NOVEL PRO-INSULIN ASPART STRUCTURE AND METHOD FOR PREPARING INSULIN ASPART

Abstract

Provided in the present invention are a novel pro-insulin aspart structure design and a method for preparing insulin aspart. The main steps comprise designing the pro-insulin aspart sequence, constructing recombinant insulin aspart engineered bacteria, inducing an insulin fusion protein expressed in the form of an inclusion body by means of the engineered bacteria, and obtaining a mature insulin aspart material drug by means of denaturing, renaturing enzymatic digestion, and separation purification, By means of changing the recombinant leading peptide and C-peptice sequences, the invention avoids the dangerous and tedious step of cleavage using cyanogen bromide. The C-peptide is shortened to one amino acid, reducing the quality loss of enzymatic digestion conversion.

Claims

1-11. (canceled)

12. A novel short-proinsulin aspart sequence for preparing recombinant insulin aspart and analogs thereof, wherein the sequence has an amino acid sequence as shown in Formula I:
R-R.sub.1-(B.sub.1-B.sub.27)-B.sub.28-B.sub.29-B.sub.30-R.sub.2-(A.sub.1-A.sub.21), wherein R-R.sub.1 is a leading peptide sequence, which meets the following conditions: a. R is one part of super oxide dismutase (SOD) homolog, which consists of 63 amino acids including active methionine; and two cysteine (C) residues are substituted by serine (S); wherein, the sequence of R is SEQ ID NO 1: TABLE-US-00011 MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHE FGDNTAGSTSAGP; b. the leading peptide does not affect refolding of short-proinsulin aspart, and can be cleaved; c. R.sub.1 is any one of Arg and Lys; R.sub.2 is a C-peptide, which consists of any one from Arg or Lys; B.sub.1-B.sub.27 denotes an amino acid sequence of B1 to B27 in B-chain of native human insulin; A.sub.1-A.sub.21 denotes an A-chain of native human insulin; B.sub.28 is Aspartic acid; B.sub.29 is Lysine; and B.sub.30 is Threonine; the sequence of (B.sub.1-B.sub.27)-B.sub.28-B.sub.29-B.sub.30-R.sub.2-(A.sub.1-A.sub.21) is one of SEQ ID NO 12 or SEQ ID NO 13; a sequence of SEQ ID NO 12 is: FVNQHLCGSHLVEALYLVCGERGFFYTDKTRGIVEQCCTSICSLYQLENYCN; and a sequence of SEQ ID NO 13 is: TABLE-US-00012 FVNQHLCGSHLVEALYLVCGERGFFYTDKTKGIVEQCCTSICSLYQLENY CN.

13. The proinsulin aspart sequence according to claim 12, wherein R1 and R2 are a same amino acid, and are any one of arginine or lysine.

14. The proinsulin aspart sequence according to claim 12, wherein the proinsulin aspart sequence is one of SEQ ID NO 14 or SEQ ID NO 15; a sequence of SEQ ID NO 14 is: TABLE-US-00013 MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHE FGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTDKTRGIVEQ CCTSICSLYQLENYCN; a sequence of SEQ ID NO 15 is: TABLE-US-00014 MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHE FGDNTAGSTSAGPKFVNQHLCGSHLVEALYLVCGERGFFYTDKTKGIVEQ CCTSICSLYQLENYCN.

15. A nucleic acid sequence, wherein the nucleic acid sequence encodes the proinsulin aspart sequence of claim 12.

16. A recombinant expression vector, wherein the recombinant expression vector comprises the nucleic acid sequence of claim 15.

17. The recombinant expression vector according to claim 16, wherein the expression vector is proinsulin aspart pET-API.

18. A microorganism, wherein the microorganism comprises the recombinant expression vector of claim 16.

19. The microorganism according to claim 18, wherein the microorganism is an E. coli, and the expression vector is proinsulin aspart pET-API.

20. A process for preparing insulin aspart, comprising following steps: (a) culturing E. coli cells under conditions suitable for expressing the short-proinsulin aspart sequence of the Formula I of claim 12; (b) disrupting the cultured E. coli cells to provide inclusion bodies containing the short-proinsulin aspart sequence of claim 12; (c) solubilizing and refolding the inclusion bodies; (d) enzymatically cleaving the refolded proinsulin aspart to be converted into insulin aspart-R.sub.2 intermediates with crude purity; (e) purifying the insulin aspart-R.sub.2 intermediates by a two-step ion exchange chromatography and a one-step RP-HPLC chromatography to obtain insulin aspart-R.sub.2 intermediates with high purity; (f) converting the purified insulin aspart-R.sub.2 intermediates with carboxypeptidase B to obtain mature insulin aspart; (g) further purifying the mature insulin aspart using the RP-HPLC chromatography to obtain the insulin aspart with high purity after carboxypeptidase B digestion; and (h) drying and crystallizing the insulin aspart with high purity to form a final API of insulin aspart.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0088] According to the present invention, FIG. 1 is a process flow diagram of the preparation of insulin aspart using an improved proinsulin aspart in Escherichia coli.

DESCRIPTION OF THE EMBODIMENTS

Detailed Ways

[0089] In order to facilitate those skilled in the art to understand the content of the present invention, the technical solutions of the present invention will be further described below in conjunction with specific embodiments, but the following content should not limit the scope of protection claimed by the claims of the present invention in any way.

[0090] The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

Example 1: Construction of an E. coli Clone Expressing Proinsulin Aspart of Formula I

[0091] A proinsulin aspart fusion protein sequence, such as Formula I, is designed for expression in E. coli. The N terminal leading amino acid sequence can enhance expression and protect proinsulin aspart from degradation by E. coli. The preferred leading amino acid sequence is

TABLE-US-00008 (SEQ ID NO: 1) MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHE FGDNTAGSTSAGP;

[0092] The C terminal of this leading amino acid sequence is connected to the B-chain of the insulin aspart by an arginine or lysine residue, which enable the cleavage of the leading peptide by Trypsin at the same time. The C-peptide of the proinsulin aspart is shortened to a single arginine or lysine residue, and the precursor sequence of the novel proinsulin aspart sequence, is:

TABLE-US-00009 (SEQ ID NO: 2) FVNQHLCGSHLVEALYLVCGERGFFYTDKTRGIVEQCCTSICSLYQLENY CN.

[0093] The full length of proinsulin aspart with leading peptide is MATHAVSVLKGDGPVQGIINFEQHESNGPVKVWGSIHGLTEGLHGFHVHEFGDNTAGSTSAGPRFVNQHLCGSHLVEALYLVCGERGFFYTDKTRGIVEQCCTSICSLYQLENYCN (SEQ ID NO: 14). To ensure efficient expression of the protein of SEQ ID NO: 14 in E. coli, the genetic codons are optimized. The optimized gene sequences are:

TABLE-US-00010 (SEQ ID NO: 18) 5′ATGGCGACGCATGCCGTGAGCGTGCTGAAGGGCGACGGCCCAGTGCAG GGCATCATCAATTTCGAGCAGCATGAAAGTAATGGACCAGTGAAGGTGTG GGGAAGCATTCATGGACTGACTGAAGGCCTGCATGGATTCCATGTTCATG AGTTTGGAGATAATACAGCTGGCTCTACCAGTGCAGGTCCGCGGTTTGTG AACCAGCATCTGTGCGGCAGCCATCTGGTGGAAGCGCTGTATCTGGTGTG CGGCGAACGCGGCTTCTTTTATACCGATAAAACCCGCGGCATTGTGGAAC AGTGCTGCACCAGCATTTGCAGCCTGTATCAGCTGGAAAACTATTGCAAC TAA3′.

[0094] A DNA fragment of SEQ ID NO: 18 is chemically synthesized by commercial CRO company. A 5′ NcoI site: CCATGG and 3′ Hind III site: AAGCTT are included in the synthesized DNA fragment. The DNA fragment encoding the entire amino acid sequence of SEQ ID NO: 14 is cleaved with the Nco I and Hind III restriction enzymes and inserted into the pET 28a expression vector, which is digested with the same restriction enzymes, to form the pET-API expression vector.

[0095] The transformed pET-API expression vectors are transfected into E. coli strain BL21 (DE3). The positive clones are selected by Kanamycin resistance and confirmed by DNA sequencing. The confirmed positive clone is cultured and amplified. Sterile medium and glycerol are added to the cells. One (1) ml aliquots of the cells are then dispensed into sterile ampoules and stored at −80° C., forming the proinsulin aspart working cell bank (WCB).

Example 2: Expression of the Proinsulin Aspart Fusion Protein

[0096] Culture the cells from the WCB of Example 1 in LB media (containing 1.5% w/v yeast extract, and 0.5% w/v NaCl) at 37° C. for 8 hours to obtain the cell seeds solution. The recovered seeds solution are inoculated into BFM media (containing 0.6% w/v (NH.sub.4).sub.2HPO.sub.4, 0.4% w/v NH.sub.4Cl, 1.35% w/w KH.sub.2PO.sub.4, 0.139% w/w MgSO.sub.4.7H.sub.2O, 0.28% w/w Citric Acid Monohydrate, 0.8% w/w Glucose, 0.3% w/w Yeast Extract Powder and 1% mL/L Trace Element Solution) to further culture for another 8 hours to obtain a second generation of seeds, which is then inoculated into fermentor in a volume ratio of 1:10. Culture for about 16 hours until the O.D.sub.600. (optical density) of the fermentation liquor reaches about 150. Then, add 1 mM IPTG to the fermentor to induce proinsulin aspart expression. Incubate for another 12 hours to finish the fermentation. The cells are collected by centrifugation.

[0097] The harvested cells, containing inclusion bodies of proinsulin aspart, are suspended in 25 mM Tris, 10 mM EDTA, pH 8.0 buffers, with a cell concentration of 200 g/L. The cells are lysed by lysozyme treatment and homogenization. Discard the supernatant of lysed cells and collect the inclusion body pellet.

[0098] Wash the inclusion bodies pellet with a buffer of 25 mM Tris, 1M Urea, 1% Tween 20, pH 8.0. Resuspend the washed inclusion bodies in a buffer of 25 mM Tris, 0.1 M EDTA and 0.5 mM L-cysteine pH is adjusted to 12 and keep the solution dissolved at 15-25° C. for 10-60 min. The solution is named the inclusion bodies dissolution solution.

Example 3: Refolding of the Proinsulin Aspart

[0099] After dissolution the inclusion body solution was filtered with a 1 μm PP filter element, the temperature was controlled to 20° C., the pH was adjusted to 11.0, and then refolded for 24-48 h to obtain refolded proinsulin aspart.

Example 4: Transformation and Purification of Trypsin Enzyme Digestion

[0100] When the refolding is complete, adjust the solution pH to 9.0. Add Trypsin to the final concentration of 0.063 mg/g protein to the refolded protein solution. Digest at 18° C. for 36 hours to cleave the leading peptides, to obtain B31Arg-insulin aspart intermediate. The process can be monitored by HPLC-RP (C18). After digestion is finished, a final concentration of 3 mM zinc is added to the solution, and the pH is adjusted to 6.0. A flocculent precipitate of B31Arg-insulin aspart is formed.

[0101] The B31Arg-insulin aspart precipitate is dissolved in a buffer of 3% isopropanol and 50 mM acetic acid, at pH 3.5. The dissolved B31Arg-insulin aspart solution is loaded onto a SP column as sample, which has been equilibrated with the same buffer for dissolving the insulin aspart. The B31Arg-insulin aspart can also be eluted by 30% isopropanol, 1 M sodium chloride in linear gradient. After two SP chromatography purifications, the purity of B31Arg-insulin aspart is about 85%. The B31Arg-insulin aspart eluate was added with 5 mM zinc chloride and the pH was adjusted to 6.5 to form a flocculated precipitate of B31Arg-insulin aspart.

[0102] Further purification can be performed with reverse phase preparation to remove impurities. The final purity of B31Arg-aspart insulin can reach more than 98%, and the content of DesB30-insulin aspart drops below 0.1%. That high-purity B31Arg-aspart insulin intermediate is obtained.

Example 5: Transformation of Carboxypeptidase B Enzyme Digestion

[0103] The purified B31Arg-insulin aspart intermediate precipitate was resuspended in 25 mM Tris and 2 mM EDTA, and dissolved at the pH 9.0. After the dissolution is completed, adjust the pH of the solution to 8.5 and add the final concentration of 0.33 mg/g protein to the dissolution solution to conduct enzyme digestion at 20° C. for 3 h. After that, the C-peptide arginine residue was removed to obtain insulin aspart. This process can be detected by HPLC-RP (C18). Otherwise, after the digestion is completed, the pH of the digestion solution is adjusted to 3.8, and the digestion is terminated.

Example 6: Final Purification and Freeze-Dry

[0104] Loading the converted insulin aspart onto the reverse phase preparative chromatography column and equilibrate the column with a solution obtained by mixing 0.1M ammonium acetate, 0.1M sodium acetate, 0.1 M Tris and pH 7.5 with acetonitrile a ratio of 9:1. The elution buffer is a solution obtained by mixing a solution of 0.1 M ammonium acetate, 0.1 M sodium acetate, 0.1 M Tris and pH 3.5 with acetonitrile in a ratio of 1:9. In addition, insulin aspart is eluted with a linear gradient of elution buffer.

[0105] Zinc chloride with a final concentration of 5 mM was added to the insulin aspart solution in the reverse phase preparation eluate, to form a precipitate. In order to control the concentration of insulin aspart to 8 mg/ml, the precipitate is dissolved in 10 mM hydrochloric acid solution. Add the final concentration of 150 mM NaCl, 50 mM trisodium citrate, 0.5% (v/v) m-cresol and 20% (v/v) absolute ethanol to the insulin aspart solution, and adjust the pH to 6.3. Finally, add zinc acetate with a final concentration of 6 mM to the solution and then leave it at 15° C. for 24 hours after stirring at room temperature for about 3 h. After crystallization is completed, the crystals are collected by suction filtration. Then use 5 ml 75% ethanol solution/(g insulin aspart) to clean the crystals, and collect the crystals after washing. Wash the crystals again with 10 ml ethanol solution/(g insulin aspart), and collect the crystals after washing. Finally, the crystals are transferred to a vacuum drying oven and dried at a temperature of 25° C. for 80 h, and the vacuum pressure is not greater than −0.08 MPa. After drying, the final insulin aspart API is obtained.