NOVEL PROINSULIN GLARGINE AND METHOD FOR PREPARING INSULIN GLARGINE THEREFROM
20230287077 · 2023-09-14
Inventors
- Chuangen TANG (Nanjing, CN)
- Jing WANG (Nanjing, CN)
- Shangshu PAN (Nanjing, CN)
- Xiaoyang FAN (Nanjing, CN)
- Xiaorui LIU (Nanjing, CN)
- Song CHEN (Nanjing, CN)
- Haoning Zhang (Nanjing, CN)
Cpc classification
C12N15/70
CHEMISTRY; METALLURGY
International classification
Abstract
The present invention discloses novel proinsulin glargine and method for for preparing insulin glargine therefrom. A sequence of the proinsulin glargine containing SOD fusion peptide subjected to site-directed mutagenesis and “0 C peptide” is designed; recombinant Escherichia coli for expressing insulin glargine are constructed; insulin glargine fusion protein in a form of an inclusion body is expressed; and denaturation, renaturation, modification, enzyme digestion, separation and purification are carried out to obtain a mature insulin glargine active pharmaceutical ingredient. According to the present invention, the SOD fusion peptide sequence is mutated to enhance the fermentation yield of the insulin glargine by 75%; and a “0 C peptide” strategy is adopted to reduce the quality loss and miscleavage impurities in the enzyme digestion transformation. The purity of the insulin glargine active pharmaceutical ingredient prepared in the present invention is up to 99.9%, and the maximum single impurity content is controlled at 0.05%.
Claims
1. Proinsulin glargine, comprising an amino acid sequence having the following structure: ##STR00003## wherein, B.sub.1-B.sub.32 is formed by adding two arginine Arg residues behind a C-terminal of a B.sub.30 site of B.sub.1-B.sub.30 in a Chain B of natural human insulin; A.sub.1-A.sub.20 is an insulin Chain A having 20 amino acids; and A.sub.21 is glycine; wherein the structure of the amino acid sequence is: R-R.sub.1-(B.sub.1-B.sub.32)-(A.sub.1-A.sub.20)-A.sub.21, wherein, R-R.sub.1 is a fusion peptide sequence, and the amino acid sequence of R is TABLE-US-00038 MATX.sub.1AVSVLKGDGPVQGIINFEQX.sub.2ESNGPVKVWGSIX.sub.3GLTEGLHGFH VHEFGDNTAGSTSAGP; X.sub.1 is proline or histidine; X.sub.2 is proline or histidine; X.sub.3 is proline or histidine; and R.sub.1 is arginine or lysine.
2. The proinsulin glargine according to claim 1, wherein the amino acid sequence of R is disclosed as SEQ ID NO: 2 or SEQ ID NO: 3.
3. An DNA for encoding proinsulin glargine according to claim 1.
4. An DNA for encoding proinsulin glargine according to claim 2.
5. An expression vector containing the DNA according to claim 3.
6. Non-plant cells for expressing the proinsulin glargine according to claim 1.
7. Non-plant cells for expressing the proinsulin glargine according to claim 2.
8. A method for producing insulin glargine, comprising the following step: fermenting recombinant Escherichia coli expressing the proinsulin glargine according to claim 1 at 35-37° C. for at least 20 hours to produce the insulin glargine.
9. A method for producing insulin glargine, comprising the following step: fermenting recombinant Escherichia coli expressing the proinsulin glargine according to claim 2 at 35-37° C. for at least 20 hours to produce the insulin glargine.
10. The method according to claim 9, wherein the fermented insulin glargine is subjected to enzyme digestion, modification, renaturation and purification.
11. The method according to claim 10, wherein trypsin is used for the enzyme digestion; and citraconic anhydride is used for the modification.
Description
BRIEF DESCRIPTION OF FIGURES
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DETAILED DESCRIPTION
[0090] The materials, reagents and the like used in the following embodiments can be obtained commercially without special instructions.
Embodiment 1: Structure Design of Novel Proinsulin Glargine
[0091] A protein sequence of proinsulin glargine shown in a formula I was designed:
##STR00002##
[0092] The improved sequence of the proinsulin glargine adopted a “0 C peptide” strategy, namely, no amino acid sequence existed between a chain B and a chain A. An N-terminal lead amino acid sequence can enhance expression, protect the proinsulin glargine and prevent the proinsulin glargine from being degraded by E. coli. The amino acid sequence of R in a fusion peptide R-R.sub.1 is
TABLE-US-00013 MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHE FGDNTAGSTSAGP
(shown as SEQ ID NO: 3). A C terminal of the amino acid sequence of the fusion peptide was connected to the chain B of the insulin glargine through arginine or lysine residues, and finally, the fusion peptide was removed through trypsin cracking.
Embodiment 2: Construction of a Recombinant Plasmid Containing a Novel Proinsulin Glargine Encoding Gene
[0093] A sequence of novel proinsulin glargine was designed according to the method in the Embodiment 1: the sequence of (B.sub.1-B.sub.32)-(A.sub.1-A.sub.20)-A.sub.21 in the novel proinsulin glargine was SEQ ID NO: 6. The complete sequence of proinsulin glargine with fusion peptide was disclosed as SEQ ID NO: 10. In order to ensure that the fusion protein disclosed as SEQ ID NO: 10 could be effectively expressed in E. coli, a genetic codon was optimized. The optimized gene sequence is disclosed as SEQ ID NO: 14, and contains a 5′ Ncol site (CCATGG) and 1 3′ Hind III site (AAGCTT).
[0094] A DNA fragment shown as SEQ ID NO: 14 was chemically synthesized by commercial CRO Company, cracked by Ncol and Hind III restriction enzymes, inserted into a pET-28a expression vector cracked by the same restriction enzyme, and connected by a ligase to form a pET-PIG-1 expression vector.
Embodiment 3: Construction of Recombinant E. coli Expressing Novel Proinsulin Glargine
[0095] The recombinant expression vector pET-PIG-1 constructed in the Embodiment 2 was transfected into E. coli BL21 (DE3) competent cells. Positive clones were screened through Kanamycin resistance and confirmed by using DNA sequencing. The positive clones were cultured and amplified at 37° C., and a sterile culture medium and glycerol were added into the cells. 1 mL of cell culture solution was transferred into a sterile ampoule, and stored at -80° C. to form a proinsulin glargine working seed bank (WCB01).
Embodiment 4: Expression of Proinsulin Glargine Fusion Protein
[0096] The WCB01 obtained in the Embodiment 3 was inoculated into an MLB culture medium (containing 15 g/L of yeast powder and 5 g/L of sodium chloride) according to the inoculation size of 0.2%, and cultured under 37° C. at 250 rpm for 6-14 h to obtain a primary seed solution. The primary seed solution was inoculated into a BFM culture medium which containing 6 g/L of diammonium hydrogen phosphate, 4 g/L of ammonium chloride, 13.5 g/L of potassium dihydrogen phosphate, 1.39 g/L of magnesium sulfate heptahydrate, 2.8 g/L of citric acid monohydrate, 8 g/L of glucose, 3 g/L of yeast powder and 1 mL/L of microelement solution (containing 10 g/L of ferrous sulfate heptahydrate, 1.1 g/L of zinc chloride, 1.0 g/L of copper sulfate pentahydrate, 0.4 g/L of manganese chloride tetrahydrate, 0.2 g/L of boric acid, 2.7 g/L of calcium chloride and 0.2 g/L of sodium molybdate) according to the inoculation size of 0.2%, and further cultured for 8-16 h to obtain a secondary seed solution. Then the secondary seed solution was inoculated into the BFM culture medium in a fermentation tank according to the volume ratio of 1:10, and continuously cultured at a growth temperature of 30-39° C. under conditions of the growth dissolved oxygen content of 10-50% and the growth pH of 6.0-7.3 for 12-18 h until the OD.sub.600 of fermentation liquid was 100-200; and IPTG with the final concentration of 0.1-1.0 mM was added into the fermentation tank to induce the expression of the proinsulin glargine, and other growth conditions were not changed. Continuous inducing was carried out for 8-16 h, and thalli were collected by using a centrifuge.
[0097] Thalli containing inclusion body of the proinsulin glargine were resuspended by using a buffer solution with pH of 8.0 and containing 25 mM of Tris and 10 mM of EDTA, and the concentration of the thalli is controlled to 200 g/L. The thalli were subjected to lysozyme treatment and high-pressure homogenization for cracking, a thallus lysate was centrifuged, the inclusion body precipitate was collected, and a supernatant was removed.
[0098] The inclusion body was washed by using a washing solution with pH of 8.0 and containing 25 mM of Tris, 1 M of urea and 1% of Tween 20. After washing, the inclusion body was resuspended by using a buffer solution containing 25 mM of Tris, 0.1 mM of EDTA and 0.5 mM of L-cysteine, the pH was regulated to 12.0, and dissolving was carried out at a temperature of 15° C. for 50 min. The dissolved solution is named as an inclusion body dissolving solution.
Embodiment 5: Renaturation of Proinsulin Glargine
[0099] The WCB01 inclusion body dissolving solution prepared in the Embodiment 4 was filtered through a 1 .Math.m PP filter element, the temperature was controlled at 20° C., the pH was regulated to 11.0, and then renaturation was carried out for 32 h to obtain renatured proinsulin glargine.
Embodiment 6: Preparation of Insulin Glargine by Enzyme Digestion Transformation and Purification
(I) Preparation of Insulin Glargine by Enzyme Digestion
[0100] After renaturation, 0.2 vol% of citraconic anhydride was added into the renaturation solution for modifying. The pH of the renaturation solution was regulated to 8.5, and the renaturation solution was stirred to modify for 2 h. After modification, ethanolamine which accounts for 60% of the citraconic anhydride for modification was added to neutralize excessive citraconic anhydride, the pH was regulated to 9.4, and neutralizing was carried out for 15 min. Bovine trypsin with the final concentration of 0.063 mg bovine trypsin/g protein was directly added, the pH was regulated to 9.0, and enzyme digestion was carried out at 20° C. for 24 h. Fusion peptide was removed to obtain citraconic anhydride modified insulin glargine. The enzyme digestion engineering was monitored by RP-HPLC (C18). After enzyme digestion, the pH was regulated to 2.0 with hydrochloric acid to terminate the enzyme digestion reaction. The pH was kept at 2.0 for 12 h, and citraconic anhydride modified lysine at a B.sub.29 site was hydrolyzed to obtain the insulin glargine. After hydrolysis, zinc chloride with the final concentration of 3 mM was added, and the pH was regulated to 6.0, so that the insulin glargine forms a flocculent precipitate.
(2) Purification of Insulin Glargine
[0101] The insulin glargine precipitate obtained after enzyme digestion and hydrolysis in step (1) was dissolved by 3 vol% acetic acid under pH of 3.5. The dissolved insulin glargine was loaded as a sample onto a cationic chromatographic column, and balanced with a buffer solution. The insulin glargine could be eluted by 30% isopropanol and 1.0 M of sodium chloride in a linear gradient manner. After cationic chromatography purification, the pH was regulated to 7.3 by zinc chloride with the final concentration of 3 mM so as to form flocculent precipitate of the insulin glargine. The above operations were repeated 2 times.
[0102] The insulin glargine purified by cationic chromatography was loaded onto a reversed-phase preparative chromatographic column. 0.1 M of ammonium dihydrogen phosphate and acetonitrile were mixed in a ratio of 9:1, and then the pH was regulated to 3.5 to obtain a solution-balanced chromatographic column. An elution buffer solution was a solution obtained by mixing a 0.1 M of diammonium hydrogen phosphate-10% acetonitrile mixed solution with the pH of 3.5 and 60% acetonitrile in different proportions. The insulin glargine was eluted by using the linear gradient of the elution buffer solution. The purity of the insulin glargine in the obtained insulin glargine eluate was 97%. After the first reversed-phase chromatographic purification, the pH was regulated to 7.3 by zinc chloride with the final concentration of 3 mM so that the insulin glargine formed the flocculent precipitate. The insulin glargine precipitate was dissolved with 3% acetic acid under the pH value of 3.5. The dissolved insulin glargine was loaded onto the reversed-phase preparative chromatographic column as a sample. 0.05 M of Tris and acetonitrile were mixed in a ratio of 9:1, and the pH was regulated to 8.5 to obtain a solution-balanced chromatographic column. The elution buffer solution was a solution obtained by mixing a 0.05 M of Tris-10% acetonitrile mixed solution with the pH of 8.5 and 60% acetonitrile in different proportions. The insulin glargine was eluted by using the linear gradient of the elution buffer solution, and the purity of the insulin glargine in the insulin glargine eluate was 99.9% by measurement. After the second reversed-phase chromatographic purification, the pH was regulated to 7.3 by 100 mM of a hydrochloric acid solution so that the insulin glargine formed the flocculent precipitate. The collected precipitate was subjected to resuspension washing by 0.3% of a sodium chloride solution (pH of 7.0) 3 times, and then centrifuged to obtain a wet insulin glargine solid.
Embodiment 7 Preparation of Insulin Glargine Active Pharmaceutical Ingredient
[0103] The wet insulin glargine solid prepared in the Embodiment 6 was dissolved by using 100 mM of a hydrochloric acid aqueous solution until the concentration was 30 mg/mL; the pH was regulated to 4.0; filtering was carried out by using a 0.22 .Math.m PES filter membrane; the filtered insulin glargine solution was transferred into a freeze dryer, and the set parameters of the freeze drying procedure were as follows: [0104] 1) shelf refrigeration before feeding: the temperature was set to be -30° C.; [0105] 2) refrigeration control: the temperature was set to be -30° C., the time was set to be 240 min, and the duration was set to be 240 min; [0106] 3) refrigeration of a water catcher: the temperature was set to be -50° C., and the duration was set to be 10 min; [0107] 4) pre-vacuumizing: pre-vacuumizing was carried out to reach 0.2000 mbar, the alarm vacuum was to be 0.5000 mbar, and the alarm vacuum duration was set to be 10 s; [0108] 5) primary drying: the temperature was set to be -10° C., the time was set to be 240 min, the duration was set to be 3,600 min, and the vacuum was set to be 0.1800 mbar; and [0109] 6) vacuum drying: the temperature was set to be 25° C., the time was set to be 480 min, the duration was set to be 240 min, and the vacuum was set to be 0.1800 mbar.
[0110] The purity of the final insulin glargine active pharmaceutical ingredient obtained by the treatment above was 99.9% or above.
Embodiment 8 Preparation of Insulin Glargine
[0111] The specific implementation is the same as the Embodiments 2-6. The difference is as follows: the complete sequence of the proinsulin glargine with the fusion peptide is disclosed as SEQ ID NO: 7, and the gene sequence for encoding the proinsulin glargine with the fusion peptide is disclosed as SEQ ID NO: 11. The expression level of the fusion protein of the proinsulin glargine produced by fermenting the constructed recombinant E. coli under the same conditions is 5.8 g/L; and the HPLC detection on the purified product indicates that the high main peak chromatographic purity is 99.42% and the maximum single impurity chromatographic purity is 0.16% (see
TABLE-US-00014 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 11.226 12.93 0.16 2 13.513 0.79 0.01 3 16.309 3.22 0.04 4 16.792 1.10 0.01 5 17.316 3.26 0.04 6 17.887 4.05 0.05 7 18.525 2.18 0.03 8 18.937 4.83 0.06 9 19.675 7828.73 99.42
Embodiment 9 Preparation of Insulin Glargine
[0112] The specific implementation is the same as the Embodiments 2-6. The difference is as follows: the complete sequence of the proinsulin glargine with the fusion peptide is disclosed as SEQ ID NO: 8, and the gene sequence for encoding the proinsulin glargine with the fusion peptide is disclosed as SEQ ID NO: 12. The expression level of the fusion protein of the proinsulin glargine produced by fermenting the constructed recombinant E. coli under the same conditions is 6.1 g/L; and the HPLC detection on the purified product indicates that the high main peak chromatographic purity is 99.59% and the maximum single impurity chromatographic purity is 0.14% (see
TABLE-US-00015 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 20.927 1134 0.02 2 21.789 6077592 99.59 3 22.388 4444 0.07 4 23.208 1540 0.03 5 25.206 8487 0.14 6 28.111 1759 0.03 7 28.604 2850 0.05 8 28.897 321 0.01 9 29.017 318 0.01 10 29.091 1534 0.03 11 29.392 1147 0.02 12 29.877 1119 0.02 13 30.001 602 0.01
Embodiment 10 Preparation of Insulin Glargine
[0113] The specific implementation is the same as the Embodiments 2-6. The difference is as follows: the complete sequence of the proinsulin glargine with the fusion peptide is disclosed as SEQ ID NO: 9, and the gene sequence for encoding the proinsulin glargine with the fusion peptide is disclosed as SEQ ID NO: 13. The expression level of the fusion protein of the proinsulin glargine produced by fermenting the constructed recombinant E. coli under the same conditions is 6.0 g/L; and the HPLC detection on the purified product indicates that the high main peak chromatographic purity is 99.80% and the maximum single impurity chromatographic purity is 0.09% (see
TABLE-US-00016 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 18.410 6105 0.09 2 17.715 1916 0.03 3 21.611 6832023 99.80 4 28.367 1690 0.02 5 29.131 2405 0.04 6 29.849 813 0.01 7 29.999 737 0.01
Comparative Example 1: Construction of Non-Optimized Recombinant E. coli of Proinsulin Glargine
[0114] The specific implementation is the same as the Embodiments 2-3. The difference is that the amino acid sequence in the fusion peptide R-R.sub.1 is replaced by
TABLE-US-00017 MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHE FGDNTAGSTSAGPR (SEQ ID NO: 1),
and a DNA segment of the whole amino acid sequence for encoding the SEQ ID NO: 1 is correspondingly adjusted to:
TABLE-US-00018 5′-ATGGCGACGAAAGCCGTGAGCGTGCTGAAGGGCGACGGCCCAGTGC AGGGCATCATCAATTTCGAGCAGAAAGAAAGTAATGGACCAGTGAAGGTG TGGGGAAGCATTAAAGGACTGACTGAAGGCCTGCATGGATTCCATGTTCA TGAGTTTGGAGATAATACAGCTGGCTCTACCAGTGCAGGTCCGAAATTTG TGAACCAGCATCTGTGCGGCAGCCATCTGGTGGAAGCGCTGTATCTGGTG TGCGGCGAACGCGGCTTCTTTTATACCCCGAAAACCCGCCGCGGCATTGT GGAACAGTGCTGCACCAGCATTTGCAGCCTGTATCAGCTGGAAAATTATT GCGGCTAA-3′ (SEQ ID NO: 22).
[0115] The complete sequence of the proinsulin glargine with the fusion protein is disclosed as
TABLE-US-00019 MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHE FGDNTAGSTSAGPKFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRGIVE QCCTSICSLYQLENYCG (SEQ ID NO: 15).
[0116] Then enzyme digestion treatment was carried out on the nucleotide sequence for encoding the above amino acid sequence and plasmid pET-28a by using Ncol and Hind III restriction enzymes, and the segment subjected to enzyme digestion was connected with a vector to obtain a recombinant expression vector pET-PIG-2.
[0117] The recombinant expression vector pET-PIG-2 was transformed into E. coli BL21 (DE3) competent cells. Positive clones were screened through Kanamycin resistance and confirmed by using DNA sequencing. The positive clones were cultured and amplified, and a sterile culture medium and glycerol were added into the cells. 1 mL of cell culture solution was transferred into a sterile ampoule, and stored at -80° C. to form a proinsulin glargine working seed bank (WCB02).
Comparative Example 2: Expression of Proinsulin Glargine Fusion Protein
[0118] The recombinant bacteria WCB02 obtained in the Comparative example 1 were cultured in three batches according to the method in the Embodiment 4, and processed to obtain an inclusion body dissolving solution; and then, renaturation was carried out according to the method in the Embodiment 5. The yields of proinsulin glargine renaturation precursors obtained by fermenting WCB01 and WCB02 were respectively detected by using HPLC. The results of three groups of parallel experiments are shown in Table 4. The WCB01 renaturation liquid chromatography is shown in the
TABLE-US-00020 Yields of proinsulin glargine renaturation precursor after different insulin glargine sequence expressions # Yield under use of WCB01 (g/L) Yield under use of WCB02 (g/L) Rate of increase of WCB01 relative to WCB02 1 6.2 3.5 77% 2 6.3 3.6 75% 3 6.6 3.7 78%
[0119] The result in Table 1 shows that by using the SOD fragment subjected to site-directed mutagenesis as the fusion peptide and adopting the preferred sequence SEQ ID NO: 10 of the “0 C peptide” strategy, more effective expression and more stable high fermentation yield can be obtained, and specifically, the fermentation yield of insulin glargine is increased by 75% or above, and is maximally increased by 78%.
TABLE-US-00021 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 5.981 3728 0.02 2 6.670 2863 0.02 3 7.336 96294 0.62 4 8.800 30923 0.20 5 9.653 63346 0.41 6 9.981 14592 0.09 7 10.447 15349 0.10 8 11.440 95758 0.62 9 11.877 132292 0.86 10 12.207 217422 1.41 11 13.066 6246235 40.40 12 13.486 2272194 14.69 13 14.046 2436344 15.76 14 14.868 472685 3.06 15 15.527 370660 2.40 16 16.669 658939 4.26 17 17.230 168242 1.09 18 17.739 917577 5.93 19 18.501 735984 4.76 20 19.546 270981 1.75 21 20.324 141662 0.92 22 21.023 98337 0.64
TABLE-US-00022 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 7.295 2536 0.02 2 7.619 1584 0.01 3 7.643 1159 0.01 4 8.441 24235 0.20 5 8.945 1332 0.01 6 9.322 46100 0.37 7 9.671 18693 0.15 8 10.116 12874 0.10 9 10.464 6401 0.05 10 11.134 100618 0.82 11 11.542 107712 0.87 12 11.924 84129 0.68 13 12.079 72595 0.59 14 12.838 6335431 51.42 15 13.381 1267742 10.29 16 13.822 1891466 15.35 17 14.168 547291 4.44 18 14.719 330588 2.68 19 15.360 275838 2.24 20 15.856 33956 0.28 21 16.134 93890 0.76 22 16.482 398809 3.24 23 16.969 95196 0.77 24 17.577 397227 3.22 25 18.013 54857 0.45 26 18.401 119467 0.97
TABLE-US-00023 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 7.290 2542 0.02 2 7.614 1385 0.01 3 7.642 1119 0.01 4 8.405 26003 0.20 5 8.947 1558 0.01 6 9.338 49743 0.38 7 9.676 21486 0.16 8 10.137 11248 0.09 9 10.509 6231 0.05 10 11.161 80814 0.62 11 11.552 115175 0.88 12 11.919 201458 1.54 13 12.833 6554868 50.05 14 13.352 1500343 11.46 15 13.827 2051646 15.67 16 14.165 580765 4.43 17 14.695 409681 3.13 18 15.405 186479 1.42 19 15.716 133971 1.02 20 16.110 89273 0.68 21 16.466 367821 2.81 22 16.971 120239 0.92 23 17.569 353694 2.70 24 17.854 50995 0.39 25 17.973 54941 0.42 26 18.382 121957 0.93
TABLE-US-00024 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 7.153 43402 0.50 2 8.547 10401 0.12 3 9.409 28985 0.34 4 10.141 3161 0.04 5 11.207 31408 0.37 6 11.584 59171 0.69 7 11.962 47234 0.55 8 12.828 3525420 40.98 9 13.249 1256739 14.61 10 13.820 1511732 17.57 11 14.646 279064 3.24 12 15.240 205973 2.39 13 16.454 370498 4.31 14 17.510 552834 6.43 15 18.300 264290 3.07 16 18.893 119583 1.39 17 19.338 137776 1.60 18 20.046 88628 1.03 19 20.821 42519 0.49 20 21.517 23344 0.27
TABLE-US-00025 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 7.105 44323 0.44 2 8.487 12383 0.12 3 9.382 25154 0.25 4 9.738 4008 0.04 5 10.096 5892 0.06 6 10.462 3146 0.03 7 11.132 39277 0.39 8 11.532 51520 0.51 9 11.867 48987 0.49 10 12.749 3568760 35.41 11 13.174 1772217 17.58 12 13.732 1090494 10.82 13 14.101 417898 4.15 14 14.555 277829 2.76 15 15.211 175373 1.74 16 16.366 535098 5.31 17 17.420 472387 4.69 18 17.448 387633 3.85 19 18.199 539587 5.35 20 19.199 410304 4.07 21 20.267 87937 0.87 22 20.763 109568 1.09
TABLE-US-00026 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 7.197 2058 0.03 2 7.586 960 0.01 3 7.627 1039 0.01 4 8.390 17399 0.22 5 8.935 1431 0.02 6 9.296 30156 0.38 7 9.673 23883 0.30 8 10.159 6689 0.09 9 10.513 4025 0.05 10 10.864 2450 0.03 11 11.120 39285 0.50 12 11.560 64445 0.82 13 11.918 141948 1.81 14 12.827 3726812 47.46 15 13.260 1169681 14.90 16 13.830 1192710 15.19 17 14.172 363760 4.63 18 14.667 241046 3.07 19 15.381 102318 1.30 20 15.673 45485 0.58 21 15.927 4970 0.06 22 16.170 36141 0.46 23 16.509 215523 2.74 24 16.941 45912 0.58 25 17.322 32154 0.41 26 17.582 166907 2.13 27 17.837 67783 0.86 28 18.439 104829 1.34
Comparative 3 Expression of Proinsulin Glargine Containing 1 Mutated Fusion Peptide Sequence
[0120] The strategy in the Embodiment 1 was adjusted, the sequence for encoding insulin glargine was designed, and was expressed in a host cell, so that the proinsulin glargine contained a fusion peptide R sequence subjected to site-directed mutagenesis on 1 site on the basis of SEQ ID NO: 15. The amino acid sequence was designed as:
TABLE-US-00027 MATKAVSVLKGDGPVQGIINFEQKESNGPVKVWGSIHGLTEGLHGFHVHE FGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRGIVE QCCTSICSLYQLENYCG (SEQ ID NO: 16);
and the nucleotide sequence for encoding the amino acid sequence was disclosed as SEQ ID NO: 19.
[0121] Renaturation was carried out according to the method in Embodiment 5. The yield of the proinsulin glargine renaturation precursor obtained by strain fermentation was detected by HPLC, and the result indicated that the yield of the proinsulin glargine renaturation precursor under the same conditions was 1.9 g/L. The renaturation liquid chromatography is respectively disclosed as
TABLE-US-00028 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 7.100 31530 0.58 2 8.481 6966 0.13 3 9.372 14681 0.27 4 9.756 2109 0.04 5 10.125 3795 0.07 6 10.475 3608 0.07 7 11.172 25035 0.46 8 11.546 42006 0.77 9 11.889 77271 1.42 10 12.789 1939000 35.64 11 13.211 814935 14.98 12 13.775 613260 11.27 13 14.144 231346 4.25 14 14.573 160525 2.95 15 15.227 96528 1.77 16 16.406 305068 5.61 17 17.445 459176 8.44 18 18.186 286812 5.27 19 19.207 180833 3.32 20 19.945 50905 0.94 21 20.343 48036 0.88 22 20.867 30012 0.55 23 21.435 15293 0.28 24 22.233 1649 0.03
Comparative Example 4 Expression of Proinsulin Glargine Containing 2 Mutated Fusion Peptide Sequences
[0122] The strategy in the Embodiment 1 was adjusted, the sequence for encoding insulin glargine was designed, and was expressed in a host cell, so that the proinsulin glargine contained a fusion peptide R sequence subjected to site-directed mutagenesis on 2 sites on the basis of SEQ ID NO: 15. The amino acid sequence was respectively designed as:
TABLE-US-00029 MATKAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHE FGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRRGIVE QCCTSICSLYQLENYCG (SEQ ID NO: 17);
and the nucleotide sequence for encoding the amino acid sequence was disclosed as SEQ ID NO: 20.
[0123] Renaturation was carried out according to the method in Embodiment 5. The yield of the proinsulin glargine renaturation precursor obtained by strain fermentation was detected by HPLC, and the result indicated that the yield of the proinsulin glargine renaturation precursor under the same conditions was 2.2 g/L. The renaturation liquid chromatography is respectively disclosed as
TABLE-US-00030 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 7.080 32442 0.54 2 8.429 7111 0.12 3 9.337 17594 0.29 4 10.104 1634 0.03 5 11.042 10294 0.17 6 11.485 36434 0.61 7 11.850 108086 1.80 8 12.720 2197231 36.59 9 13.132 1050997 17.50 10 13.712 931607 15.51 11 14.506 172841 2.88 12 15.214 174204 2.90 13 16.344 273268 4.55 14 17.415 359878 5.99 15 17.744 148993 2.48 16 18.227 137118 2.28 17 18.752 106493 1.77 18 19.230 109246 1.82 19 19.905 82369 1.37 20 20.807 25772 0.43 21 21.341 21575 0.36
Comparative Example 5 Expression of Proinsulin Glargine Containing C Peptide
[0124] The strategy in the Embodiment 1 was adjusted, and the sequence for encoding insulin glargine was designed, and was expressed in a host cell, so that the proinsulin glargine contained “C peptide (EAR)”; the amino acid sequence was designed as:
TABLE-US-00031 MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHE FGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTPKTRREARG IVEQCCTSICSLYQLENYCG (SEQ ID NO: 18);
and the nucleotide sequence for encoding the amino acid sequence was disclosed as SEQ ID NO: 21.
[0125] Renaturation was carried out according to the method in Embodiment 5. The yield of the proinsulin glargine renaturation precursor obtained by strain fermentation was detected by HPLC. The result is disclosed as Table 8. The renaturation liquid chromatography is disclosed as
TABLE-US-00032 Yields of proinsulin glargine renaturation precursor after different insulin glargine sequence expressions Yield under use of WCB01 (g/L) Yield of strain expressing SEQ ID NO: 19 (g/L) Yield of strain expressing SEQ ID NO: 20 (g/L) Yield of strain expressing SEQ ID NO: 21 (g/L) 6.2 1.9 2.2 1.8
[0126] The result of Table 6 indicates that the fermentation yield obtained by using the fusion peptide sequence subjected to site-directed mutagenesis on 1 or 2 sites and the fusion peptide sequence adopting the “C peptide” (REA) strategy is far lower than the yield of WCB01.
TABLE-US-00033 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 7.094 30377 0.57 2 8.487 7761 0.15 3 9.382 14594 0.27 4 9.743 2603 0.05 5 10.105 4467 0.08 6 10.482 3140 0.06 7 11.115 25287 0.48 8 11.541 33463 0.63 9 11.862 75536 1.42 10 12.763 1878930 35.33 11 13.179 848037 15.95 12 13.740 611936 11.51 13 14.113 245888 4.62 14 14.557 172803 3.25 15 15.205 112415 2.11 16 16.377 259256 4.87 17 17.439 418883 7.88 18 18.243 305379 5.74 19 19.245 131360 2.47 20 20.040 73644 1.44 21 20.787 59947 1.13
Comparative Example 6: Renaturation, Enzyme Digestion Transformation And Purification
[0127] The inclusion body dissolving solutions prepared in the Comparative example 2 were respectively renatured according to the conditions in the Embodiment 5. Modification, enzyme digestion and purification were carried out on the renatured sample according to the method in Embodiment 6. The liquid chromatograms of the recombinant bacterium WCB01 and the recombinant bacterium WCB02 in the Comparative example 2 after sample purification are respectively disclosed as
TABLE-US-00034 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 19.960 6005658 99.93 2 21.681 3011 0.05 3 29.279 1181 0.02
TABLE-US-00035 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 6.450 753 0.01 2 7.657 420 0.01 3 80184 609 0.01 4 8.438 538 0.01 5 8.510 268 0.00 6 8.576 256 0.00 7 8.709 792 0.01 8 8.816 316 0.00 9 8.879 360 0.00 10 8.972 987 0.01 11 9.162 449 0.01 12 9.186 268 0.00 13 9.304 365 0.00 14 9.410 1019 0.01 15 9.598 450 0.01 16 9.694 625 0.01 17 9.804 741 0.01 18 9.880 559 0.01 19 10.156 2983 0.04 20 10.475 739 0.01 21 11.001 119807 1.49 22 11.531 2151 0.03 23 12.047 1591 0.02 24 12.292 1474 0.02 25 12.465 2309 0.03 26 12.752 14885 0.18 27 12.949 15308 0.19 28 13.220 17592 0.22 29 13.536 52838 0.66 30 13.878 85586 1.06 31 14.381 8191 0.10 32 15.065 10546 0.13 33 16.135 8074 0.10 34 16.763 173201 2.15 35 17.114 21542 0.27 36 17.819 1731 0.02 37 19.010 20447 0.25 38 19.736 7428281 92.25 39 20.859 50936 0.63 40 22.476 53 0.00 41 22.666 1348 0.02 42 23.631 109 0.00 43 23.721 434 0.01 44 29.584 191 0.00
Comparative 7: Enzyme Digestion Transformation Using Porcine Trypsin
[0128] The WCB01 renatured solution prepared in the Embodiment 3 was subjected to enzyme digestion transformation through porcine trypsin according to the enzyme digestion method in Embodiment 4. The liquid chromatograms of the enzyme digestion transformation respectively utilizing porcine trypsin and bovine trypsin are respectively disclosed as
TABLE-US-00036 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 6.123 25974 0.09 2 6.576 32496 0.12 3 7.150 18545 0.07 4 7.344 2774 0.01 5 7.593 36611 0.13 6 7.897 26753 0.10 7 8.428 65286 0.23 8 8.607 16521 0.06 9 8.825 15502 0.06 10 9.011 92535 0.33 11 9.435 27660 0.10 12 9.628 10516 0.04 13 9.787 5496 0.02 14 9.894 5612 0.02 15 10.066 9195 0.03 16 10.212 16404 0.06 17 10.423 6520 0.02 18 10.570 39115 0.14 19 10.880 29368 0.10 20 11.451 70108 0.25 21 12.002 3610530 12.83 22 12.403 98065 0.35 23 12.783 20620 0.07 24 130.84 125933 0.45 25 13.728 31204 0.11 26 14.421 134964 0.48 27 15.341 58382 0.21 28 15.609 18801 0.07 29 16.793 115174 0.41 30 17.311 2486 0.04 31 17.509 12968 0.05 32 18.124 111013 0.39 33 18.809 115239 0.41 34 19.372 70992 0.25 35 20.447 9569327 34.00 36 21.527 2327425 8.27 37 22.216 5483055 19.48 38 23.270 395684 1.41 39 24.581 2141960 7.61 40 24.992 611732 2.17 41 26.320 440323 1.56 42 26.819 2029210 7.21 43 28.805 61812 0.22 44 29.604 4203 0.01
TABLE-US-00037 Main peak areas and retention time in chromatography detection # Retention time Peak area % peak area 1 6.141 96963 0.29 2 6.648 41805 0.13 3 7.202 38587 0.12 4 7.374 14189 0.04 5 7.629 21517 0.06 6 7.928 54454 0.16 7 8.446 73781 0.22 8 8.653 45482 0.14 9 8.868 21845 0.07 10 9.061 100605 0.30 11 9.229 18851 0.06 12 9.486 30588 0.09 13 9.675 14226 0.04 14 9.821 7601 0.02 15 9.973 11309 0.03 16 10.093 8934 0.03 17 10.252 18843 0.06 18 10.435 6463 0.02 19 10.618 37421 0.11 20 10.929 24440 0.07 21 11.504 79186 0.24 22 12.104 475734 1.42 23 12.472 78628 0.24 24 12.870 33249 0.10 25 13.180 160861 0.48 26 13.862 35587 0.11 27 14.570 76881 0.23 28 14.817 73991 0.22 29 15.508 75852 0.23 30 16.301 20875 0.06 31 17.032 106983 0.32 32 17.735 29030 0.09 33 18.272 198861 0.60 34 18.860 131756 0.39 35 19.604 78034 0.23 36 20.730 11142487 33.37 37 21.832 2758566 8.26 38 22.481 9109465 27.28 39 23.599 505312 1.51 40 24.919 2404115 7.20 41 25.339 1005576 3.01 42 27.172 4038121 12.09 43 29.079 87112 0.26
[0129] Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone who is familiar with the technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be subject to that defined in the claims.