KR102017540B1 - Method of preparing glucagon like peptide-1 or analogues using fusion polypeptide - Google Patents

Abstract

The present invention provides a method for producing GLP-1 or an analog thereof using a fusion polypeptide comprising an N-terminal fusion partner and glucagon-like peptide-1 or an analog thereof. The fusion polypeptide according to the present invention can improve the yield in the production of GLP-1 or its analogs by genetic recombination technology, and can stably express GLP-1 or its analogs by minimizing degradation from proteolytic enzymes of host cells. have.

Description

METHODS OF PREPARING GLUCAGON LIKE PEPTIDE-1 OR ANALOGUES USING FUSION POLYPEPTIDE}

The present invention relates to a method for producing GLP-1 or an analog thereof using a fusion polypeptide comprising an N-terminal fusion partner and glucagon-like peptide-1 (GLP-1) or an analog thereof.

Diabetes is a metabolic disorder in which blood sugar levels are elevated due to a deficiency in insulin secretion, a deficiency in insulin action, or both. Diabetes is largely divided into two types, and is divided into insulin-dependent type 1 diabetes and insulin-independent (insulin resistant) type 2 diabetes.

Type 2 diabetes now accounts for 90% to 95% of all diabetes. Type 2 diabetes is a complex disease whose mechanism is unclear, but may occur during puberty, but usually occurs in adults. Patients with type 2 diabetes have complications of macrovascular and microvascular, including coronary artery disease, stroke, peripheral vascular disease, hypertension, kidney disease, neuropathy and retinopathy. In this regard, despite advances in diabetes treatment, hypoglycemic shock is often a limiting factor in achieving optimal glycemic control.

Meanwhile, glucagon-like peptide-1 (GLP-1) is used as one of the active substances in the body for the treatment and prevention of diabetes. GLP-1 is a hormone that stimulates glucose dependent insulin secretion while inhibiting glucagon secretion. Various methods are currently proposed for producing GLP-1 or an analog thereof as a therapeutic agent for type 2 diabetes. Recently, attempts have been made to mass-produce GLP-1 using genetic recombination technology.

Genetic recombination technology is a method of producing a target protein by cloning the nucleic acid of several important proteins to an expression vector to obtain a recombinant expression vector, and transduced into a suitable host cell culture. However, when the target protein is degraded by the degradation enzyme inherent in the host cell, the yield is low, or when the fusion partner is used, the size of the peptide is too large compared with the size of the target protein to be prepared, and thus the yield is significantly reduced.

Therefore, in the case of mass production of the GLP-1 or its analogues using genetic recombination technology, it is important to develop a fusion partner capable of stably expressing the polypeptide while improving production yield.

Accordingly, the present inventors have studied to develop a fusion partner capable of stably expressing GLP-1 or an analog thereof and improve the production yield, and thus include an N-terminal fusion partner and GLP-1 or an analog thereof according to the present invention. When the production of GLP-1 or an analog thereof using the fusion polypeptide, the present invention was completed by confirming that the production yield of GLP-1 or an analog thereof is increased.

In order to achieve the above object, one aspect of the present invention, (a) N-terminal fusion partner consisting of the amino acid sequence represented by the general formula (1); Glucagon like peptide-1 (GLP-1) or analogs thereof; And preparing an expression vector comprising a nucleotide encoding a fusion polypeptide comprising a linker between the N-terminal fusion partner and the GLP-1 or an analog thereof; (b) transducing the expression vector into a host cell; (c) culturing the host cell; (d) purifying the fusion polypeptide expressed in the host cell; And (e) culturing the purified fusion polypeptide with a cleavage enzyme to recover GLP-1 or an analog thereof.

[Formula 1]

Met-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6- (Z) _n

In the general formula 1,

Xaa1 to Xaa6, independently of each other, isoleucine (Ile, I), glycine (Gly, G), alanine (Ala, A), proline (Pro, P), valine (Val, V), leucine (Leu, L ), Methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W), asparagine (Asn, N), serine (Ser, S), threonine (Thr, T) Cysteine (Cys, C), Glutamine (Gln, Q), Arginine (Arg, R), Lysine (Lys, K), Histidine (His, H), Asphalt acid (Asp, D) and Glutamic acid (Glu, E) Selected from the group consisting of

Z is 1 to 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345,

N is an integer of 0 or 1.

Fusion polypeptides according to the present invention include novel fusion partners having a lower molecular weight than known fusion partners. By using such a low molecular weight fusion partner, the production yield of GLP-1 or an analog thereof can be improved. In addition, the fusion polypeptide according to the present invention has the advantage of stably producing GLP-1 or an analog thereof by preventing expression from proteolytic enzymes of the host cell by inducing expression in the inclusion body form in the host cell. Thus, it is possible to provide a method for producing GLP-1 or an analog thereof with improved stability and yield than with conventional fusion partners.

Figure 1 shows the result of SDS-PAGE analysis of the total protein produced in recombinant E. coli (lane M: marker protein, lane 1: H6TEV-GLP-1K28R (strain number PG005), lane 2: PG07-H6TEV-GLP-1K28R) (Strain number PG006), lane 3: PG15-H6TEV-GLP-1K28R (strain number PG007), lane 4: PG22-H6TEV-GLP-1K28R (strain number PG008), lane 5: PG29-H6TEV-GLP-1K28R (strain Number PG009), lane 6: PG36-H6TEV-GLP-1K28R (strain number PG010), lane 7: PG43-H6TEV-GLP-1K28R (strain number PG011)).
Figure 2 shows the result of analysis by SDS-PAGE after separating the total cell fraction of recombinant E. coli into soluble and insoluble fraction (lane M: marker protein, lane T: total fraction, lane S: soluble fraction, lane I: insoluble fraction) Lane 1: H6TEV-GLP-1K28R (strain number PG005), lane 2: PG07-H6TEV-GLP-1K28R (strain number PG006), lane 3: PG15-H6TEV-GLP-1K28R (strain number PG007), lane 4: PG22-H6TEV-GLP-1K28R (strain number PG008).
Figure 3 shows the result of analysis by SDS-PAGE after separating the total cell fraction of recombinant E. coli into soluble and insoluble fraction (lane M: marker protein, lane T: total fraction, lane S: soluble fraction, lane I: insoluble fraction) Lane 5: PG29-H6TEV-GLP-1K28R (strain number PG009), lane 6: PG36-H6TEV-GLP-1K28R (strain number PG010), lane 7: PG43-H6TEV-GLP-1K28R (strain number PG011).
4 is a GLP-1K28R fusion poly substituted with isoleucine (I), asparagine (N), arginine (R), and asphalt acid (D) for the second or third amino acid residue of PG43 in PG43-H6TEV-GLP-1K28R. The result of analyzing the variants of the peptides by SDS-PAGE.
FIG. 5 shows a GLP-1K28R fusion poly substituted with isoleucine (I), asparagine (N), arginine (R), and asphalt acid (D) for the fourth or fifth amino acid residue of PG43 in PG43-H6TEV-GLP-1K28R. The result of analyzing the variants of the peptides by SDS-PAGE.
FIG. 6 shows GLP-1K28R fusion poly substituted with isoleucine (I), asparagine (N), arginine (R), and asphalt acid (D) with the sixth or seventh amino acid residue of PG43 in PG43-H6TEV-GLP-1K28R. The result of analyzing the variants of the peptides by SDS-PAGE.
FIG. 7 shows the results of purification of the PG43-H6TEV-GLP-1K28R fusion polypeptide in the insoluble fraction by chromatography (blue and green lines in the chromatogram, respectively, indicate absorbance at 280 nm wavelength, elution buffer ratio).
8 is a result of SDS-PAGE analysis of the purified PG43-H6TEV-GLP-1K28R fusion polypeptide by chromatography (lane M: marker protein, lane S: sample before purification, FT: flow-through fraction, lane E). Eluent fraction). Arrows indicate PG43-H6TEV-GLP-1 K28R fusion polypeptides.
9 is a result of analyzing the fraction by SDS-PAGE after cleavage of purified PG43-H6TEV-GLP-1K28R fusion polypeptide using TEV protease (lane M: marker protein, lane C: sample not treated with TEV protease, Lane T: sample treated with TEV protease).
Figure 10 shows the result of measuring the molecular weight of GLP-1K28R purified according to the present invention.

Hereinafter, the present invention will be described in detail.

One aspect of the invention, (a) N-terminal fusion partner consisting of the amino acid sequence represented by the general formula (1); Glucagon like peptide-1 (GLP-1) or analogs thereof; And preparing an expression vector comprising a nucleotide encoding a fusion polypeptide comprising a linker between the N-terminal fusion partner and the GLP-1 or an analog thereof; (b) transducing the expression vector into a host cell; (c) culturing the host cell; (d) purifying the fusion polypeptide expressed in the host cell; And (e) culturing the purified fusion polypeptide with a cleavage enzyme to recover GLP-1 or an analog thereof.

[Formula 1]

Met-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6- (Z) _n

In the general formula 1,

Xaa1 to Xaa6, independently of each other, isoleucine (Ile, I), glycine (Gly, G), alanine (Ala, A), proline (Pro, P), valine (Val, V), leucine (Leu, L ), Methionine (Met, M), phenylalanine (Phe, F), tyrosine (Tyr, Y), tryptophan (Trp, W), asparagine (Asn, N), serine (Ser, S), threonine (Thr, T) Cysteine (Cys, C), Glutamine (Gln, Q), Arginine (Arg, R), Lysine (Lys, K), Histidine (His, H), Asphalt acid (Asp, D) and Glutamic acid (Glu, E) Selected from the group consisting of

Z is 1 to 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345,

N is an integer of 0 or 1.

First, in the step (a), N-terminal fusion partner consisting of the amino acid sequence represented by the formula (1); Glucagon like peptide-1 (GLP-1) or analogs thereof; And a nucleotide encoding a fusion polypeptide comprising a linker between the N-terminal fusion partner and the GLP-1 or an analog thereof.

The glucagon-like peptide-1 (GLP-1) is a polypeptide of 31 amino acids. The GLP-1 is a hormone with functions such as stimulating insulin secretion, inhibiting glucagon secretion, inhibiting gastric or intestinal motility, and inducing weight loss. Specifically, the GLP-1 may be composed of the amino acid sequence of SEQ ID NO: 151. The GLP-1 may be encoded by a nucleotide sequence represented by SEQ ID NO: 293.

In addition, an analog of GLP-1 is a peptide having a function similar to that of GLP-1. For example, it may be a peptide having a variation in substitution, addition and / or deletion of amino acids as compared to native GLP-1, within the range that the activity is maintained or improved.

For example, liraglutide is an analog of GLP-1, in which the 28th lysine of GLP-1 is substituted with arginine (K28R) and N-Palmitoyl-L- consisting of palmitic acid and glutamic acid in the amino group of the 20th lysine residue. It has a form in which glutamic acid is bound. The liraglutide may be used as a therapeutic agent for type 2 diabetes or obesity, and in general, the liraglutide produces a liraglutide precursor peptide (GLP-1K28R) to which N-Palmitoyl-L-glutamic acid is not bound. It can be produced by the process of binding N-Palmitoyl-L-glutamic acid to its 20th lysine residue (Dunweber, Jensen et al . 2007). Specifically, the GLP-1K28R may be composed of the amino acid sequence of SEQ ID NO: 152. The GLP-1K28R may be encoded by the nucleotide sequence represented by SEQ ID NO: 294.

When the target polypeptide is produced using a recombinant microbial system, it may be degraded by an enzyme in the host cell according to the properties of the target material. In addition, the target polypeptide may be expressed at a low level, and there is a concern that the yield of the target protein may be lowered due to improper protein folding.

In order to solve the above problem, there is a problem that a fusion partner is used but still needs to be improved. For example, MBP (maltose binding protein), glutathione S-transferase, thiorredoxin, SUMO, ubiquitin, etc., which are conventionally used as fusion partners, are 397, 216, 106, and 101, respectively. Although having 76 amino acids, there was a problem in that the yield was not good when producing a relatively low molecular weight target polypeptide.

In contrast, the N-terminal fusion partner is a low molecular weight peptide consisting of 7 to 43 amino acids. When applied to produce GLP-1 or an analog thereof, GLP-1 or an analog thereof can be obtained with improved yield than when utilizing existing fusion partners. For example, the ratio of GLP-1K28R as an analog of GLP-1 in a recombinant fusion polypeptide is shown in Table 1 below.

Fusion partner Amino acid count Molecular Weight (kDa) GLP-1K28R Rate (%) * MBP (Maltose binding protein) 397 44.2 7 Glutathione S-transferase 216 23.8 12 Thioredoxin 106 11.7 21 SUMO 101 11.1 22 Ubiquitin 76 8.4 27 N-terminal fusion partner comprising any of the amino acid sequences of SEQ ID NOs: 9, 31, 53, 75, 97 or 119 7-43 0.8-4.7 60-35

* Calculation including linker part

As shown in Table 1, conventional fusion partners have a ratio of 7% to 27% of GLP-1K28R in the fusion polypeptide, whereas the ratio of GLP-1K28R in the fusion polypeptide to which the fusion partner according to the present invention is fused. Is identified as 35% to 60%. Thus, the final production yield can be improved since the amount of GLP-1K28R obtainable from the same concentration of fusion polypeptide is higher than that of conventional fusion partners.

In addition, the fusion partner may induce insoluble expression of the fusion polypeptide, causing the fusion polypeptide to accumulate in high concentration into the host cell as an insoluble inclusion body. Therefore, there is an advantage that it is easy to obtain GLP-1 or an analog thereof, which may be degraded or cleaved in whole or in part by proteases and peptidase in host cells such as Escherichia coli .

In the N-terminal fusion partner, the Xaa1 to Xaa6, independently of each other, isoleucine (Ile, I), proline (Pro, P), leucine (Leu, L), asparagine (Asn, N), arginine ( Arg, R), histidine (His, H) and asphalt acid (Asp, D).

Further, in the N-terminal fusion partner, when n is an integer of 0, the N-terminal fusion partner may be composed of seven amino acids. In addition, when n is an integer of 1, Z is 1, 2, 3, 4, 5, 6, 7 starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345. , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 Dog, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 amino acids. Specifically, when n is an integer of 1, Z may be 8, 15, 22, 29, or 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345.

For example, the N-terminal fusion partner may be composed of the amino acid sequence represented by the following formula (2).

[Formula 2]

Met-Xaa1-Ile-Arg-Pro-Leu-His- (Z) _n

In the general formula 2,

Xaa1 is isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine, tryptophan, asparagine, serine, threonine, cysteine, glutamine, arginine, lysine, histidine, asphalt acid or glutamic acid,

Z is 1 to 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345,

N is an integer of 0 or 1.

In addition, the Xaa1 may be selected from the group consisting of isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine and tryptophan. Specifically, Xaa1 may be selected from the group consisting of isoleucine (Ile, I), asparagine (Asn, N), arginine (Arg, R), and asphalt acid (Asp, D).

When n is an integer of 0, the N-terminal fusion partner may be composed of seven amino acids. In addition, when n is an integer of 1, Z is 1, 2, 3, 4, 5, 6, 7 starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345. , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 Dog, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 amino acids. Specifically, when n is an integer of 1, Z may be 8, 15, 22, 29, or 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345.

 In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 2 may be composed of the amino acid sequence of SEQ ID NO: 8, 30, 52, 74, 96 or 118. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by Formula 2 may be composed of the amino acid sequence of SEQ ID NO: 161, 183, 205, 227, 249 or 271.

In addition, the Xaa1 may be selected from the group consisting of asparagine, serine, threonine, cysteine and glutamine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 2 may be composed of the amino acid sequence of SEQ ID NO: 9, 31, 53, 75, 97 or 119. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by Formula 2 may be composed of the amino acid sequence of SEQ ID NO: 162, 184, 206, 228, 250 or 272.

In addition, the Xaa1 may be selected from the group consisting of arginine, lysine and histidine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 2 may be composed of the amino acid sequence of SEQ ID NO: 10, 32, 54, 76, 98 or 120. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by Formula 2 may be composed of the amino acid sequence of SEQ ID NO: 163, 185, 207, 229, 251 or 273.

In addition, the Xaa1 may be asphalt acid or glutamic acid. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 2 may be composed of the amino acid sequence of SEQ ID NO: 11, 33, 55, 77, 99 or 121. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by Formula 2 may be composed of the amino acid sequence of SEQ ID NO: 164, 186, 208, 230, 252 or 274.

In addition, the N-terminal fusion partner may be composed of the amino acid sequence represented by the following general formula (3).

[Formula 3]

Met-Asn-Xaa2-Arg-Pro-Leu-His- (Z) _n

In the general formula 3,

Xaa2 is isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine, tryptophan, asparagine, serine, threonine, cysteine, glutamine, arginine, lysine, histidine, asphalt acid or glutamic acid,

Z is 1 to 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345,

N is an integer of 0 or 1.

Specifically, Xaa2 may be selected from the group consisting of isoleucine (Ile, I), asparagine (Asn, N), arginine (Arg, R), and asphalt acid (Asp, D).

When n is an integer of 0, the N-terminal fusion partner may be composed of seven amino acids. In addition, when n is an integer of 1, Z is 1, 2, 3, 4, 5, 6, 7 starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345. , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 Dog, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 amino acids. Specifically, when n is an integer of 1, Z may be 8, 15, 22, 29, or 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345.

In addition, the Xaa2 may be selected from the group consisting of isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine and tryptophan.

In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 3 may be composed of the amino acid sequence of SEQ ID NO: 9, 31, 53, 75, 97 or 119. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 3 may be composed of the amino acid sequence of SEQ ID NO: 162, 184, 206, 228, 250 or 272.

In addition, the Xaa2 may be selected from the group consisting of asparagine, serine, threonine, cysteine and glutamine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 3 may be composed of the amino acid sequence of SEQ ID NO: 12, 34, 56, 78, 100 or 122. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 3 may be composed of the amino acid sequence of SEQ ID NO: 165, 187, 209, 231, 253 or 275.

In addition, the Xaa2 may be selected from the group consisting of arginine, lysine and histidine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 3 may be composed of the amino acid sequence of SEQ ID NO: 13, 35, 57, 79, 101 or 123. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by Formula 3 may be composed of the amino acid sequence of SEQ ID NO: 166, 188, 210, 232, 254 or 276.

In addition, Xaa2 may be asphalt acid or glutamic acid. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 3 may be composed of the amino acid sequence of SEQ ID NO: 14, 36, 58, 80, 102 or 124. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by Formula 3 may be composed of the amino acid sequence of SEQ ID NO: 167, 189, 211, 233, 255 or 277.

In addition, the N-terminal fusion partner may be composed of the amino acid sequence represented by the general formula (4).

[Formula 4]

Met-Asn-Ile-Xaa3-Pro-Leu-His- (Z) _n

In the general formula 4,

Xaa3 is isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine, tryptophan, asparagine, serine, threonine, cysteine, glutamine, arginine, lysine, histidine, asphalt acid or glutamic acid,

Z is 1 to 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345,

N is an integer of 0 or 1.

In addition, the Xaa3 may be selected from the group consisting of isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine and tryptophan. Specifically, Xaa3 may be selected from the group consisting of isoleucine (Ile, I), asparagine (Asn, N), arginine (Arg, R), and asphalt acid (Asp, D).

When n is an integer of 0, the N-terminal fusion partner may be composed of seven amino acids. In addition, when n is an integer of 1, Z is 1, 2, 3, 4, 5, 6, 7 starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345. , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 Dog, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 amino acids. Specifically, when n is an integer of 1, Z may be 8, 15, 22, 29, or 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345.

In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 4 may be composed of the amino acid sequence of SEQ ID NO: 15, 37, 59, 81, 103 or 125. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by Formula 4 may be composed of the amino acid sequence of SEQ ID NO: 168, 190, 212, 234, 256 or 278.

In addition, the Xaa 3 may be selected from the group consisting of asparagine, serine, threonine, cysteine and glutamine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 4 may be composed of the amino acid sequence of SEQ ID NO: 16, 38, 60, 82, 104 or 126. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 4 may be composed of the amino acid sequence of SEQ ID NO: 169, 191, 213, 235, 257 or 279.

In addition, the Xaa 3 may be selected from the group consisting of arginine, lysine and histidine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 4 may be composed of the amino acid sequence of SEQ ID NO: 9, 31, 53, 75, 97 or 119. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 4 may be composed of the amino acid sequence of SEQ ID NO: 162, 184, 206, 228, 250 or 272.

In addition, the Xaa 3 may be asphalt acid or glutamic acid. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 4 may be composed of the amino acid sequence of SEQ ID NO: 17, 39, 61, 83, 105 or 127. In addition, the fusion polypeptide including the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 4 may be composed of the amino acid sequence of SEQ ID NO: 170, 192, 213, 236, 258 or 280.

In addition, the N-terminal fusion partner may be composed of the amino acid sequence represented by the general formula (5).

[Formula 5]

Met-Asn-Ile-Arg-Xaa4-Leu-His- (Z) _n

In the general formula 5,

Xaa4 is isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine, tryptophan, asparagine, serine, threonine, cysteine, glutamine, arginine, lysine, histidine, asphalt acid or glutamic acid,

Z is 1 to 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345,

N is an integer of 0 or 1.

In addition, the Xaa4 may be selected from the group consisting of isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine and tryptophan. Specifically, Xaa4 may be selected from the group consisting of isoleucine (Ile, I), asparagine (Asn, N), arginine (Arg, R), and asphalt acid (Asp, D).

When n is an integer of 0, the N-terminal fusion partner may be composed of seven amino acids. In addition, when n is an integer of 1, Z is 1, 2, 3, 4, 5, 6, 7 starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345. , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 Dog, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 amino acids. Specifically, when n is an integer of 1, Z may be 8, 15, 22, 29, or 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345.

In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 5 may be composed of the amino acid sequence of SEQ ID NO: 8, 40, 62, 84, 106 or 128. In addition, the fusion polypeptide including the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 5 may be composed of the amino acid sequence of SEQ ID NO: 171, 193, 215, 237, 259 or 281.

In addition, the Xaa4 may be selected from the group consisting of asparagine, serine, threonine, cysteine and glutamine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 5 may be composed of the amino acid sequence of SEQ ID NO: 19, 41, 63, 85, 107 or 129. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 5 may be composed of the amino acid sequence of SEQ ID NO: 172, 194, 216, 238, 260 or 282.

In addition, the Xaa4 may be selected from the group consisting of arginine, lysine and histidine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 5 may be composed of the amino acid sequence of SEQ ID NO: 20, 42, 64, 86, 108 or 130. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 5 may be composed of the amino acid sequence of SEQ ID NO: 173, 195, 217, 239, 261 or 283.

In addition, Xaa4 may be asphalt acid or glutamic acid. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 5 may be composed of the amino acid sequence of SEQ ID NO: 21, 43, 65, 87, 190 or 131. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 5 may be composed of the amino acid sequence of SEQ ID NO: 174, 196, 218, 240, 262 or 284.

In addition, the N-terminal fusion partner may be composed of the amino acid sequence represented by the following formula (6).

[Formula 6]

Met-Asn-Ile-Arg-Pro-Xaa5-His- (Z) _n

In the general formula 6,

Xaa5 is isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine, tryptophan, asparagine, serine, threonine, cysteine, glutamine, arginine, lysine, histidine, asphalt acid or glutamic acid,

Z is 1 to 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345,

N is an integer of 0 or 1.

In addition, the Xaa5 may be selected from the group consisting of isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine and tryptophan. Specifically, Xaa5 may be selected from the group consisting of isoleucine (Ile, I), asparagine (Asn, N), arginine (Arg, R), and asphalt acid (Asp, D).

When n is an integer of 0, the N-terminal fusion partner may be composed of seven amino acids. In addition, when n is an integer of 1, Z is 1, 2, 3, 4, 5, 6, 7 starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345. , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 Dog, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 amino acids. Specifically, when n is an integer of 1, Z may be 8, 15, 22, 29, or 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345.

In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 6 may be composed of the amino acid sequence of SEQ ID NO: 22, 44, 66, 88, 110 or 132. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by Formula 6 may be composed of the amino acid sequence of SEQ ID NO: 175, 197, 219, 241, 263 or 285.

In addition, the Xaa5 may be selected from the group consisting of asparagine, serine, threonine, cysteine and glutamine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 6 may be composed of the amino acid sequence of SEQ ID NO: 23, 45, 67, 89, 111 or 135. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by Formula 6 may be composed of the amino acid sequence of SEQ ID NO: 176, 198, 220, 242, 264 or 286.

In addition, the Xaa5 may be selected from the group consisting of arginine, lysine and histidine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 6 may be composed of the amino acid sequence of SEQ ID NO: 24, 46, 68, 90, 112 or 134. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by Formula 6 may be composed of the amino acid sequence of SEQ ID NO: 177, 199, 221, 243, 265 or 287.

In addition, Xaa5 may be asphalt acid or glutamic acid. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 6 may be composed of the amino acid sequence of SEQ ID NO: 25, 47, 69, 91, 113 or 135. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by Formula 6 may be composed of the amino acid sequence of SEQ ID NO: 178, 200, 222, 244, 266 or 288.

In addition, the N-terminal fusion partner may be composed of the amino acid sequence represented by the following general formula (7).

[Formula 7]

Met-Asn-Ile-Arg-Pro-Leu-Xaa6- (Z) _n

In the general formula 7,

Xaa6 is isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine, tryptophan, asparagine, serine, threonine, cysteine, glutamine, arginine, lysine, histidine, asphalt acid or glutamic acid,

Z is 1 to 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345,

N is an integer of 0 or 1.

In addition, the Xaa6 may be selected from the group consisting of isoleucine, glycine, alanine, proline, valine, leucine, methionine, phenylalanine, tyrosine and tryptophan. Specifically, Xaa6 may be selected from the group consisting of isoleucine (Ile, I), asparagine (Asn, N), arginine (Arg, R), and asphalt acid (Asp, D).

When n is an integer of 0, the N-terminal fusion partner may be composed of seven amino acids. In addition, when n is an integer of 1, Z is 1, 2, 3, 4, 5, 6, 7 starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345. , 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 Dog, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36 amino acids. Specifically, when n is an integer of 1, Z may be 8, 15, 22, 29, or 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345.

In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 7 may be composed of the amino acid sequence of SEQ ID NO: 26, 48, 70, 92, 114 or 136. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 7 may be composed of the amino acid sequence of SEQ ID NO: 179, 201, 223, 245, 267 or 289.

In addition, the Xaa6 may be selected from the group consisting of asparagine, serine, threonine, cysteine and glutamine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 7 may be composed of the amino acid sequence of SEQ ID NO: 27, 49, 71, 93, 115 or 137. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 7 may be composed of the amino acid sequence of SEQ ID NO: 180, 202, 224, 246, 268 or 290.

In addition, the Xaa6 may be selected from the group consisting of arginine, lysine and histidine. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 7 may be composed of the amino acid sequence of SEQ ID NO: 28, 50, 72, 94, 116 or 138. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 7 may be composed of the amino acid sequence of SEQ ID NO: 181, 203, 225, 247, 269 or 291.

In addition, the Xaa6 may be asphalt acid or glutamic acid. In one embodiment, the N-terminal fusion partner consisting of the amino acid sequence represented by the general formula 7 may be composed of the amino acid sequence of SEQ ID NO: 29, 51, 73, 95, 117 or 139. In addition, the fusion polypeptide comprising the N-terminal fusion partner comprising the amino acid sequence represented by the general formula 7 may be composed of the amino acid sequence of SEQ ID NO: 182, 204, 226, 248, 270 or 292.

In Formulas 1 to 7, wherein n is an integer of 0, the N-terminal fusion partner may be composed of seven amino acids, in the present invention the N-terminal fusion partner consisting of seven amino acids "PG07" Named. In addition, when n is an integer of 1, Z may be 8, 15, 22, 29, or 36 amino acids starting from the amino acid at position 1 of the amino acid sequence represented by SEQ ID NO: 345. In this case, the N-terminal fusion partner may be composed of 15, 22, 29, 36 or 43 amino acids, in the present invention 15, 22, 29, 36 or 43 amino acids The resulting N-terminal fusion partners were named "PG15", "PG22", "PG29", "PG36", and "PG43" in order.

The N-terminal fusion partner may be an N-terminal derivative of chaperonin 10 (GroES proteon). In addition, the N-terminal fusion partner is a peptide having 7 to 43 amino acids, it may be composed of 7 to 43 consecutive amino acids from the N- terminal to the C- terminal of SEQ ID NO: 119.

Specifically, the N-terminal fusion partner may be composed of any one amino acid sequence of SEQ ID NO: 8 to 139. The number of amino acids of the fusion partner can be adjusted according to the properties of the polypeptide of interest. For example, 7, 8, 9, 10, 13, 15, 17, 22, 25, 27, 29, 30, 33, 38, 40, 43 Dogs and the like. In one embodiment, the N-terminal fusion partner may be composed of the amino acid sequence represented by SEQ ID NO: 9, 31, 53, 75, 97 or 119.

 The fusion polypeptide may comprise a linker between the fusion partner and GLP-1 or an analog thereof, wherein the linker may comprise an affinity tag.

As used herein, the term “affinity tag” can be used for a variety of purposes as a peptide or nucleic acid sequence that can be introduced into a recombinant fusion polypeptide or a nucleic acid encoding the same, for example, to enhance the purification efficiency of GLP-1 or an analog thereof. It may be for. Affinity tags that can be used in the present invention can be used as desired with any suitable material known in the art. For example, the affinity tag used in the present invention may be a polyhistidine tag (H6 or H10; SEQ ID NO: 140 or 141), a polylysine tag (K6 or K10; SEQ ID NO: 142 or 143), or a polyarginine tag (R6 or R10; SEQ ID NO: 144 or 145).

In addition, the linker may comprise a protease recognition sequence. A protease is a protease that recognizes a specific amino acid sequence and cleaves a peptide bond in the recognized sequence or a peptide bond with the first amino acid of the polypeptide fused with the last amino acid of the sequence. The fusion polypeptide according to the present invention comprises a linker having a protease recognition sequence, so that, in the final step, an amino terminus (affinity tag in the case of using an affinity tag) containing a cleavage enzyme recognition sequence in purifying GLP-1 or an analog thereof And the N-terminus of GLP-1 or an analog thereof, to recover GLP-1 or an analog thereof.

Specifically, the protease recognition sequence may be selected from the group consisting of tobacco etching virus protease recognition sequence, enterokinase recognition sequence, ubiquitin hydrolase recognition sequence, factor Xa recognition sequence, purine recognition sequence, and combinations thereof.

Specifically, the protease recognition sequence may be selected from the group consisting of tobacco etching virus protease recognition sequence, enterokinase recognition sequence, ubiquitin hydrolase recognition sequence, factor Xa recognition sequence, purine recognition sequence, and combinations thereof.

Specifically, the protease recognition sequence may be a recognition sequence selected from the group consisting of tobacco etching virus protease, enterokinase, ubiquitin hydrolase, factor Xa, purine, and combinations thereof. For example, the protease recognition sequence may include any one of the amino acid sequences of SEQ ID NOs: 146 to 150. In addition, the novel fusion polypeptide comprising the fusion partner, linker and GLP-1 or an analog thereof may be composed of the amino acid sequence of any one of the amino acid sequence of SEQ ID NOs: 161 to 292.

Another aspect of the present invention may provide a nucleotide encoding the aforementioned fusion polypeptide, for example, may encode any one of the amino acid sequences of SEQ ID NOs: 161 to 292, wherein the nucleotide is SEQ ID NO: 296 or It may be composed of any one of the nucleotide sequence of SEQ ID NO: 301.

In step (b), the host cell is transduced with an expression vector comprising a nucleotide encoding the fusion polypeptide prepared in step (a).

As used herein, the term “vector” may be introduced into a host cell to be recombined and inserted into the host cell genome. Alternatively, the vector refers to a nucleic acid means comprising a nucleotide sequence capable of spontaneously replicating as an episome. The vector may include linear nucleic acids, plasmids, phagemids, cosmids, RNA vectors, viral vectors and analogs thereof. Examples of viral vectors include, but are not limited to, retroviruses, adenoviruses, and adeno-associated viruses. In addition, the plasmid may comprise a selection marker, such as an antibiotic resistance gene, and the host cell holding the plasmid may be cultured under selective conditions.

The term "host cell" as used herein refers to prokaryotic or eukaryotic cells into which a recombinant expression vector can be introduced.

As used herein, the term “transduction” means the introduction of nucleic acids (eg, vectors) into cells using techniques known in the art.

The host cell may be transformed to include a nucleotide encoding the fusion polypeptide according to the present invention and used for expression and / or secretion of GLP-1 or an analog thereof. Preferred host cells that can be used in the present invention are E. coli cells, immortal 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 E. coli BL21 (DE3), and the genes and methods of their use are known in the art.

In the step (c), the host cell comprising an expression vector having a nucleotide encoding the fusion polypeptide according to the present invention is cultured.

At this time, the host cell may be cultured by any fermentation method, for example, batch, fed-batch, semi-continuous and continuous fermentation method may be used. In an embodiment, the fermentation medium may be selected from combination media or restriction media. In certain embodiments, restriction media is selected. Restriction media may be supplemented with low levels of amino acids, vitamins such as thiamine or other ingredients. Detailed descriptions of culture procedures and inorganic salt media useful in the methods of the present invention are described in Riesenberg, Schulz et al. 1991.

For example, production of fusion polypeptides can be accomplished in fermentor culture. For example, it can be incubated at a temperature of 37 ° C. in a fermentor containing 2 L of restriction medium and maintained at pH 6.8 through addition of hydrochloric acid or ammonia. Dissolved oxygen can be maintained in excess by increasing the stirring rate and air flow rate into the fermentor, or via pure oxygen injection as needed. In order to cultivate the cells in the fermentor at a high concentration, a feeding solution containing glucose or glycerol may be delivered to the culture during cell culture.

In addition, when the above conditions are maintained and the optical density of the target culture for induction is reached, for example, at a specific value of optical density (A600) at a wavelength of 600 nm, IPTG is added to express the fusion polypeptide. This may be disclosed. Optimal expression conditions can be determined by varying the optical density, IPTG concentration, pH, temperature, dissolved oxygen level, of each cell upon induction. In addition, the optical density of cells upon induction can be varied in the range of A600 30 to 300. The IPTG concentration can vary from 0.01 mM to 1.0 mM, the pH range from 5.5 to 7.5, the temperature range from 15 ° C. to 37 ° C., and the air flow rate from 1 vvm (liters of air per liter of medium per minute) to 5 vvm. have. After 4 to 48 hours after induction, the cells from the fermentor are centrifuged to harvest the cells and the cell pellet can be frozen at -80 ° C. The culture sample may be analyzed by SDS-PAGE or the like to analyze the expression level of the recombinant fusion polypeptide.

On the other hand, the host cell culture may be carried out at a temperature of 15 ℃ to 40 ℃ and pH conditions of about 5.5 to about 7.5. When expression constructs with Lac family promoters are used, expression can be induced by adding IPTG to the culture at a final concentration of about 0.01 mM to about 1.0 mM.

After addition of the inducing agent, the culture may be incubated for a period of time, for example about 12 hours, during which the recombinant protein is expressed. After addition of the inducing agent, the culture may be incubated for about 4 to 48 hours.

The cell culture can be centrifuged to remove the medium without the cells (supernatant) and recover the cells. For example, the cell culture solution may be centrifuged at 12,000 rpm for 30 minutes (4 ° C.) and the supernatant removed to obtain an insoluble fraction. Insoluble fractions can be resuspended in buffer containing chaotropic agents such as urea or guanidine hydrochloride for solubilization of recombinant fusion polypeptides present in the form of insoluble inclusion bodies in the insoluble fraction obtained by centrifugation. In an embodiment, the cells are disrupted using a high pressure mechanical cell disruption device (eg, Microfluidizer). Resuspended cells can be disrupted using, for example, sonication.

Any method known in the art that is suitable for lysing the cells can be used to recover the accumulated inclusions in the cells. For example, in embodiments, chemical and / or enzymatic cell lysis reagents such as cell wall lysozymes and EDTA can be used.

In step (d), the fusion polypeptide expressed in the host cell cultured in step (c) is purified.

At this time, the insoluble fraction mainly contains a fusion polypeptide expressed in the form of an insoluble inclusion body. Solubilization of the inclusion bodies present in the insoluble fraction can be carried out under denaturing conditions with chaotropic agents. Inclusion body solubilization conditions may include the use of a buffer containing chaotropic agents, and the inclusion body solubilization buffer may include urea or guanidine hydrochloride, sodium phosphate or tris buffer and sodium chloride as chaotropic agents. In addition, when affinity chromatography is performed by immobilized metal affinity chromatography (IMAC), the inclusion body solubilization buffer may comprise imidazole. In an embodiment, the inclusion body solubilization buffer may comprise 4 M to 10 M urea or 3 M to 8 M guanidine hydrochloride. Inclusion body solubilization buffer may also comprise 5 mM to 100 mM sodium phosphate or tris (pH 7-9). Inclusion body solubilization buffer may comprise 0 M to 1 M sodium chloride. In addition, the inclusion body solubilization buffer for IMAC may comprise 0 mM to 50 mM of imidazole. In this case, the inclusion body solubilization buffer contains 8 M urea, 20 mM Tris, 500 mM sodium chloride, 50 mM imidazole, pH 7.4, and resuspends the insoluble fraction obtained by centrifugation of the lysed cells to fusion poly of the insoluble fraction. Inclusion bodies of peptides can be solubilized.

For example, when the insoluble fraction of the inclusion body is solubilized with the inclusion body solubilization buffer, shaking culture for 1 hour to 6 hours at a temperature of 2 ° C. to 8 ° C., followed by 30 minutes (1 ° C.) at 12,000 rpm (12,000Хg) Centrifugation to remove crushed cell debris present in the insoluble fraction and obtain a supernatant containing the solubilized fusion polypeptide. The supernatant is filtered through a depth filter and a membrane filter to remove insoluble and solid components so that they can be injected into the purification column.

Furthermore, recombinant fusion polypeptides or target polypeptides that are solubilized after expression in the form of insoluble inclusion bodies are isolated or purified from other protein and cell debris by size exclusion, anion or cation exchange, hydrophobic interaction, or affinity chromatography methods. can do.

For example, the fusion polypeptide of the present invention may be purified using a HisTrap FF 5 mL column (GE Healthcare) filled with Ni-sepharose FF because it contains a polyhistidine tag. Solubilized recombinant fusion polypeptides were equilibrated with inclusion body solubilization buffer (8 M urea, 20 mM Tris, 500 mM sodium chloride, 50 mM imidazole, pH 7.4) using an S9 sample pump mounted on an AKTA pure 25 chromatography system. The column was injected into a HisTrap FF 5 mL column and washed with the inclusion body solubilization buffer followed by stepwise increase of the ratio of elution buffer (8 M urea, 20 mM Tris, 500 mM sodium chloride, 500 mM imidazole, pH 7.4) to 100%. The fusion polypeptide bound to can be eluted and fractions collected.

On the other hand, the fusion polypeptide according to the present invention can be easily purified in high purity by having a different isoelectric point between the fusion partner and the target polypeptide GLP-1 or an analog thereof. The isoelectric point (pI) of a protein is a pH at which the protein does not carry a net charge, and the protein can be separated according to its isoelectric point.

For example, the pI value of a fusion partner consisting of the amino acid sequence amino acid sequence of SEQ ID NOs: 9, 31, 53, 75, 97 or 119 of the present invention may be 9.52, 11.72, 10.27, 10.27, 10.43, 10.42, respectively. In addition, a fusion partner having an amino acid sequence of SEQ ID NOs. 154 to 159 in which a purification tag (SEQ ID NO: 140) and a tobacco etching virus protease recognition sequence (SEQ ID NO: 146) are fused to each of the N-terminal fusion partners C-terminus. The pI values are 7.06, 10.27, 9.52, 9.52, 9.82 and 9.30, respectively, and the pI values of GLP-1 or GLP-1K28R are 5.53. That is, the pI value of GLP-1 or an analog thereof and the pI value of the N-terminal fusion partner and fusion partners comprising the same are substantially different. Thus, purification of the target GLP-1 or an analog thereof from a fusion partner can be facilitated using methods such as ion exchange chromatography, isoelectric point precipitation, etc. using methods such as ion exchange chromatography, isoelectric point precipitation and the like.

In step (e), the fusion polypeptide purified by the above-described method is incubated with a cleavage enzyme to recover GLP-1 or an analog thereof (GLP-1K28R).

At this time, the fusion polypeptide may be suitably cleaved by a cleavage enzyme, thereby releasing GLP-1 or GLP-1K28R into a suitable form. Since 8 M of urea is added to the purified fusion polypeptide fraction, it is preferable to dilute to 1 M using dilution buffer (20 mM Tris, pH 7.4) to prevent denaturation of the cleavage enzyme. The fusion polypeptide may be diluted to a urea concentration of 1 M after purification and present in a buffer containing 20 mM Tris, pH 7.4, 1 M urea, 62.5 mM sodium chloride, 62.5 mM imidazole. Recombinant fusion polypeptides are cleaved with GLP-1 or GLP-1K28R with an amino terminal fusion partner containing an affinity tag and a cleavage recognition sequence in response to the cleavage enzyme. Protease cleavage methods can be any suitable method known in the art and described in the literature, including manufacturer's instructions. Preferably, after purification, TEV protease is added to a fusion polypeptide diluted to 1 M to a final concentration of 500 nM and the cleavage reaction can proceed for at least 6 hours at room temperature. For example, the TEV protease cleaves from about 60% to about 100% of the recombinant fusion polypeptide.

Yields of the recombinant fusion polypeptide, GLP-1 or GLP-1K28R can be determined by methods known to those skilled in the art, for example, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) or Western blot analysis. . Gels electrophoresed with SDS-PAGE undergo staining, destaining, and digital imaging to allow for quantitative and qualitative analysis of recombinant fusion polypeptides, GLP-1 or GLP-1K28R.

In addition, the concentration of the purified fusion polypeptide, GLP-1 or GLP-1K28R, can be determined by absorbance spectroscopy by methods known in the art and described in the literature.

Western blot analysis to determine the yield or purity of the purified fusion polypeptide, GLP-1 or GLP-1K28R, transfers proteins isolated on SDS-PAGE gels to nitrocellulose membranes and is specific for GLP-1 or GLP-1K28R. It can be carried out according to a suitable method known in the art using a specific antibody. In an embodiment, an enzyme-linked immunosorbent assay (ELISA) method may be used as one of methods for measuring purity of GLP-1 or GLP-1K28R.

The yield of the purified fusion polypeptide, GLP-1 or GLP-1K28R is determined by the amount of purified fusion polypeptide or the desired polypeptide per volume of culture (eg, culture volume of g / mg protein / liter), fusion polypeptide Percentages (eg, amount of recombinant fusion polypeptide / total cell protein), and percentages or ratios to dry cell weight. The measure of yield of a polypeptide described herein is based on the amount of polypeptide of interest expressed in its complete form.

If the yield is expressed in terms of the amount of purified fusion polypeptide, GLP-1 or GLP-1K28R relative to the culture volume, the cultured cell density or cell concentration may be considered in determining the yield.

As such, one embodiment of the present invention obtains GLP-1 or GLP-1K28R using a fusion polypeptide comprising a fusion partner consisting of the amino acid sequence represented by the general formula (1). Through this, GLP-1 or GLP-1K28R can be provided by a method for mass production having high yield characteristics by minimizing problems such as degradation by enzymes or inappropriate folding in cells. An example of the specific production method will be described by way of examples.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1 Preparation and Production of Liraglutide Precursor Peptide (GLP-1K28R) Fusion Polypeptide

Example 1.1. Construction of GLP-1K28R Fusion Polypeptide Expression Plasmids

Genes for the GLP-1K28R fusion polypeptide were synthesized using the overlap extension polymerase chain reaction (OE-PCR) method. Wherein the GLP-1K28R fusion polypeptide is PG07 (SEQ ID NO: 9), PG15 (SEQ ID NO: 31), PG22 (SEQ ID NO: 53), PG29 (SEQ ID NO: 75), PG36 (SEQ ID NO: 97) and the amino terminal fusion partner. One of PG43 (SEQ ID NO: 119) and 6 histidine tag (SEQ ID NO: 140), TEV protease recognition sequence (SEQ ID NO: 146), and the amino acid sequence of GLP-1K28R (SEQ ID NO: 151) are included.

The GLP-1K28R fusion polypeptide (H6TEV-GLP-1K28R) used as a control did not contain an amino terminal fusion partner, 6 histidine tags (SEQ ID NO: 140), TEV protease recognition sequence (SEQ ID NO: 146), and GLP-1K28R Amino acid sequence of SEQ ID NO: 152 is included. The gene of each fusion polypeptide contains the recognition sequences of the restriction enzymes NdeI, NcoI and XhoI and one stop codon. The nucleotide sequence encoding the GLP-1K28R fusion polypeptide corresponds to SEQ ID NOs: 296 to 301, respectively, and the control corresponds to SEQ ID NO: 295.

To prepare GLP-1K28R fusion polypeptide expression plasmids pSGK530, pSGK495, pSGK496, pSGK500, pSGK501, pSGK502 and pSGK497 as described in Table 2 below, the GLP-1K28R fusion polypeptide gene fragment synthesized with OE-PCR was used as NdeI and XhoI. Restriction enzymes were cleaved and cloned into expression vector pET26b capable of expression regulation by IPTG, including the T7 promoter, lac effector and LacI gene.

Strain Host cell Plasmid Recombinant fusion polypeptides PG005 E. coli BL21 (DE3) pSGK530 H6TEV-GLP-1K28R PG006 E. coli BL21 (DE3) pSGK495 PG07-H6TEV-GLP-1K28R PG007 E. coli BL21 (DE3) pSGK496 PG15-H6TEV-GLP-1K28R PG008 E. coli BL21 (DE3) pSGK500 PG22-H6TEV-GLP-1K28R PG009 E. coli BL21 (DE3) pSGK501 PG29-H6TEV-GLP-1K28R PG010 E. coli BL21 (DE3) pSGK502 PG36-H6TEV-GLP-1K28R PG011 E. coli BL21 (DE3) pSGK497 PG43-H6TEV-GLP-1K28R

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

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

Expression of GLP-1K28R Fusion Polypeptide Stored at −80 ° C. The plasmids of the transformed E. coli were dissolved at room temperature and 50 μl of 5 ml of LB liquid medium containing kanamycin at a concentration of 50 μg / ml. The seed was added to the test tube and cultured for 12 hours in a shaker at 37 ° C. Spawn cultured 2 ml of E. coli was added to a flask containing 200 ml of LB liquid medium containing kanamycin at a concentration of 50 μg / ml and cultured in a shaker at 37 ° C. After about 3 hours of culture, when the optical density (OD600) of the cell was about 1.0, IPTG was added to a final concentration of 0.1 mM to induce the expression of the GLP-1K28R fusion polypeptide. 4 hours after expression induction, the optical density was measured to determine the optical density of the cells.

Example 1.3. Preparation of Samples for Comparative Expression Level Analysis

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

Example 1.4. Identification of GLP-1K28R Produced by SDS-PAGE Analysis

50 μl of total cells, soluble and insoluble fractions were respectively mixed in 50 μl of 2 × concentrated SDS sample buffer (SDS sample buffer 2 × concentration, Sigma), and then heated at 95 ° C. for 5 minutes to obtain protein of each sample. It can be denatured. Denatured proteins in the samples were allowed to separate in the gel by molecular weight size using 16% SDS-PAGE gel and TANK buffer. After SDS-PAGE, the gel was stained with staining buffer containing Coomassie blue R-250, and then stained with destaining buffer so that only the stained protein was visible. The results are shown in FIGS. 1 and 2.

Referring to FIG. 1, the band of H6TEV-GLP-1K28R (molecular weight of 5.1 kDa) without the fusion partner comprising the amino acid sequence of SEQ ID NO: 1 as a control was not detected in the SDS-PAGE gel, and thus the intracellular protein after expression. It seems to have been degraded by the enzyme. Expression of the GLP-1K28R fusion polypeptide in the SDS-PAGE gel was confirmed from PG07-H6TEV-GLP-1K28R (molecular weight of 6.1 kDa) fused to PG07, the smallest amino terminal fusion partner.

The expression level of PG15-H6TEV-GLP-1K28R (molecular weight of 7.1 kDa) to which the amino terminal fusion partner PG15 was fused increased compared to PG07-H6TEV-GLP-1K28R. PG15-H6TEV-GLP-1K28R (molecular weight of 7.1 kDa), PG22-H6TEV-GLP-1K28R (7.9 kDa molecular weight), GLP-1K28R fusion polypeptide fused with amino terminal fusion partners PG15, PG22, PG29, PG36 and PG43 ), PG29-H6TEV-GLP-1K28R (molecular weight of 8.4 kDa), PG36-H6TEV-GLP-1K28R (molecular weight of 9.1 kDa), and PG43-H6TEV-GLP-1K28R (molecular weight of 11.7 kDa) were the control (H6TEV -GLP-1K28R) was found to be very improved.

Densitometer analysis showed that the expression levels of fusion polypeptides fused with PG22, PG29, PG36, and PG43 in PG07, PG15, PG22, PG29, PG36, and PG43 were similar and were significantly higher than those of fused polypeptide with PG07, PG15. It was confirmed (FIGS. 2 and 3).

2, all GLP-1K28R fusion polypeptides, including the control group, are not observed in the soluble fraction but all appear in the insoluble fraction (lane 1: H6TEV-GLP-1K28R (strain number PG005) and lanes). 2: For PG07-H6TEV-GLP-1K28R (strain number PG006), the solubility test was not performed because the target peptide was not expressed or the expression level was low.

For reference, the molecular weight, isoelectric point, average hydrophobicity and molar extinction coefficient of the polypeptide used in the examples are shown in Table 3 below. The molar extinction coefficient was calculated with Expasy's ProtParam Tool (http://web.expasy.org/protparam/).

Polypeptide Protein Name Molecular Weight (Da) Isoelectric point (pI) Average hydrophobicity
(GRAVY) Molar absorption coefficient PG07 880.08 9.52 -0.371 - PG15 1875.31 11.72 -0.667 - PG22 2620.11 10.27 -0.745 - PG29 3217.82 10.27 -0.200 - PG36 3834.50 10.43 -0.183 - PG43 4559.35 10.42 -0.023 - H6 840.86 7.21 -3.200 - H10 1389.43 7.35 -3.200 - K6 787.06 10.70 -3.900 - K10 1299.76 10.95 -3.900 - R6 955.14 12.70 -4.500 - R10 1579.89 12.95 -4.500 - Tobacco etch virus (TEV) protease 812.88 4.00 -0.867 1490 Enterokinase 606.54 3.77 -3.580 - Ubiquitin carboxyl-terminal hydrolase 557.65 12.00 -1.200 - Glucagon-like peptide-1 (GLP-1) 3355.71 5.53 -0.235 6990 Liraglutide Precursor Peptides 3383.72 5.53 -0.255 6990 H6TEV 1766.92 6.56 -1.731 1490 PG07-H6TEV 2781.11 7.06 -1.405 6990 PG15-H6TEV 3776.35 10.27 -1.272 6990 PG22-H6TEV 4521.15 9.52 -1.203 6990 PG29-H6TEV 5118.86 9.52 -0.760 6990 PG36-H6TEV 5735.53 9.82 -0.670 6990 PG43-H6TEV 8361.43 9.30 -0.772 13980 H6TEV-GLP-1K28R 5132.63 6.34 -0.691 8480 PG07-H6TEV-GLP-1K28R 6146.82 6.69 -0.719 13980 PG15-H6TEV-GLP-1K28R 7142.06 9.40 -0.747 13980 PG22-H6TEV-GLP-1K28R 7886.86 8.23 -0.764 13980 PG29-H6TEV-GLP-1K28R 8484.57 8.23 -0.549 13980 PG36-H6TEV-GLP-1K28R 9101.24 9.22 -0.511 13980 PG43-H6TEV-GLP-1K28R 11727.14 8.23 -0.615 20970

Example 1.5. Changes in Expression Levels of GLP-1K28R Fusion Polypeptides by Amino Acid Substitution of N-terminal Fusion Partners

To determine the effect of 6 amino acid residue changes from PG43 2 to 7 in PG43-H6TEV-GLP-1K28R on the expression level of the GLP-1K28R fusion polypeptide, each amino acid residue was assigned to isoleucine, asparagine, and arginine. A total of 22 variants of GLP-1K28R fusion polypeptides substituted with asphalt acid were prepared and compared with the expression levels of PG43-H6TEV-GLP-1K28R.

Plasmid DNA for variant expression of the GLP-1K28R fusion polypeptide was constructed using site-directed mutagenesis. As template for the positional mutation, pSGK497, a PG43-H6TEV-GLP-1K28R expression plasmid, was used. The primers used forward and reverse single-stranded DNA oligomers in which the nucleotide sequence of the amino acid substitution site of each variant was changed. Primers used in the experiments are shown in Table 4 below.

No. PG43 mutants Oligomer sequence SEQ ID NO: One PG43-N2I F-primer GGAGATATACATATG ATT ATTCGTCCATTGCAT SEQ ID NO: 303 R-primer ATGCAATGGACGAAT AAT CATATGTATATCTCC SEQ ID NO: 304 2 PG43-N2N F-primer - R-primer - 3 PG43-N2R F-primer GGAGATATACATATG CGC ATTCGTCCATTGCAT SEQ ID NO: 305 R-primer ATGCAATGGACGAAT GCG CATATGTATATCTCC SEQ ID NO: 306 4 PG43-N2D F-primer GGAGATATACATATG GAT ATTCGTCCATTGCAT SEQ ID NO: 307 R-primer ATGCAATGGACGAAT ATC CATATGTATATCTCC SEQ ID NO: 308 5 PG43-I3I F-primer - R-primer - 6 PG43-I3N F-primer GATATACATATGAAT AAC CGTCCATTGCATGAT SEQ ID NO: 309 R-primer ATCATGCAATGGACG GTT ATTCATATGTATATC SEQ ID NO: 310 7 PG43-I3R F-primer GATATACATATGAAT CGC CGTCCATTGCATGAT SEQ ID NO: 311 R-primer ATCATGCAATGGACG GCG ATTCATATGTATATC SEQ ID NO: 312 8 PG43-I3D F-primer GATATACATATGAAT GAT CGTCCATTGCATGAT SEQ ID NO: 313 R-primer ATCATGCAATGGACG ATC ATTCATATGTATATC SEQ ID NO: 314 9 PG43-R4I F-primer ATACATATGAATATT ATT CCATTGCATGATCGC SEQ ID NO: 315 R-primer GCGATCATGCAATGG AAT AATATTCATATGTAT SEQ ID NO: 316 10 PG43-R4N F-primer ATACATATGAATATT AAC CCATTGCATGATCGC SEQ ID NO: 317 R-primer GCGATCATGCAATGG GTT AATATTCATATGTAT SEQ ID NO: 318 11 PG43-R4R F-primer - R-primer - 12 PG43-R4D F-primer ATACATATGAATATT GAT CCATTGCATGATCGC SEQ ID NO: 319 R-primer GCGATCATGCAATGG ATC AATATTCATATGTAT SEQ ID NO: 320 13 PG43-P5I F-primer CATATGAATATTCGT ATT TTGCATGATCGCGTG SEQ ID NO: 321 R-primer CACGCGATCATGCAA AAT ACGAATATTCATATG SEQ ID NO: 322 14 PG43-P5N F-primer CATATGAATATTCGT AAC TTGCATGATCGCGTG SEQ ID NO: 323 R-primer CACGCGATCATGCAA GTT ACGAATATTCATATG SEQ ID NO: 324 15 PG43-P5R F-primer CATATGAATATTCGT CGC TTGCATGATCGCGTG SEQ ID NO: 325 R-primer CACGCGATCATGCAA GCG ACGAATATTCATATG SEQ ID NO: 326 16 PG43-P5D F-primer CATATGAATATTCGT GAT TTGCATGATCGCGTG SEQ ID NO: 327 R-primer CACGCGATCATGCAA ATC ACGAATATTCATATG SEQ ID NO: 328 17 PG43-L6I F-primer ATGAATATTCGTCCA ATT CATGATCGCGTGATC SEQ ID NO: 329 R-primer GATCACGCGATCATG AAT TGGACGAATATTCAT SEQ ID NO: 330 18 PG43-L6N F-primer ATGAATATTCGTCCA AAC CATGATCGCGTGATC SEQ ID NO: 331 R-primer GATCACGCGATCATG GTT TGGACGAATATTCAT SEQ ID NO: 332 19 PG43-L6R F-primer ATGAATATTCGTCCA CGC CATGATCGCGTGATC SEQ ID NO: 333 R-primer GATCACGCGATCATG GCG TGGACGAATATTCAT SEQ ID NO: 334 20 PG43-L6D F-primer ATGAATATTCGTCCA GAT CATGATCGCGTGATC SEQ ID NO: 335 R-primer GATCACGCGATCATG ATC TGGACGAATATTCAT SEQ ID NO: 336 21 PG43-H7I F-primer AATATTCGTCCATTG ATT GATCGCGTGATCGTC SEQ ID NO: 337 R-primer GACGATCACGCGATC AAT CAATGGACGAATATT SEQ ID NO: 338 22 PG43-H7N F-primer AATATTCGTCCATTG AAC GATCGCGTGATCGTC SEQ ID NO: 339 R-primer GACGATCACGCGATC GTT CAATGGACGAATATT SEQ ID NO: 340 23 PG43-H7R F-primer AATATTCGTCCATTG CGC GATCGCGTGATCGTC SEQ ID NO: 341 R-primer GACGATCACGCGATC GCG CAATGGACGAATATT SEQ ID NO: 342 24 PG43-H7D F-primer AATATTCGTCCATTG GAT GATCGCGTGATCGTC SEQ ID NO: 343 R-primer GACGATCACGCGATC ATC CAATGGACGAATATT SEQ ID NO: 344

Expression plasmids obtained after the positional mutations for each variant were confirmed for correct cloning through DNA sequencing.

E. coli BL21 (DE3) cells were transformed by chemical method using calcium chloride using the mutant expression plasmids of the prepared GLP-1K28R fusion polypeptide. E. coli transformed with the expression plasmids of the GLP-1K28R fusion polypeptide formed colonies in LB solid medium containing kanamycin at a concentration of 50 μg / ml. The transformed Escherichia coli were cultured in LB liquid medium containing kanamycin at a concentration of 50 μg / ml, respectively, and then 50% glycerol was added to the same volume as the culture medium to make a cell concentrate, which was stored in a freezer at -80 ° C. It was.

Variant expression plasmids of GLP-1K28R fusion polypeptides stored at −80 ° C. were dissolved at room temperature, and 50 μl of LB liquid medium containing kanamycin at a concentration of 50 μg / ml was dissolved. Addition to the included test tube was incubated for 12 hours in a shaker at 37 ℃ temperature. 2 ml of spawn cultured E. coli was added to a flask containing 200 ml of LB liquid medium containing kanamycin at a concentration of 50 µg / ml and cultured in a shaker at 37 ° C. When the optical density (OD600) of the cell was about 1.0 after 3 hours of incubation, the final concentration of IPTG was added to 0.1 mM to induce the expression of the GLP-1K28R fusion polypeptide. 4 hours after expression induction, the optical density was measured to determine the optical density of the cells.

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

50 μl of total cells, soluble and insoluble fractions were respectively mixed in 50 μl of 2 × concentrated SDS sample buffer (SDS sample buffer 2 × concentration, Sigma), and then heated at 95 ° C. for 5 minutes to obtain protein of each sample. It can be denatured. Denatured proteins in the samples were allowed to separate in the gel by molecular weight size using a 16% SDS-PAGE gel and TANK buffer. After SDS-PAGE, the gel was stained with staining buffer containing Coomassie blue R-250, and then stained with destaining buffer so that only the stained protein was visible.

As shown in Figures 4 to 6, expression by the change of 6 amino acid residues from 2 to 7 of PG43 in PG43-H6TEV-GLP-1K28R compared to the expression level of the control group PG43-H6TEV-GLP-1K28R Improved and reduced variants were identified. In particular, the mutant strain in which the second residue was substituted with asphalt acid and the mutant strain in which the seventh residue was replaced with isoleucine, asparagine, arginine, and asphalt acid through densitometer analysis showed that expression levels were improved by two to three times or more compared with the control group. Confirmed.

Example 2. Recovery and Purification of GLP-1K28R Fusion Polypeptide

Example 2.1. Cell disruption and insoluble inclusions recovery

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

Example 2.2. Insoluble Inclusion Solubilization

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

Example 2.3. GLP-1K28R Fusion Polypeptide Purification

The highest expression level of PG43-H6TEV-GLP-1K28R among 7 GLP-1K28R fusion polypeptides was purified. First, AKTA pure 25 chromatography system equipped with S9 sample pump (S9) and F9-C fraction collector (Fraction collector F9-C) for purification of GLP-1K28R fusion polypeptide in solubilized insoluble fraction (GE Healthcare) ) Was used. Solubilized insoluble fraction samples were injected into a HisTrap FF 1 mL column (GE Healthcare) previously equilibrated with inclusion body solubilization buffer (8 M urea, 20 mM Tris, 500 mM sodium chloride, 50 mM imidazole, pH 7.4).

After the injection is complete, the column is washed with a 5-fold column volume of equilibration buffer and 100% stepwise addition of 5-fold column volume of elution buffer (8 M urea, 20 mM Tris, 500 mM sodium chloride, 500 mM imidazole, pH 7.4). GLP-1K28R fusion polypeptide bound to the resin of the column was eluted to increase. The results of the fractions obtained after elution are shown in the figures, respectively (FIGS. 7 and 8). Most of the GLP-1K28R fusion polypeptide in the solubilized insoluble fraction sample injected into the column was eluted after binding to the resin in the column, and showed more than 95% purity.

 Example 3. Linker Sequence Cleavage Through Protease Treatment

Fractions of about 5 ml of purified GLP-1K28R fusion polypeptide were combined and diluted to 1 M by adding 140 ml of dilution buffer (20 mM Tris, pH 7.4). TEV protease was added to the diluted recombinant fusion polypeptide to a final concentration of 500 nM and the cleavage reaction was allowed to proceed for 12 hours at room temperature.

In order to confirm the cleavage by the TEV protease, SDS-PAGE analysis was performed after cleavage and the results are shown in the figure (FIG. 9). SDS-PAGE analysis of before and after cleavage of GLP-1K28R fusion polypeptide (PG43-H6TEV-GLP-1K28R) by TEV protease showed that the 7.9 kDa GLP-1K28R fusion polypeptide was nearly 100% in amino yield. It was confirmed that the terminal fusion partner and the 6 histidine tag and TEV protease recognition sequence fusion PG43-H6TEV and the target polypeptide GLP-1K28R.

Example 4. Molecular weight analysis of GLP-1K28R after cleavage

MALTI-TOF MS was used to confirm the expression of the complete form of GLP-1K28R fusion polypeptide (PG43-H6TEV-GLP-1K28R), the correct cleavage by TEV protease and the modification of GLP-1K28R obtained after cleavage. The molecular weight analysis used was performed. The result of measuring the molecular weight of GLP-1K28R obtained according to the present invention is shown in the figure.

Referring to FIG. 10, the molecular weight of GLP-1K28R obtained from PG43-H6TEV-GLP-1K28R was measured to be 3382.59 Da, which was within the margin of error with the theoretical molecular weight of 3383.72 Da, which is a proteolytic enzyme in E. coli. It was confirmed that the complete form was expressed without partial cleavage or degradation of amino or carboxy terminus by.

Therefore, the TEV protease recognizes the ENLFQ sequence, which is the recognition sequence in PG43-H6TEV-GLP-1K28R, and is believed to precisely cleave the peptide bond between Q (glutamine), the last amino acid, and H (histidine), the first amino acid of GLP-1K28R. .

<110> PEPGENE INC. <120> METHOD OF PREPARING GLUCAGON LIKE PEPTIDE-1 OR ANALOGUES USING          FUSION POLYPEPTIDE <130> FPD201809-0085 <150> KR 10-2018-0018255 <151> 2018-02-14 <160> 345 <170> KoPatentIn 3.0 <210> 1 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner <400> 1 Met Xaa Xaa Xaa Xaa Xaa Xaa   1 5 <210> 2 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner <400> 2 Met Xaa Ile Arg Pro Leu His   1 5 <210> 3 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner <400> 3 Met Asn Xaa Arg Pro Leu His   1 5 <210> 4 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner <400> 4 Met Asn Ile Xaa Pro Leu His   1 5 <210> 5 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner <400> 5 Met Asn Ile Arg Xaa Leu His   1 5 <210> 6 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner <400> 6 Met Asn Ile Arg Pro Xaa His   1 5 <210> 7 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner <400> 7 Met Asn Ile Arg Pro Leu Xaa   1 5 <210> 8 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 8 Met Ile Ile Arg Pro Leu His   1 5 <210> 9 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 9 Met Asn Ile Arg Pro Leu His   1 5 <210> 10 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 10 Met Arg Ile Arg Pro Leu His   1 5 <210> 11 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 11 Met Asp Ile Arg Pro Leu His   1 5 <210> 12 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 12 Met Asn Asn Arg Pro Leu His   1 5 <210> 13 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 13 Met Asn Arg Arg Pro Leu His   1 5 <210> 14 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 14 Met Asn Asp Arg Pro Leu His   1 5 <210> 15 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 15 Met Asn Ile Ile Pro Leu His   1 5 <210> 16 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 16 Met Asn Ile Asn Pro Leu His   1 5 <210> 17 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 17 Met Asn Ile Asp Pro Leu His   1 5 <210> 18 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 18 Met Asn Ile Arg Ile Leu His   1 5 <210> 19 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 19 Met Asn Ile Arg Asn Leu His   1 5 <210> 20 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 20 Met Asn Ile Arg Arg Leu His   1 5 <210> 21 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 21 Met Asn Ile Arg Asp Leu His   1 5 <210> 22 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 22 Met Asn Ile Arg Pro Ile His   1 5 <210> 23 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 23 Met Asn Ile Arg Pro Asn His   1 5 <210> 24 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 24 Met Asn Ile Arg Pro Arg His   1 5 <210> 25 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 25 Met Asn Ile Arg Pro Asp His   1 5 <210> 26 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 26 Met Asn Ile Arg Pro Leu Ile   1 5 <210> 27 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 27 Met Asn Ile Arg Pro Leu Asn   1 5 <210> 28 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 28 Met Asn Ile Arg Pro Leu Arg   1 5 <210> 29 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG07) <400> 29 Met Asn Ile Arg Pro Leu Asp   1 5 <210> 30 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 30 Met Ile Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 31 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 31 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 32 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 32 Met Arg Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 33 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 33 Met Asp Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 34 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 34 Met Asn Asn Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 35 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 35 Met Asn Arg Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 36 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 36 Met Asn Asp Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 37 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 37 Met Asn Ile Ile Pro Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 38 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 38 Met Asn Ile Asn Pro Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 39 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 39 Met Asn Ile Asp Pro Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 40 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 40 Met Asn Ile Arg Ile Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 41 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 41 Met Asn Ile Arg Asn Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 42 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 42 Met Asn Ile Arg Arg Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 43 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 43 Met Asn Ile Arg Asp Leu His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 44 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 44 Met Asn Ile Arg Pro Ile His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 45 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 45 Met Asn Ile Arg Pro Asn His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 46 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 46 Met Asn Ile Arg Pro Arg His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 47 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 47 Met Asn Ile Arg Pro Asp His Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 48 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 48 Met Asn Ile Arg Pro Leu Ile Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 49 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 49 Met Asn Ile Arg Pro Leu Asn Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 50 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 50 Met Asn Ile Arg Pro Leu Arg Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 51 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG15) <400> 51 Met Asn Ile Arg Pro Leu Asp Asp Arg Val Ile Val Lys Arg Lys   1 5 10 15 <210> 52 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 52 Met Ile Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 53 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 53 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              20 <210> 54 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 54 Met Arg Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 55 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 55 Met Asp Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 56 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 56 Met Asn Asn Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 57 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 57 Met Asn Arg Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 58 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 58 Met Asn Asp Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 59 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 59 Met Asn Ile Ile Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 60 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 60 Met Asn Ile Asn Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 61 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 61 Met Asn Ile Asp Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 62 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 62 Met Asn Ile Arg Ile Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 63 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 63 Met Asn Ile Arg Asn Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 64 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 64 Met Asn Ile Arg Arg Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 65 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 65 Met Asn Ile Arg Asp Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 66 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 66 Met Asn Ile Arg Pro Ile His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 67 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 67 Met Asn Ile Arg Pro Asn His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 68 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 68 Met Asn Ile Arg Pro Arg His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 69 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 69 Met Asn Ile Arg Pro Asp His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 70 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 70 Met Asn Ile Arg Pro Leu Ile Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 71 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 71 Met Asn Ile Arg Pro Leu Asn Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 72 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 72 Met Asn Ile Arg Pro Leu Arg Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 73 <211> 22 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG22) <400> 73 Met Asn Ile Arg Pro Leu Asp Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala              20 <210> 74 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 74 Met Ile 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              20 25 <210> 75 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 75 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              20 25 <210> 76 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 76 Met Arg 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              20 25 <210> 77 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 77 Met Asp 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              20 25 <210> 78 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 78 Met Asn Asn 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              20 25 <210> 79 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 79 Met Asn Arg 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              20 25 <210> 80 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 80 Met Asn Asp 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              20 25 <210> 81 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 81 Met Asn Ile Ile 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              20 25 <210> 82 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 82 Met Asn Ile Asn 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              20 25 <210> 83 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 83 Met Asn Ile Asp 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              20 25 <210> 84 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 84 Met Asn Ile Arg Ile 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              20 25 <210> 85 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 85 Met Asn Ile Arg Asn 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              20 25 <210> 86 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 86 Met Asn Ile Arg Arg 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              20 25 <210> 87 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 87 Met Asn Ile Arg Asp 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              20 25 <210> 88 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 88 Met Asn Ile Arg Pro Ile 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              20 25 <210> 89 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 89 Met Asn Ile Arg Pro Asn 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              20 25 <210> 90 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 90 Met Asn Ile Arg Pro Arg 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              20 25 <210> 91 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 91 Met Asn Ile Arg Pro Asp 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              20 25 <210> 92 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 92 Met Asn Ile Arg Pro Leu Ile 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              20 25 <210> 93 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 93 Met Asn Ile Arg Pro Leu Asn 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              20 25 <210> 94 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 94 Met Asn Ile Arg Pro Leu Arg 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              20 25 <210> 95 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG29) <400> 95 Met Asn Ile Arg Pro Leu Asp 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              20 25 <210> 96 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 96 Met Ile 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          35 <210> 97 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 97 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          35 <210> 98 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 98 Met Arg 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          35 <210> 99 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 99 Met Asp 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          35 <210> 100 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 100 Met Asn Asn 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          35 <210> 101 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 101 Met Asn Arg 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          35 <210> 102 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 102 Met Asn Asp 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          35 <210> 103 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 103 Met Asn Ile Ile 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          35 <210> 104 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 104 Met Asn Ile Asn 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          35 <210> 105 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 105 Met Asn Ile Asp 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          35 <210> 106 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 106 Met Asn Ile Arg Ile 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          35 <210> 107 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 107 Met Asn Ile Arg Asn 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          35 <210> 108 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 108 Met Asn Ile Arg Arg 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          35 <210> 109 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 109 Met Asn Ile Arg Asp 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          35 <210> 110 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 110 Met Asn Ile Arg Pro Ile 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          35 <210> 111 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 111 Met Asn Ile Arg Pro Asn 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          35 <210> 112 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 112 Met Asn Ile Arg Pro Arg 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          35 <210> 113 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 113 Met Asn Ile Arg Pro Asp 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          35 <210> 114 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 114 Met Asn Ile Arg Pro Leu Ile 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          35 <210> 115 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 115 Met Asn Ile Arg Pro Leu Asn 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          35 <210> 116 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 116 Met Asn Ile Arg Pro Leu Arg 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          35 <210> 117 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG36) <400> 117 Met Asn Ile Arg Pro Leu Asp 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          35 <210> 118 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 118 Met Ile 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> 119 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 119 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> 120 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 120 Met Arg 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> 121 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 121 Met Asp 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> 122 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 122 Met Asn Asn 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> 123 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 123 Met Asn Arg 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> 124 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 124 Met Asn Asp 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> 125 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 125 Met Asn Ile Ile 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> 126 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 126 Met Asn Ile Asn 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> 127 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 127 Met Asn Ile Asp 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> 128 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 128 Met Asn Ile Arg Ile 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> 129 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 129 Met Asn Ile Arg Asn 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> 130 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 130 Met Asn Ile Arg Arg 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> 131 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 131 Met Asn Ile Arg Asp 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> 132 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 132 Met Asn Ile Arg Pro Ile 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> 133 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <133> 133 Met Asn Ile Arg Pro Asn 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> 134 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 134 Met Asn Ile Arg Pro Arg 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> 135 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 135 Met Asn Ile Arg Pro Asp 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> 136 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 136 Met Asn Ile Arg Pro Leu Ile 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> 137 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 137 Met Asn Ile Arg Pro Leu Asn 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> 138 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 138 Met Asn Ile Arg Pro Leu Arg 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 <139> <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner (PG43) <400> 139 Met Asn Ile Arg Pro Leu Asp 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> 140 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6 <400> 140 His His His His His His   1 5 <210> 141 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H10 <400> 141 His His His His His His His His His His   1 5 10 <210> 142 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for K6 <400> 142 Lys Lys Lys Lys Lys Lys Lys   1 5 <210> 143 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for K10 <400> 143 Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys Lys   1 5 10 <210> 144 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for R6 <400> 144 Arg Arg Arg Arg Arg Arg   1 5 <210> 145 <211> 10 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for R10 <400> 145 Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg Arg   1 5 10 <210> 146 <211> 6 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Tobacco etch virus (TEV) protease <400> 146 Glu Asn Leu Tyr Phe Gln   1 5 <210> 147 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Enterokinase <400> 147 Asp Asp Asp Asp Lys   1 5 <210> 148 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Ubiquitin carboxyl-terminal hydrolase <400> 148 Arg Leu Arg Gly Gly   1 5 <210> 149 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Factor Xa <400> 149 Ile Glu Gly Arg   One <210> 150 <211> 4 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for Furin <400> 150 Arg Lys Arg Arg   One <210> 151 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for GLP-1 <400> 151 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 Lys Gly Arg Gly              20 25 30 <210> 152 <211> 31 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for GLP-1K28R <400> 152 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> 153 <211> 13 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV <400> 153 Met His His His His His His Glu Asn Leu Tyr Phe Gln   1 5 10 <210> 154 <211> 21 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV <400> 154 Met Asn Ile Arg Pro Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln              20 <210> 155 <211> 29 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV <400> 155 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln              20 25 <210> 156 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV <400> 156 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 Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln          35 <210> 157 <211> 43 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV <400> 157 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln          35 40 <210> 158 <211> 50 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV <400> 158 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln      50 <210> 159 <211> 57 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV <400> 159 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> 160 <211> 44 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for H6TEV-GLP-1K28R <400> 160 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> 161 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 161 Met Ile Ile Arg Pro Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 162 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 162 Met Asn Ile Arg Pro Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 163 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 163 Met Arg Ile Arg Pro Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 164 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 164 Met Asp Ile Arg Pro Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 165 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 165 Met Asn Asn Arg Pro Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 166 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 166 Met Asn Arg Arg Pro Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 167 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 167 Met Asn Asp Arg Pro Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 168 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 168 Met Asn Ile Ile Pro Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 169 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 169 Met Asn Ile Asn Pro Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 170 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 170 Met Asn Ile Asp Pro Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 171 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 171 Met Asn Ile Arg Ile Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 172 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 172 Met Asn Ile Arg Asn Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 173 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 173 Met Asn Ile Arg Arg Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 174 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 174 Met Asn Ile Arg Asp Leu His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <175> 175 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 175 Met Asn Ile Arg Pro Ile His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 176 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 176 Met Asn Ile Arg Pro Asn His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 177 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 177 Met Asn Ile Arg Pro Arg His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 178 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 178 Met Asn Ile Arg Pro Asp His Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 179 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 179 Met Asn Ile Arg Pro Leu Ile Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 180 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 180 Met Asn Ile Arg Pro Leu Asn Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 181 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 181 Met Asn Ile Arg Pro Leu Arg Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 182 <211> 52 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG07-H6TEV-GLP-1K28R <400> 182 Met Asn Ile Arg Pro Leu Asp Pro Trp His His His His His His Glu   1 5 10 15 Asn Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser              20 25 30 Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val          35 40 45 Arg Gly Arg Gly      50 <210> 183 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 183 Met Ile Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 184 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 184 Met Asn Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 185 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 185 Met Arg Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 186 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 186 Met Asp Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 187 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 187 Met Asn Asn Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 188 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 188 Met Asn Arg Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 189 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 189 Met Asn Asp Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 190 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 190 Met Asn Ile Ile Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 191 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 191 Met Asn Ile Asn Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 192 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 192 Met Asn Ile Asp Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 193 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 193 Met Asn Ile Arg Ile Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 194 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 194 Met Asn Ile Arg Asn Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 195 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 195 Met Asn Ile Arg Arg Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 196 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 196 Met Asn Ile Arg Asp Leu His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 197 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 197 Met Asn Ile Arg Pro Ile His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 198 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 198 Met Asn Ile Arg Pro Asn His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 199 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 199 Met Asn Ile Arg Pro Arg His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 200 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 200 Met Asn Ile Arg Pro Asp His Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 201 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 201 Met Asn Ile Arg Pro Leu Ile Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 202 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 202 Met Asn Ile Arg Pro Leu Asn Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 203 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 203 Met Asn Ile Arg Pro Leu Arg Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 204 <211> 60 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG15-H6TEV-GLP-1K28R <400> 204 Met Asn Ile Arg Pro Leu Asp Asp Arg Val Ile Val Lys Arg Lys Pro   1 5 10 15 Trp His His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu              20 25 30 Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala          35 40 45 Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg Gly      50 55 60 <210> 205 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 205 Met Ile Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <206> 206 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 206 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 Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 207 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 207 Met Arg Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 208 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 208 Met Asp Ile Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 209 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 209 Met Asn Asn Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 210 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 210 Met Asn Arg Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 211 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 211 Met Asn Asp Arg Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 212 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 212 Met Asn Ile Ile Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 213 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 213 Met Asn Ile Asn Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 214 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 214 Met Asn Ile Asp Pro Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 215 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 215 Met Asn Ile Arg Ile Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 216 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 216 Met Asn Ile Arg Asn Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 217 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 217 Met Asn Ile Arg Arg Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 218 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 218 Met Asn Ile Arg Asp Leu His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 219 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 219 Met Asn Ile Arg Pro Ile His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 220 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 220 Met Asn Ile Arg Pro Asn His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 221 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 221 Met Asn Ile Arg Pro Arg His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 222 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 222 Met Asn Ile Arg Pro Asp His Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 223 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 223 Met Asn Ile Arg Pro Leu Ile Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 224 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 224 Met Asn Ile Arg Pro Leu Asn Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 225 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 225 Met Asn Ile Arg Pro Leu Arg Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 226 <211> 67 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG22-H6TEV-GLP-1K28R <400> 226 Met Asn Ile Arg Pro Leu Asp Asp Arg Val Ile Val Lys Arg Lys Glu   1 5 10 15 Val Glu Thr Lys Ser Ala Pro Trp His His His His His His Glu Asn              20 25 30 Leu Tyr Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser          35 40 45 Tyr Leu Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg      50 55 60 Gly Arg Gly  65 <210> 227 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 227 Met Ile 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 228 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 228 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 229 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 229 Met Arg 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 230 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 230 Met Asp 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 231 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 231 Met Asn Asn 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 232 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 232 Met Asn Arg 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 233 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 233 Met Asn Asp 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 234 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 234 Met Asn Ile Ile 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 235 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 235 Met Asn Ile Asn 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 236 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 236 Met Asn Ile Asp 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 237 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 237 Met Asn Ile Arg Ile 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 238 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 238 Met Asn Ile Arg Asn 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 239 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 239 Met Asn Ile Arg Arg 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 240 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 240 Met Asn Ile Arg Asp 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 241 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 241 Met Asn Ile Arg Pro Ile 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 242 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 242 Met Asn Ile Arg Pro Asn 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 243 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 243 Met Asn Ile Arg Pro Arg 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 244 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 244 Met Asn Ile Arg Pro Asp 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 245 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 245 Met Asn Ile Arg Pro Leu Ile 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 246 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 246 Met Asn Ile Arg Pro Leu Asn 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 247 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 247 Met Asn Ile Arg Pro Leu Arg 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 248 <211> 74 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG29-H6TEV-GLP-1K28R <400> 248 Met Asn Ile Arg Pro Leu Asp 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 Pro Trp His              20 25 30 His His His His His Glu Asn Leu Tyr Phe Gln His Ala Glu Gly Thr          35 40 45 Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Gly Gln Ala Ala Lys Glu      50 55 60 Phe Ile Ala Trp Leu Val Arg Gly Arg Gly  65 70 <210> 249 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 249 Met Ile 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 250 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 250 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 251 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 251 Met Arg 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 252 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 252 Met Asp 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 253 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 253 Met Asn Asn 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 254 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 254 Met Asn Arg 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 255 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 255 Met Asn Asp 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 256 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 256 Met Asn Ile Ile 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 257 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 257 Met Asn Ile Asn 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 258 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 258 Met Asn Ile Asp 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 259 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 259 Met Asn Ile Arg Ile 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 260 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 260 Met Asn Ile Arg Asn 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 261 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 261 Met Asn Ile Arg Arg 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 262 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 262 Met Asn Ile Arg Asp 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 263 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 263 Met Asn Ile Arg Pro Ile 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 264 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 264 Met Asn Ile Arg Pro Asn 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 265 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 265 Met Asn Ile Arg Pro Arg 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 266 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 266 Met Asn Ile Arg Pro Asp 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 267 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 267 Met Asn Ile Arg Pro Leu Ile 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 268 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 268 Met Asn Ile Arg Pro Leu Asn 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 269 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 269 Met Asn Ile Arg Pro Leu Arg 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 270 <211> 81 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG36-H6TEV-GLP-1K28R <400> 270 Met Asn Ile Arg Pro Leu Asp 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 Pro Trp His His His His His Glu Asn Leu Tyr          35 40 45 Phe Gln His Ala Glu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu      50 55 60 Glu Gly Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val Arg Gly Arg  65 70 75 80 Gly     <210> 271 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 271 Met Ile 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> 272 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 272 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> 273 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 273 Met Arg 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> 274 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 274 Met Asp 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> 275 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 275 Met Asn Asn 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> 276 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 276 Met Asn Arg 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> 277 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 277 Met Asn Asp 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> 278 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 278 Met Asn Ile Ile 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> 279 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 279 Met Asn Ile Asn 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> 280 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 280 Met Asn Ile Asp 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> 281 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 281 Met Asn Ile Arg Ile 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> 282 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 282 Met Asn Ile Arg Asn 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> 283 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 283 Met Asn Ile Arg Arg 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> 284 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 284 Met Asn Ile Arg Asp 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> 285 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 285 Met Asn Ile Arg Pro Ile 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> 286 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 286 Met Asn Ile Arg Pro Asn 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> 287 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 287 Met Asn Ile Arg Pro Arg 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> 288 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 288 Met Asn Ile Arg Pro Asp 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> 289 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 289 Met Asn Ile Arg Pro Leu Ile 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> 290 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 290 Met Asn Ile Arg Pro Leu Asn 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> 291 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 291 Met Asn Ile Arg Pro Leu Arg 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> 292 <211> 88 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for PG43-H6TEV-GLP-1K28R <400> 292 Met Asn Ile Arg Pro Leu Asp 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> 293 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for GLP-1 <400> 293 catgcggaag gcacctttac cagcgatgtg agcagctatc tggaaggcca ggcggcgaaa 60 gaatttattg cgtggctggt gaaaggccgc ggctaa 96 <210> 294 <211> 96 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for GLP-1K28R <400> 294 catgcggaag gcacctttac cagcgatgtg agcagctatc tggaaggcca ggcggcgaaa 60 gaatttattg cgtggctggt gcgtggccgc ggctaa 96 <210> 295 <211> 135 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for H6TEV-GLP-1K28R <400> 295 atgcatcatc atcaccacca cgaaaacctg tatttccagc atgcggaagg cacctttacc 60 agcgatgtga gcagctatct ggaaggccag gcggcgaaag aatttattgc gtggctggtg 120 cgtggccgcg gctaa 135 <210> 296 <211> 159 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for PG07-H6TEV-GLP-1K28R <400> 296 atgaatattc gtccattgca tccatggcat catcatcacc accacgaaaa cctgtatttc 63 cagcatgcgg aaggcacctt taccagcgat gtgagcagct atctggaagg ccaggcggcg 123 aaagaattta ttgcgtggct ggtgcgtggc cgcggctaa 162 <210> 297 <211> 183 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for PG15-H6TEV-GLP-1K28R <400> 297 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaaccatg gcatcatcat 60 caccaccacg aaaacctgta tttccagcat gcggaaggca cctttaccag cgatgtgagc 120 agctatctgg aaggccaggc ggcgaaagaa tttattgcgt ggctggtgcg tggccgcggc 180 taa 183 <210> 298 <211> 204 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for PG22-H6TEV-GLP-1K28R <400> 298 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctccat ggcatcatca tcaccaccac gaaaacctgt atttccagca tgcggaaggc 120 acctttacca gcgatgtgag cagctatctg gaaggccagg cggcgaaaga atttattgcg 180 tggctggtgc gtggccgcgg ctaa 204 <210> 299 <211> 225 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for PG29-H6TEV-GLP-1K28R <400> 299 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 60 tctgctggcg gcatcgttct gaccggccca tggcatcatc atcaccacca cgaaaacctg 120 tatttccagc atgcggaagg cacctttacc agcgatgtga gcagctatct ggaaggccag 180 gcggcgaaag aatttattgc gtggctggtg cgtggccgcg gctaa 225 <210> 300 <211> 246 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for PG36-H6TEV-GLP-1K28R <400> 300 atgaatattc gtccattgca tgatcgcgtg atcgtcaagc gtaaagaagt tgaaactaaa 63 tctgctggcg gcatcgttct gaccggctct gcagcggcta aatccacccc atggcatcat 123 catcaccacc acgaaaacct gtatttccag catgcggaag gcacctttac cagcgatgtg 183 agcagctatc tggaaggcca ggcggcgaaa gaatttattg cgtggctggt gcgtggccgc 243 ggctaa 249 <210> 301 <211> 267 <212> DNA <213> Artificial Sequence <220> <223> nucleotide seqeunce for PG43-H6TEV-GLP-1K28R <400> 301 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> 302 <211> 9 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for N-terminal fusion partner PG15 (2-7) <400> 302 Met Asp Arg Val Ile Val Lys Arg Lys   1 5 <210> 303 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for PG43-N2I <400> 303 ggagatatac atatgattat tcgtccattg cat 33 <210> 304 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for PG43-N2I <400> 304 atgcaatgga cgaataatca tatgtatatc tcc 33 <210> 305 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-N2R <400> 305 ggagatatac atatgcgcat tcgtccattg cat 33 <210> 306 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-N2R <400> 306 atgcaatgga cgaatgcgca tatgtatatc tcc 33 <210> 307 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-N2D <400> 307 ggagatatac atatggatat tcgtccattg cat 33 <210> 308 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-N2D <400> 308 atgcaatgga cgaatatcca tatgtatatc tcc 33 <210> 309 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-I3N <400> 309 gatatacata tgaataaccg tccattgcat gat 33 <210> 310 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-I3N <400> 310 atcatgcaat ggacggttat tcatatgtat atc 33 <210> 311 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-I3R <400> 311 gatatacata tgaatcgccg tccattgcat gat 33 <210> 312 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-I3R <400> 312 atcatgcaat ggacggcgat tcatatgtat atc 33 <210> 313 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-I3D <400> 313 gatatacata tgaatgatcg tccattgcat gat 33 <210> 314 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-I3D <400> 314 atcatgcaat ggacgatcat tcatatgtat atc 33 <210> 315 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-R4I <400> 315 atacatatga atattattcc attgcatgat cgc 33 <210> 316 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-R4I <400> 316 gcgatcatgc aatggaataa tattcatatg tat 33 <210> 317 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-R4N <400> 317 atacatatga atattaaccc attgcatgat cgc 33 <210> 318 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-R4N <400> 318 gcgatcatgc aatgggttaa tattcatatg tat 33 <210> 319 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-R4D <400> 319 atacatatga atattgatcc attgcatgat cgc 33 <210> 320 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-R4D <400> 320 gcgatcatgc aatggatcaa tattcatatg tat 33 <210> 321 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-P5I <400> 321 catatgaata ttcgtatttt gcatgatcgc gtg 33 <210> 322 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-P5I <400> 322 cacgcgatca tgcaaaatac gaatattcat atg 33 <210> 323 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-P5N <400> 323 catatgaata ttcgtaactt gcatgatcgc gtg 33 <210> 324 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-P5N <400> 324 cacgcgatca tgcaagttac gaatattcat atg 33 <210> 325 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-P5R <400> 325 catatgaata ttcgtcgctt gcatgatcgc gtg 33 <210> 326 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-P5R <400> 326 cacgcgatca tgcaagcgac gaatattcat atg 33 <210> 327 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-P5D <400> 327 catatgaata ttcgtgattt gcatgatcgc gtg 33 <210> 328 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-P5D <400> 328 cacgcgatca tgcaaatcac gaatattcat atg 33 <210> 329 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-L6I <400> 329 atgaatattc gtccaattca tgatcgcgtg atc 33 <210> 330 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-L6I <400> 330 gatcacgcga tcatgaattg gacgaatatt cat 33 <210> 331 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-L6N <400> 331 atgaatattc gtccaaacca tgatcgcgtg atc 33 <210> 332 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-L6N <400> 332 gatcacgcga tcatggtttg gacgaatatt cat 33 <210> 333 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-L6R <400> 333 atgaatattc gtccacgcca tgatcgcgtg atc 33 <210> 334 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-L6R <400> 334 gatcacgcga tcatggcgtg gacgaatatt cat 33 <210> 335 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-L6D <400> 335 atgaatattc gtccagatca tgatcgcgtg atc 33 <210> 336 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-L6D <400> 336 gatcacgcga tcatgatctg gacgaatatt cat 33 <210> 337 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-H7I <400> 337 aatattcgtc cattgattga tcgcgtgatc gtc 33 <210> 338 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-H7I <400> 338 gacgatcacg cgatcaatca atggacgaat att 33 <210> 339 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-H7N <400> 339 aatattcgtc cattgaacga tcgcgtgatc gtc 33 <210> 340 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-H7N <400> 340 gacgatcacg cgatcgttca atggacgaat att 33 <210> 341 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-H7R <400> 341 aatattcgtc cattgcgcga tcgcgtgatc gtc 33 <210> 342 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-H7R <400> 342 gacgatcacg cgatcgcgca atggacgaat att 33 <210> 343 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> forward primer for PG43-H7D <400> 343 aatattcgtc cattggatga tcgcgtgatc gtc 33 <210> 344 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for PG43-H7D <400> 344 gacgatcacg cgatcatcca atggacgaat att 33 <210> 345 <211> 36 <212> PRT <213> Artificial Sequence <220> <223> amino acid sequence for 8-43 aa fragment of N-terminal fusion          partner (PG43) <400> 345 Asp Arg Val Ile Val Lys Arg Lys Glu Val Glu Thr Lys Ser Ala Gly   1 5 10 15 Gly Ile Val Leu Thr Gly Ser Ala Ala Ala Lys Ser Thr Arg Gly Glu              20 25 30 Val Leu Ala Val          35

Claims (14)

(a) an N-terminal fusion partner consisting of the amino acid sequence represented by the following general formula (1);
Glucagon like peptide-1 (GLP-1) or analogs thereof; And
Preparing an expression vector comprising a nucleotide encoding a fusion polypeptide comprising a linker between said N-terminal fusion partner and said GLP-1 or an analog thereof;
(b) transducing the expression vector into a host cell;
(c) culturing the host cell;
(d) purifying the fusion polypeptide expressed in the host cell; And
(e) culturing the purified fusion polypeptide with a cleavage enzyme to recover GLP-1 or an analog thereof, the method of producing GLP-1 or an analog thereof:
[Formula 1]
Met-Xaa1-Xaa2-Xaa3-Xaa4-Xaa5-Xaa6- (Z) _n
Formula 1 is one selected from the group consisting of SEQ ID NO: 119, SEQ ID NO: 121, SEQ ID NO: 136, SEQ ID NO: 137, SEQ ID NO: 138, and SEQ ID NO: 139. delete delete delete delete delete delete delete The method of claim 1,
Wherein the linker comprises an affinity tag, GLP-1 or an analog thereof. The method of claim 1,
The linker comprises a protease recognition sequence, GLP-1 or a method for producing an analog thereof. The method of claim 10,
The protease recognition sequence is selected from the group consisting of tobacco etch virus protease recognition sequence, enterokinase recognition sequence, ubiquitin carboxy terminal hydrolase recognition sequence, factor Xa recognition sequence, purine recognition sequence, and combinations thereof, GLP-1 Method of production of analogs. The method of claim 1,
Wherein the GLP-1 comprises the amino acid sequence of SEQ ID NO: 151, a method for producing GLP-1 or an analog thereof. The method of claim 1,
The analog of GLP-1 comprises the amino acid sequence of SEQ ID NO: 152, a method for producing GLP-1 or an analog thereof. The method of claim 1,
The fusion polypeptide is composed of the amino acid sequence of any one of the amino acid sequence of SEQ ID NO: 161 to 292, GLP-1 or a method for producing an analog thereof.

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