

In the PG97-H6TEV-GLP-1K28R fusion polypeptide, an S9 sample pump (Sample pump S9) and F9-C fraction collector (F9-C) are equipped for purification of GLP-1K28R released by TEV protease cleavage. AKTA pure 25 chromatography system (GE Healthcare) was used. Samples digested with TEV protease were injected onto a Resource RPC 3 ml column (GE Healthcare) pre-equilibrated with binding buffer (10 mM potassium phosphate, 2% acetonitrile pH 7.0). PG97-H6TEV did not bind to the column, so a flow-through fraction was collected, and an elution buffer (10 mM potassium phosphate, 70% acetonitrile pH 7.0) was passed through the column to 50%, thereby weakly binding to the resin of the column. PG97-H6TEV-GLP-1K28R was removed, and the elution buffer was passed through the column to 100% to purify GLP-1K28R bound to the resin of the column. The flow-through and elution fractions of the fraction collector were analyzed by the SDS-PAGE method described above.

Referring to FIG. 5 , it was confirmed that GLP-1K28R was eluted by a high concentration of acetonitrile after binding to the Resource RPC resin. Therefore, since PG97-H6TEV-GLP-1K28R and PG97-H6TEV can be easily separated by controlling the concentrations of GLP-1K28R and acetonitrile, the purification of GLP-1K28R can be facilitated using reversed-phase chromatography.

* Example 5. Molecular weight analysis of purified GLP-1K28R after cleavage

Molecular weight analysis using MALTI-TOF MS was performed to confirm the presence or absence of expression of the complete form of PG97-H6TEV-GLP-1K28R and the presence or absence of modification of GLP-1K28R obtained after accurate cleavage and cleavage by TEV protease. The results of measuring the molecular weight of the chemically synthesized GLP-1K28R standard material and the molecular weight of GLP-1K28R obtained according to the present invention are shown in FIGS. 6 and 7 .

6 and 7, the molecular weight (3382.9 Da) of GLP-1K28R obtained from PG97-H6TEV-GLP-1K28R was exactly the same as the molecular weight of chemically synthesized GLP-1K28R (3382.8 Da), so proteolysis in E. coli It was confirmed that it was expressed in a complete form without partial cleavage or degradation of the amino or carboxy terminus by an enzyme (proteolytic enzyme). Therefore, the TEV protease recognizes the ENLFQ sequence, which is the recognition sequence in PG97-H6TEV-GLP-1K28R, and it is judged to precisely cut the peptide bond between Q (glutamine), the last amino acid, and H (histidine), the first amino acid of GLP-1K28R. .

Example 6. Reversed-phase HPLC analysis of purified GLP-1K28R

Chemically synthesized GLP-1K28R and recombinant GLP-1K28R produced through the present invention were injected into a C18 high performance liquid chromatography (HPLC) column equilibrated in buffer A (0.1% trifluoroacetic acid, 2% acetonitrile), followed by buffer B ( Retention time and purity of chemically synthesized GLP-1K28R and recombinant GLP-1K28R were analyzed by reverse-phase HPLC method in which the concentration of 0.1% trifluoroacetic acid, 80% acetonitrile) was linearly increased. As a result, both chemical synthesis and recombinant GLP-1K28R showed the same retention time, and the purity of recombinant GLP-1K28R was confirmed to be 95% or more (FIG. 8).

The description of the present invention described above is for illustration, and those of ordinary skill in the art to which the present invention pertains can understand that it can be easily modified into other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive. For example, each component described as a single type may be implemented in a dispersed form, and likewise components described as distributed may also be implemented in a combined form.

The scope of the present invention is indicated by the following claims, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention.

<110> PEPGENE INC. <120> METHOD FOR PRODUCTION OF GLUCAGON-LIKE PEPTIDE-1 OR ANALOGUES WITH GROES PUSION <130> YP-21-104 <150> KR 10-2019-0087637 <151> 2019-07-19 <160> 89 <170> KoPatentIn 3.0 <210> 1 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43 <400> 1 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val 35 40 <210> 2 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG57 <400> 2 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu 50 55 <210> 3 <211> 68 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG68 <400> 3 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn 65 <210> 4 <211> 79 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG79 <400> 4 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp 65 70 75 <210> 5 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG88 <400> 5 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu 85 <210> 6 <211> 97 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG97 <400> 6 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala <210> 7 <211> 98 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for SUMO <400> 7 Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro 1 5 10 15 Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser 20 25 30 Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu 35 40 45 Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg 50 55 60 Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp 65 70 75 80 Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile 85 90 95 Gly Gly <210> 8 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6 <400> 8 His His His His His His 1 5 <210> 9 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> Tobacco etch virus (TEV) protease recognition site <400> 9 Glu Asn Leu Tyr Phe Gln 1 5 <210> 10 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV <400> 10 Met His His His His His His Glu Asn Leu Tyr Phe Gln 1 5 10 <210> 11 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV <400> 11 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Pro Trp His His His 35 40 45 His His His Glu Asn Leu Tyr Phe Gln 50 55 <210> 12 <211> 71 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG57-H6TEV <400> 12 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Pro Trp His His His His His 50 55 60 His Glu Asn Leu Tyr Phe Gln 65 70 <210> 13 <211> 82 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG68-H6TEV <400> 13 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Pro Trp His His His His His His Glu Asn Leu Tyr 65 70 75 80 Phe Gln <210> 14 <211> 93 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG79-H6TEV <400> 14 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Pro 65 70 75 80 Trp His His His His His His His Glu Asn Leu Tyr Phe Gln 85 90 <210> 15 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG88-H6TEV <400> 15 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Pro Trp His His His His His His 85 90 95 Glu Asn Leu Tyr Phe Gln 100 <210> 16 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG97-H6TEV <400> 16 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala His His His His His His His Glu Asn Leu Tyr Phe Gln 100 105 <210> 17 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Liraglutide precursor peptide (GLP-1K28R) <400> 17 His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 20 25 30 <210> 18 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV-GLP-1K28R <400> 18 Met His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu 1 5 10 15 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala 20 25 30 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 35 40 <210> 19 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 19 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Pro Trp His His His 35 40 45 His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr 50 55 60 Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile 65 70 75 80 Ala Trp Leu Val Arg Gly Arg Gly 85 <210> 20 <211> 102 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG57-H6TEV-GLP-1K28R <400> 20 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Pro Trp His His His His His 50 55 60 His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp 65 70 75 80 Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp 85 90 95 Leu Val Arg Gly Arg Gly 100 <210> 21 <211> 113 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG68-H6TEV-GLP-1K28R <400> 21 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Pro Trp His His His His His His Glu Asn Leu Tyr 65 70 75 80 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu 85 90 95 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg 100 105 110 Gly <210> 22 <211> 124 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG79-H6TEV-GLP-1K28R <400> 22 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Pro 65 70 75 80 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu 85 90 95 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala 100 105 110 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 115 120 <210> 23 <211> 133 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG88-H6TEV-GLP-1K28R <400> 23 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Pro Trp His His His His His His 85 90 95 Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val 100 105 110 Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu 115 120 125 Val Arg Gly Arg Gly 130 <210> 24 <211> 140 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG97-H6TEV-GLP-1K28R <400> 24 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala His His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu 100 105 110 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala 115 120 125 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 130 135 140 <210> 25 <211> 141 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for SUMO-H6TEV-GLP-1K28R <400> 25 Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro 1 5 10 15 Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser 20 25 30 Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu 35 40 45 Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg 50 55 60 Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp 65 70 75 80 Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile 85 90 95 Gly Gly His His His His His His His Glu Asn Leu Tyr Phe Gln His Ala 100 105 110 Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala 115 120 125 Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly 130 135 140 <210> 26 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for hPTH1-34 (Teriparatide) <400> 26 Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn Phe <210> 27 <211> 47 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV-hPTH1-34 <400> 27 Met His His His His His His Glu Asn Leu Tyr Phe Gln Ser Val Ser 1 5 10 15 Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu 20 25 30 Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe 35 40 45 <210> 28 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG97-H6TEV-hPTH1-34 <400> 28 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala His His His His His His His Glu Asn Leu Tyr Phe Gln Ser Val Ser 100 105 110 Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met Glu 115 120 125 Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe 130 135 140 <210> 29 <211> 144 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for SUMO-H6TEV-hPTH1-34 <400> 29 Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro 1 5 10 15 Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser 20 25 30 Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu 35 40 45 Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg 50 55 60 Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp 65 70 75 80 Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile 85 90 95 Gly Gly His His His His His His His Glu Asn Leu Tyr Phe Gln Ser Val 100 105 110 Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys His Leu Asn Ser Met 115 120 125 Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His Asn Phe 130 135 140 <210> 30 <211> 33 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Teduglutide (GLP-2A2G) <400> 30 His Gly Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn 1 5 10 15 Leu Ala Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr 20 25 30 Asp <210> 31 <211> 46 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV-GLP-2A2G <400> 31 Met His His His His His His His Glu Asn Leu Tyr Phe Gln His Gly Asp 1 5 10 15 Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn Leu Ala Ala 20 25 30 Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr Asp 35 40 45 <210> 32 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG97-H6TEV-GLP-2A2G <400> 32 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala His His His His His His His Glu Asn Leu Tyr Phe Gln His Gly Asp 100 105 110 Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn Leu Ala Ala 115 120 125 Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr Asp 130 135 140 <210> 33 <211> 143 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for SUMO-H6TEV-GLP-2A2G <400> 33 Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro 1 5 10 15 Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser 20 25 30 Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu 35 40 45 Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg 50 55 60 Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp 65 70 75 80 Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile 85 90 95 Gly Gly His His His His His His His Glu Asn Leu Tyr Phe Gln His Gly 100 105 110 Asp Gly Ser Phe Ser Asp Glu Met Asn Thr Ile Leu Asp Asn Leu Ala 115 120 125 Ala Arg Asp Phe Ile Asn Trp Leu Ile Gln Thr Lys Ile Thr Asp 130 135 140 <210> 34 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Ecallantide <400> 34 Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys 1 5 10 15 Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys 20 25 30 Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu 35 40 45 Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 60 <210> 35 <211> 73 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV-Ecallantide <400> 35 Met His His His His His His His Glu Asn Leu Tyr Phe Gln Glu Ala Met 1 5 10 15 His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala 20 25 30 His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe 35 40 45 Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu 50 55 60 Glu Cys Lys Lys Met Cys Thr Arg Asp 65 70 <210> 36 <211> 169 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG97-H6TEV-Ecallantide <400> 36 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala His His His His His His His Glu Asn Leu Tyr Phe Gln Glu Ala Met 100 105 110 His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala 115 120 125 His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe 130 135 140 Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu 145 150 155 160 Glu Cys Lys Lys Met Cys Thr Arg Asp 165 <210> 37 <211> 170 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for SUMO-H6TEV-Ecallantide <400> 37 Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro 1 5 10 15 Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser 20 25 30 Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu 35 40 45 Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg 50 55 60 Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp 65 70 75 80 Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile 85 90 95 Gly Gly His His His His His His His Glu Asn Leu Tyr Phe Gln Glu Ala 100 105 110 Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala 115 120 125 Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu 130 135 140 Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu 145 150 155 160 Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 165 170 <210> 38 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Nesiritide <400> 38 Ser Pro Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp 1 5 10 15 Arg Ile Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 20 25 30 <210> 39 <211> 45 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV-Nesiritide <400> 39 Met His His His His His His His Glu Asn Leu Tyr Phe Gln Ser Pro Lys 1 5 10 15 Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser 20 25 30 Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 35 40 45 <210> 40 <211> 141 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG97-H6TEV-Nesiritide <400> 40 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala His His His His His His His Glu Asn Leu Tyr Phe Gln Ser Pro Lys 100 105 110 Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile Ser 115 120 125 Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 130 135 140 <210> 41 <211> 142 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for SUMO-H6TEV-Nesiritide <400> 41 Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro 1 5 10 15 Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser 20 25 30 Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu 35 40 45 Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg 50 55 60 Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp 65 70 75 80 Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile 85 90 95 Gly Gly His His His His His His His Glu Asn Leu Tyr Phe Gln Ser Pro 100 105 110 Lys Met Val Gln Gly Ser Gly Cys Phe Gly Arg Lys Met Asp Arg Ile 115 120 125 Ser Ser Ser Ser Gly Leu Gly Cys Lys Val Leu Arg Arg His 130 135 140 <210> 42 <211> 53 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Human Epidermal growth factor (hEGF) <400> 42 Asn Ser Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His 1 5 10 15 Asp Gly Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn 20 25 30 Cys Val Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys 35 40 45 Trp Trp Glu Leu Arg 50 <210> 43 <211> 66 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV-hEGF <400> 43 Met His His His His His His Glu Asn Leu Tyr Phe Gln Asn Ser Asp 1 5 10 15 Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val 20 25 30 Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val 35 40 45 Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu 50 55 60 Leu Arg 65 <210> 44 <211> 162 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG97-H6TEV-hEGF <400> 44 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala His His His His His His His Glu Asn Leu Tyr Phe Gln Asn Ser Asp 100 105 110 Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly Val 115 120 125 Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val Val 130 135 140 Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp Glu 145 150 155 160 Leu Arg <210> 45 <211> 163 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for SUMO-H6TEV-hEGF <400> 45 Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro 1 5 10 15 Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser 20 25 30 Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu 35 40 45 Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg 50 55 60 Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp 65 70 75 80 Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile 85 90 95 Gly Gly His His His His His His His Glu Asn Leu Tyr Phe Gln Asn Ser 100 105 110 Asp Ser Glu Cys Pro Leu Ser His Asp Gly Tyr Cys Leu His Asp Gly 115 120 125 Val Cys Met Tyr Ile Glu Ala Leu Asp Lys Tyr Ala Cys Asn Cys Val 130 135 140 Val Gly Tyr Ile Gly Glu Arg Cys Gln Tyr Arg Asp Leu Lys Trp Trp 145 150 155 160 Glu Leu Arg <210> 46 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Interferon beta 1b (INF-beta1b) <400> 46 Ser Tyr Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Ser 1 5 10 15 Gln Lys Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys 20 25 30 Asp Arg Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln 35 40 45 Phe Gln Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn 50 55 60 Ile Phe Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn Glu 65 70 75 80 Thr Ile Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His 85 90 95 Leu Lys Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg 100 105 110 Gly Lys Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile 115 120 125 Leu His Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile 130 135 140 Val Arg Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr 145 150 155 160 Gly Tyr Leu Arg Asn 165 <210> 47 <211> 178 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV-INF-beta1b <400> 47 Met His His His His His His His Glu Asn Leu Tyr Phe Gln Ser Tyr Asn 1 5 10 15 Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Ser Gln Lys Leu 20 25 30 Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys Asp Arg Met 35 40 45 Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln Phe Gln Lys 50 55 60 Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn Ile Phe Ala 65 70 75 80 Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn Glu Thr Ile Val 85 90 95 Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His Leu Lys Thr 100 105 110 Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg Gly Lys Leu 115 120 125 Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile Leu His Tyr 130 135 140 Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile Val Arg Val 145 150 155 160 Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr Gly Tyr Leu 165 170 175 Arg Asn <210> 48 <211> 274 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG97-H6TEV-INF-beta1b <400> 48 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala His His His His His His His Glu Asn Leu Tyr Phe Gln Ser Tyr Asn 100 105 110 Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Ser Gln Lys Leu 115 120 125 Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys Asp Arg Met 130 135 140 Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln Phe Gln Lys 145 150 155 160 Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn Ile Phe Ala 165 170 175 Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn Glu Thr Ile Val 180 185 190 Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His Leu Lys Thr 195 200 205 Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg Gly Lys Leu 210 215 220 Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile Leu His Tyr 225 230 235 240 Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile Val Arg Val 245 250 255 Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr Gly Tyr Leu 260 265 270 Arg Asn <210> 49 <211> 275 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for SUMO-H6TEV-INF-beta1b <400> 49 Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro 1 5 10 15 Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser 20 25 30 Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu 35 40 45 Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg 50 55 60 Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp 65 70 75 80 Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile 85 90 95 Gly Gly His His His His His His His Glu Asn Leu Tyr Phe Gln Ser Tyr 100 105 110 Asn Leu Leu Gly Phe Leu Gln Arg Ser Ser Asn Phe Gln Ser Gln Lys 115 120 125 Leu Leu Trp Gln Leu Asn Gly Arg Leu Glu Tyr Cys Leu Lys Asp Arg 130 135 140 Met Asn Phe Asp Ile Pro Glu Glu Ile Lys Gln Leu Gln Gln Phe Gln 145 150 155 160 Lys Glu Asp Ala Ala Leu Thr Ile Tyr Glu Met Leu Gln Asn Ile Phe 165 170 175 Ala Ile Phe Arg Gln Asp Ser Ser Ser Thr Gly Trp Asn Glu Thr Ile 180 185 190 Val Glu Asn Leu Leu Ala Asn Val Tyr His Gln Ile Asn His Leu Lys 195 200 205 Thr Val Leu Glu Glu Lys Leu Glu Lys Glu Asp Phe Thr Arg Gly Lys 210 215 220 Leu Met Ser Ser Leu His Leu Lys Arg Tyr Tyr Gly Arg Ile Leu His 225 230 235 240 Tyr Leu Lys Ala Lys Glu Tyr Ser His Cys Ala Trp Thr Ile Val Arg 245 250 255 Val Glu Ile Leu Arg Asn Phe Tyr Phe Ile Asn Arg Leu Thr Gly Tyr 260 265 270 Leu Arg Asn 275 <210> 50 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for human growth hormone (hGH) <400> 50 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185 190 <210> 51 <211> 204 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV-hGH <400> 51 Met His His His His His His Glu Asn Leu Tyr Phe Gln Phe Pro Thr 1 5 10 15 Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg 20 25 30 Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr 35 40 45 Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser 50 55 60 Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr 65 70 75 80 Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile 85 90 95 Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn 100 105 110 Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys 115 120 125 Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly 130 135 140 Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp 145 150 155 160 Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu 165 170 175 Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile 180 185 190 Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 195 200 <210> 52 <211> 300 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG97-H6TEV-hGH <400> 52 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala His His His His His His His Glu Asn Leu Tyr Phe Gln Phe Pro Thr 100 105 110 Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His Arg 115 120 125 Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala Tyr 130 135 140 Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr Ser 145 150 155 160 Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu Thr 165 170 175 Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu Ile 180 185 190 Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala Asn 195 200 205 Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu Lys 210 215 220 Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp Gly 225 230 235 240 Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe Asp 245 250 255 Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu Leu 260 265 270 Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg Ile 275 280 285 Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 290 295 300 <210> 53 <211> 301 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for SUMO-H6TEV-hGH <400> 53 Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro 1 5 10 15 Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser 20 25 30 Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu 35 40 45 Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg 50 55 60 Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp 65 70 75 80 Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile 85 90 95 Gly Gly His His His His His His His Glu Asn Leu Tyr Phe Gln Phe Pro 100 105 110 Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg Ala His 115 120 125 Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu Glu Ala 130 135 140 Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro Gln Thr 145 150 155 160 Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg Glu Glu 165 170 175 Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu Leu Leu 180 185 190 Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val Phe Ala 195 200 205 Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp Leu Leu 210 215 220 Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu Glu Asp 225 230 235 240 Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser Lys Phe 245 250 255 Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr Gly Leu 260 265 270 Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe Leu Arg 275 280 285 Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 290 295 300 <210> 54 <211> 86 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Insulin glargine precursor (INSG) <400> 54 Phe Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr 1 5 10 15 Leu Val Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg 20 25 30 Glu Ala Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro 35 40 45 Gly Ala Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys 50 55 60 Arg Gly Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln 65 70 75 80 Leu Glu Asn Tyr Cys Gly 85 <210> 55 <211> 99 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV-INSG <400> 55 Met His His His His His His His Glu Asn Leu Tyr Phe Gln Phe Val Asn 1 5 10 15 Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys 20 25 30 Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu 35 40 45 Asp Leu Gln Val Gly Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly 50 55 60 Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile 65 70 75 80 Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn 85 90 95 Tyr Cys Gly <210> 56 <211> 195 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG97-H6TEV-INSG <400> 56 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu 1 5 10 15 Val Glu Thr Lys Ser Ala Gly Gly Ile Val Leu Thr Gly Ser Ala Ala 20 25 30 Ala Lys Ser Thr Arg Gly Glu Val Leu Ala Val Gly Asn Gly Arg Ile 35 40 45 Leu Glu Asn Gly Glu Val Lys Pro Leu Asp Val Lys Val Gly Asp Ile 50 55 60 Val Ile Phe Asn Asp Gly Tyr Gly Val Lys Ser Glu Lys Ile Asp Asn 65 70 75 80 Glu Glu Val Leu Ile Met Ser Glu Ser Asp Ile Leu Ala Ile Val Glu 85 90 95 Ala His His His His His His His Glu Asn Leu Tyr Phe Gln Phe Val Asn 100 105 110 Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val Cys 115 120 125 Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala Glu 130 135 140 Asp Leu Gln Val Gly Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala Gly 145 150 155 160 Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly Ile 165 170 175 Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu Asn 180 185 190 Tyr Cys Gly 195 <210> 57 <211> 196 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for SUMO-H6TEV-INSG <400> 57 Met Ser Asp Ser Glu Val Asn Gln Glu Ala Lys Pro Glu Val Lys Pro 1 5 10 15 Glu Val Lys Pro Glu Thr His Ile Asn Leu Lys Val Ser Asp Gly Ser 20 25 30 Ser Glu Ile Phe Phe Lys Ile Lys Lys Thr Thr Pro Leu Arg Arg Leu 35 40 45 Met Glu Ala Phe Ala Lys Arg Gln Gly Lys Glu Met Asp Ser Leu Arg 50 55 60 Phe Leu Tyr Asp Gly Ile Arg Ile Gln Ala Asp Gln Thr Pro Glu Asp 65 70 75 80 Leu Asp Met Glu Asp Asn Asp Ile Ile Glu Ala His Arg Glu Gln Ile 85 90 95 Gly Gly His His His His His His His Glu Asn Leu Tyr Phe Gln Phe Val 100 105 110 Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Tyr Leu Val 115 120 125 Cys Gly Glu Arg Gly Phe Phe Tyr Thr Pro Lys Thr Arg Arg Glu Ala 130 135 140 Glu Asp Leu Gln Val Gly Gln Val Glu Leu Gly Gly Gly Pro Gly Ala 145 150 155 160 Gly Ser Leu Gln Pro Leu Ala Leu Glu Gly Ser Leu Gln Lys Arg Gly 165 170 175 Ile Val Glu Gln Cys Cys Thr Ser Ile Cys Ser Leu Tyr Gln Leu Glu 180 185 190 Asn Tyr Cys Gly 195 <210> 58 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for H6TEV-GLP-1K28R <400> 58 atgcatcatc atcaccacca cgaaaacctg tatttccagc atgcggaagg cacctttacc 60 agcgatgtga gcagctatct ggaaggccag gcggcgaaag aatttattgc gtggctggtg 120 cgtggccgcg gctaa 135 <210> 59 <211> 267 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG43-H6TEV-GLP-1K28R <400> 59 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcc catggcatca tcatcaccac cacgaaaacc tgtatttcca gcatgcggaa 180 ggcaccttta ccagcgatgt gagcagctat ctggaaggcc aggcggcgaa agaatttatt 240 gcgtggctgg tgcgtggccg cggctaa 267 <210> 60 <211> 309 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG57-H6TEV-GLP-1K28R <400> 60 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct gccatggcat 180 catcatcacc accacgaaaa cctgtatttc cagcatgcgg aaggcacctt taccagcgat 240 gtgagcagct atctggaagg ccaggcggcg aaagaattta ttgcgtggct ggtgcgtggc 300 cgcggctaa 309 <210> 61 <211> 342 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG68-H6TEV-GLP-1K28R <400> 61 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacccatgg catcatcatc accaccacga aaacctgtat 240 ttccagcatg cggaaggcac ctttaccagc gatgtgagca gctatctgga aggccaggcg 300 gcgaaagaat ttattgcgtg gctggtgcgt ggccgcggct aa 342 <210> 62 <211> 375 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG79-H6TEV-GLP-1K28R <400> 62 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgaccca 240 tggcatcatc atcaccacca cgaaaacctg tatttccagc atgcggaagg cacctttacc 300 agcgatgtga gcagctatct ggaaggccag gcggcgaaag aatttattgc gtggctggtg 360 cgtggccgcg gctaa 375 <210> 63 <211> 402 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG88-H6TEV-GLP-1K28R <400> 63 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat 240 gaagaagtgt tgatcatgtc cgaaccatgg catcatcatc accaccacga aaacctgtat 300 ttccagcatg cggaaggcac ctttaccagc gatgtgagca gctatctgga aggccaggcg 360 gcgaaagaat ttattgcgtg gctggtgcgt ggccgcggct aa 402 <210> 64 <211> 423 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG97-H6TEV-GLP-1K28R <400> 64 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat 240 gaagaagtgt tgatcatgtc cgaaagcgac attctggcaa ttgttgaagc gcatcatcat 300 caccaccacg aaaacctgta tttccagcat gcggaaggca cctttaccag cgatgtgagc 360 agctatctgg aaggccaggc ggcgaaagaa tttattgcgt ggctggtgcg tggccgcggc 420 taa 423 <210> 65 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for SUMO-H6TEV-GLP-1K28R <400> 65 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcatcat 300 catcaccacc acgaaaacct gtatttccag catgcggaag gcacctttac cagcgatgtg 360 agcagctatc tggaaggcca ggcggcgaaa gaatttattg cgtggctggt gcgtggccgc 420 ggcta 426 <210> 66 <211> 144 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for H6TEV-hPTH1-34 <400> 66 atgcatcatc atcaccacca cgaaaacctg tatttccagt ctgtgagtga aatacagctt 60 atgcataacc tgggaaaaca tctgaactcg atggagagag tagaatggct gcgtaagaag 120 ctgcaggatg tgcacaattt ttaa 144 <210> 67 <211> 531 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG97-H6TEV-hPTH1-34 <400> 67 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcatcat 300 catcaccacc acgaaaacct gtatttccag catgcggaag gcacctttac cagcgatgtg 360 agcagctatc tggaaggcca ggcggcgaaa gaatttattg cgtggctggt gcgtggccgc 420 ggctaaagcg tgagcgaaat tcagctgatg cataacctgg gcaaacatct gaacagcatg 480 gaacgcgtgg aatggctgcg caaaaaactg caggatgtgc ataactttta a 531 <210> 68 <211> 435 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for SUMO-H6TEV-hPTH1-34 <400> 68 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcatcat 300 catcaccacc acgaaaacct gtatttccag agcgtgagcg aaattcagct gatgcataac 360 ctgggcaaac atctgaacag catggaacgc gtggaatggc tgcgcaaaaa actgcaggat 420 gtgcataact tttaa 435 <210> 69 <211> 141 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for H6TEV-GLP-2A2G <400> 69 atgcatcatc atcaccacca cgaaaacctg tatttccagc atggcgatgg cagctttagc 60 gatgaaatga acaccattct ggataacctg gcggcgcgcg attttattaa ctggctgatt 120 cagaccaaaa ttaccgatta a 141 <210> 70 <211> 429 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG97-H6TEV-GLP-2A2G <400> 70 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat 240 gaagaagtgt tgatcatgtc cgaaagcgac attctggcaa ttgttgaagc gcatcatcat 300 caccaccacg aaaacctgta tttccagcat ggcgatggca gctttagcga tgaaatgaac 360 accattctgg ataacctggc ggcgcgcgat tttattaact ggctgattca gaccaaaatt 420 accgattaa 429 <210> 71 <211> 432 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for SUMO-H6TEV-GLP-2A2G <400> 71 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcatcat 300 catcaccacc acgaaaacct gtatttccag catggcgatg gcagctttag cgatgaaatg 360 aacaccattc tggataacct ggcggcgcgc gattttatta actggctgat tcagaccaaa 420 attaccgatt aa 432 <210> 72 <211> 222 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for H6TEV-Ecallantide <400> 72 atgcatcatc atcaccacca cgaaaacctg tatttccagg aagcgatgca tagcttttgc 60 gcgtttaaag cggatgatgg cccgtgccgc gcggcgcatc cgcgctggtt ttttaacatt 120 tttacccgcc agtgcgaaga atttatttat ggcggctgcg aaggcaacca gaaccgcttt 180 gaaagcctgg aagaatgcaa aaaaatgtgc acccgcgatt aa 222 <210> 73 <211> 510 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG97-H6TEV-Ecallantide <400> 73 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat 240 gaagaagtgt tgatcatgtc cgaaagcgac attctggcaa ttgttgaagc gcatcatcat 300 caccaccacg aaaacctgta tttccaggaa gcgatgcata gcttttgcgc gtttaaagcg 360 gatgatggcc cgtgccgcgc ggcgcatccg cgctggtttt ttaacatttt tacccgccag 420 tgcgaagaat ttatttatgg cggctgcgaa ggcaaccaga accgctttga aagcctggaa 480 gaatgcaaaa aaatgtgcac ccgcgattaa 510 <210> 74 <211> 513 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for SUMO-H6TEV-Ecallantide <400> 74 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcatcat 300 catcaccacc acgaaaacct gtatttccag gaagcgatgc atagcttttg cgcgtttaaa 360 gcggatgatg gcccgtgccg cgcggcgcat ccgcgctggt tttttaacat ttttacccgc 420 cagtgcgaag aatttattta tggcggctgc gaaggcaacc agaaccgctt tgaaagcctg 480 gaagaatgca aaaaaatgtg cacccgcgat taa 513 <210> 75 <211> 138 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for H6TEV-Nesiritide <400> 75 atgcatcatc atcaccacca cgaaaacctg tatttccaga gcccgaaaat ggtgcagggc 60 agcggctgct ttggccgcaa aatggatcgc attagcagca gcagcggcct gggctgcaaa 120 gtgctgcgcc gccattaa 138 <210> 76 <211> 426 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG97-H6TEV-Nesiritide <400> 76 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat 240 gaagaagtgt tgatcatgtc cgaaagcgac attctggcaa ttgttgaagc gcatcatcat 300 caccaccacg aaaacctgta tttccagagc ccgaaaatgg tgcagggcag cggctgcttt 360 ggccgcaaaa tggatcgcat tagcagcagc agcggcctgg gctgcaaagt gctgcgccgc 420 cattaa 426 <210> 77 <211> 429 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for SUMO-H6TEV-Nesiritide <400> 77 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcatcat 300 catcaccacc acgaaaacct gtatttccag agcccgaaaa tggtgcaggg cagcggctgc 360 tttggccgca aaatggatcg cattagcagc agcagcggcc tgggctgcaa agtgctgcgc 420 cgccatta 429 <210> 78 <211> 201 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for H6TEV-hEGF <400> 78 atgcatcatc atcaccacca cgaaaacctg tatttccaga actccgattc tgagtgccca 60 ctttcccacg atggatactg ccttcatgat ggcgtgtgca tgtacatcga ggctctcgat 120 aagtacgcct gcaactgcgt tgtgggttac attggagaga gatgccagta cagggatctc 180 aagtggtggg agcttaggta a 201 <210> 79 <211> 489 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG97-H6TEV-hEGF <400> 79 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat 240 gaagaagtgt tgatcatgtc cgaaagcgac attctggcaa ttgttgaagc gcatcatcat 300 caccaccacg aaaacctgta tttccagaac tccgattctg agtgcccact ttccccacgat 360 ggatactgcc ttcatgatgg cgtgtgcatg tacatcgagg ctctcgataa gtacgcctgc 420 aactgcgttg tgggttacat tggagagaga tgccagtaca gggatctcaa gtggtgggag 480 cttaggtaa 489 <210> 80 <211> 492 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for SUMO-H6TEV-hEGF <400> 80 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcatcat 300 catcaccacc acgaaaacct gtatttccag aactccgatt ctgagtgccc actttcccac 360 gatggatact gccttcatga tggcgtgtgc atgtacatcg aggctctcga taagtacgcc 420 tgcaactgcg ttgtgggtta cattggagag agatgccagt acagggatct caagtggtgg 480 gagcttaggt aa 492 <210> 81 <211> 537 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for H6TEV-INF-beta1b <400> 81 atgcatcatc atcaccacca cgaaaacctg tatttccaga gctataacct gctgggcttt 60 ctgcagcgca gcagcaactt tcagagccag aaactgctgt ggcagctgaa cggccgcctg 120 gaatattgcc tgaaagatcg catgaacttt gatattccgg aagaaattaa acagctgcag 180 cagtttcaga aagaagatgc ggcgctgacc atttatgaaa tgctgcagaa catttttgcg 240 atttttcgcc aggatagcag cagcaccggc tggaacgaaa ccattgtgga aaacctgctg 300 gcgaacgtgt atcatcagat taaccatctg aaaaccgtgc tggaagaaaa actggaaaaa 360 gaagatttta cccgcggcaa actgatgagc agcctgcatc tgaaacgcta ttatggccgc 420 attctgcatt atctgaaagc gaaagaatat agccattgcg cgtggaccat tgtgcgcgtg 480 gaaattctgc gcaactttta ttttattaac cgcctgaccg gctatctgcg caactaa 537 <210> 82 <211> 825 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG97-H6TEV-INF-beta1b <400> 82 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat 240 gaagaagtgt tgatcatgtc cgaaagcgac attctggcaa ttgttgaagc gcatcatcat 300 caccaccacg aaaacctgta tttccagagc tataacctgc tgggctttct gcagcgcagc 360 agcaactttc agagccagaa actgctgtgg cagctgaacg gccgcctgga atattgcctg 420 aaagatcgca tgaactttga tattccggaa gaaattaaac agctgcagca gtttcagaaa 480 gaagatgcgg cgctgaccat ttatgaaatg ctgcagaaca tttttgcgat ttttcgccag 540 gatagcagca gcaccggctg gaacgaaacc attgtggaaa acctgctggc gaacgtgtat 600 catcagatta accatctgaa aaccgtgctg gaagaaaaac tggaaaaaga agattttacc 660 cgcggcaaac tgatgagcag cctgcatctg aaacgctatt atggccgcat tctgcattat 720 ctgaaagcga aagaatatag ccattgcgcg tggaccattg tgcgcgtgga aattctgcgc 780 aacttttatt ttattaaccg cctgaccggc tatctgcgca actaa 825 <210> 83 <211> 828 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for SUMO-H6TEV-INF-beta1b <400> 83 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcatcat 300 catcaccacc acgaaaacct gtatttccag agctataacc tgctgggctt tctgcagcgc 360 agcagcaact ttcagagcca gaaactgctg tggcagctga acggccgcct ggaatattgc 420 ctgaaagatc gcatgaactt tgatattccg gaagaaatta aacagctgca gcagtttcag 480 aaagaagatg cggcgctgac catttatgaa atgctgcaga acatttttgc gatttttcgc 540 caggatagca gcagcaccgg ctggaacgaa accattgtgg aaaacctgct ggcgaacgtg 600 tatcatcaga ttaaccatct gaaaaccgtg ctggaagaaa aactggaaaa agaagatttt 660 acccgcggca aactgatgag cagcctgcat ctgaaacgct attatggccg cattctgcat 720 tatctgaaag cgaaagaata tagccattgc gcgtggacca ttgtgcgcgt ggaaattctg 780 cgcaactttt attttattaa ccgcctgacc ggctatctgc gcaactaa 828 <210> 84 <211> 612 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for H6TEV-hGH <400> 84 catcatcatc accaccacga aaacctgtat ttccagtttc caaccattcc actgtctcgc 60 ctgtttgata atgcaatgct gcgtgcacat cgtctgcatc agctggcatt tgatacctat 120 caggaatttg aagaagcata tattccaaaa gaacagaaat attctttcct gcaaaatcca 180 cagacctctc tgtgtttttc tgaatctatt ccaaccccat ccaatcgtga agagacccaa 240 cagaaatcta atctggaact gctgcgtatc tctctgttac tgattcagtc ttggctggaa 300 ccagtgcagt tcctgcgttc tgtgttcgca aattctctgg tttatggtgc atctgattct 360 aatgtttatg atctgctgaa agatttagag gaaggaattc agaccctgat gggtcgtctg 420 gaagatggtt ctccacgtac cggtcagatt tttaaacaga cctattctaa atttgatacc 480 aattctcata atgacgatgc actgctgaaa aactatggtc tgctgtactg ctttcgtaaa 540 gatatggata aagttgaaac ctttctgcgt attgttcagt gtcgttctgt tgaaggttct 600 tgtggttttt aa 612 <210> 85 <211> 903 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG97-H6TEV-hGH <400> 85 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat 240 gaagaagtgt tgatcatgtc cgaaagcgac attctggcaa ttgttgaagc gcatcatcat 300 caccaccacg aaaacctgta tttccagttt ccaaccattc cactgtctcg cctgtttgat 360 aatgcaatgc tgcgtgcaca tcgtctgcat cagctggcat ttgataccta tcaggaattt 420 gaagaagcat atattccaaa agaacagaaa tattctttcc tgcaaaatcc acagacctct 480 ctgtgttttt ctgaatctat tccaacccca tccaatcgtg aagagaccca acagaaatct 540 aatctggaac tgctgcgtat ctctctgtta ctgattcagt cttggctgga accagtgcag 600 ttcctgcgtt ctgtgttcgc aaattctctg gtttatggtg catctgattc taatgtttat 660 gatctgctga aagatttaga ggaaggaatt cagaccctga tgggtcgtct ggaagatggt 720 tctccacgta ccggtcagat ttttaaacag acctattcta aatttgatac caattctcat 780 aatgacgatg cactgctgaa aaactatggt ctgctgtact gctttcgtaa agatatggat 840 aaagttgaaa cctttctgcg tattgttcag tgtcgttctg ttgaaggttc ttgtggtttt 900 taa 903 <210> 86 <211> 906 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for SUMO-H6TEV-hGH <400> 86 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcatcat 300 catcaccacc acgaaaacct gtatttccag tttccaacca ttccactgtc tcgcctgttt 360 gataatgcaa tgctgcgtgc acatcgtctg catcagctgg catttgatac ctatcaggaa 420 tttgaagaag catatattcc aaaagaacag aaatattctt tcctgcaaaa tccacagacc 480 tctctgtgtt tttctgaatc tattccaacc ccatccaatc gtgaagagac ccaacagaaa 540 tctaatctgg aactgctgcg tatctctctg ttactgattc agtcttggct ggaaccagtg 600 cagttcctgc gttctgtgtt cgcaaattct ctggtttatg gtgcatctga ttctaatgtt 660 tatgatctgc tgaaagattt agaggaagga attcagaccc tgatgggtcg tctggaagat 720 ggttctccac gtaccggtca gatttttaaa cagacctatt ctaaatttga taccaattct 780 cataatgacg atgcactgct gaaaaactat ggtctgctgt actgctttcg taaagatatg 840 gataaagttg aaacctttct gcgtattgtt cagtgtcgtt ctgttgaagg ttcttgtggt 900 ttttaa 906 <210> 87 <211> 300 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for H6TEV-INSG <400> 87 atgcatcatc atcaccacca cgaaaacctg tatttccagt ttgtgaacca gcatctgtgc 60 ggcagccatc tggtggaagc gctgtatctg gtgtgcggcg aacgcggctt tttttatacc 120 ccgaaaaccc gccgcgaagc ggaagatctg caggtgggcc aggtggaact gggcggcggc 180 ccgggcgcgg gcagcctgca gccgctggcg ctggaaggca gcctgcagaa acgcggcatt 240 gtggaacagt gctgcaccag catttgcagc ctgtatcagc tggaaaacta ttgcggctaa 300 300 <210> 88 <211> 588 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for PG97-H6TEV-INSG <400> 88 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccg cggcgaagtg 120 ctggctgtcg gcaatggccg tatccttgaa aatggcgaag tgaagccgct ggatgtgaaa 180 gttggcgaca tcgttatttt caacgatggc tacggtgtga aatctgagaa gatcgacaat 240 gaagaagtgt tgatcatgtc cgaaagcgac attctggcaa ttgttgaagc gcatcatcat 300 caccaccacg aaaacctgta tttccagttt gtgaaccagc atctgtgcgg cagccatctg 360 gtggaagcgc tgtatctggt gtgcggcgaa cgcggctttt tttatacccc gaaaacccgc 420 cgcgaagcgg aagatctgca ggtgggccag gtggaactgg gcggcggccc gggcgcgggc 480 agcctgcagc cgctggcgct ggaaggcagc ctgcagaaac gcggcattgt ggaacagtgc 540 tgcaccagca tttgcagcct gtatcagctg gaaaactatt gcggctaa 588 <210> 89 <211> 591 <212> DNA <213> Artificial Sequence <220> <223> nucleotide sequence for SUMO-H6TEV-INSG <400> 89 atgtcggact cagaagtcaa tcaagaagct aagccagagg tcaagccaga agtcaagcct 60 gagactcaca tcaatttaaa ggtgtccgat ggatcttcag agatcttctt caagatcaaa 120 aagaccactc ctttaagaag gctgatggaa gcgttcgcta aaagacaggg taaggaaatg 180 gactccttaa gattcttgta cgacggtatt agaattcaag ctgatcagac ccctgaagat 240 ttggacatgg aggataacga tattattgag gctcacagag aacagattgg tggtcatcat 300 catcaccacc acgaaaacct gtatttccag tttgtgaacc agcatctgtg cggcagccat 360 ctggtggaag cgctgtatct ggtgtgcggc gaacgcggct ttttttatac cccgaaaacc 420 cgccgcgaag cggaagatct gcaggtgggc caggtggaac tgggcggcgg cccgggcgcg 480 ggcagcctgc agccgctggc gctggaaggc agcctgcaga aacgcggcat tgtggaacag 540 tgctgcacca gcatttgcag cctgtatcag ctggaaaact attgcggcta a 591

Claims (9)

a fusion tag consisting of the amino acid sequence of SEQ ID NO: 6; linker; a tobacco etch virus (TEV) protease recognition sequence consisting of the amino acid sequence of SEQ ID NO: 9; An expression vector comprising a polynucleotide encoding a fusion polypeptide in which a liraglutide precursor peptide (GLP-1K28R) consisting of the amino acid sequence of SEQ ID NO: 17 is sequentially positioned from the N-terminus to the C-terminus (a) to prepare a;
After the step (a), introducing the expression vector into E. coli to prepare a transformed E. coli (b);
After the step (b), culturing the transformed E. coli (c);
(d) isolating and solubilizing the fusion polypeptide expressed in the form of an inclusion body in the transformed E. coli after step (c); and
After step (d), the solubilized fusion polypeptide is treated with tobacco etch virus (TEV) protease to recover a liraglutide precursor peptide (GLP-1K28R) consisting of the amino acid sequence of SEQ ID NO: 17 ( e); Liraglutide (liraglutide) precursor peptide (GLP-1K28R) production method comprising the amino acid sequence of SEQ ID NO: 17.
delete delete delete According to claim 1,
The linker is
A method for producing a liraglutide precursor peptide (GLP-1K28R) consisting of the amino acid sequence of SEQ ID NO: 17, comprising a histidine affinity tag. delete delete delete delete

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