Fusion polypeptide and method for preparing target polypeptide through fusion polypeptide

Abstract

The present application provides a fusion polypeptide comprising a structure of formula I: Z2-SerX1HisXnX2Arg-Z1 (I) wherein Z2 and Z1 are independently selected from a polypeptide or an amino acid residue, X1 is any one amino acid residue, Xn is absent or Xn is a peptide having 1 to 5 amino acid residues, X2 is Lys or Arg, and wherein Ser is linked to the N-terminus of Z2 and Arg is linked to the C-terminus of Z1. The invention further provides a method for preparing the target polypeptide by fusing the polypeptide.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a fusion polypeptide and a method for preparing a target polypeptide using the fusion polypeptide. Background Art

[0002] At present, recombinant protein expression technology makes it possible to express a large number of proteins with medicinal value. Such proteins are usually expressed as fusion proteins in microbial host cells, such as Escherichia coli. The target protein desired to be obtained is connected to a chaperone protein or amino acids in the fusion protein, so as to improve the expression level, promote secretion, increase solubility or promote protein folding. In order to obtain the target protein from the fusion protein, it is usually necessary to remove the chaperone protein in the fusion protein by proteolysis, which means that the fusion protein must contain a processing site for the protease used for hydrolysis.

[0003] However, it is often difficult to achieve satisfactory results in terms of both substrate specificity and processing efficiency when only proteases are used for protein cleavage. For example, when using an E. coli expression system as a means of expressing recombinant proteins, although the target polypeptide can be obtained by enzyme cleavage, the yield of the polypeptide after enzyme cleavage is low, which seriously limits the industrialization of polypeptide drugs. Summary of the invention

[0004] In order to solve the technical problems existing in the prior art, the first aspect of the present application provides a fusion polypeptide, characterized in that the fusion polypeptide comprises a structure of formula I,

[0005] Z2-SerX1HisX n X2Arg-Z1(I),

[0006] Wherein, Z2 and Z1 are independently selected from polypeptides or amino acid residues, X1 is any amino acid residue, n Does not exist or X n is a peptide having 1 to 5 amino acid residues, X2 is Lys or Arg, and wherein Ser is linked to the N-terminus of Z2, and Arg is linked to the C-terminus of Z1.

[0007] The fusion polypeptide according to the present invention can obtain a target polypeptide, such as Z2 and / or Z1, under the dual action of nickel ions and proteases (such as Kex2), and the C-terminus and N-terminus of the target polypeptide do not contain additional residues, thereby not only significantly improving the quality and yield of the target polypeptide, but also greatly simplifying the subsequent purification steps. The fusion polypeptide according to the present invention can have a higher expression level without fusing other tags, reducing the waste of resources expressed by the fusion tag. In addition, the fusion polypeptide according to the present invention can regulate inclusion body expression and soluble expression. In a preferred embodiment, the target polypeptide is a pharmaceutical polypeptide, and thus, the target polypeptide obtained by the fusion polypeptide according to the present invention can significantly improve the safety of the product and reduce immunotoxicity.

[0008] According to some embodiments of the present application, X n is any amino acid residue. In some embodiments, X n is a peptide having any two amino acid residues, in some embodiments, X n is a peptide having any 3 amino acid residues, in some embodiments, X n is a peptide having any 4 amino acid residues, in some embodiments, X n is a peptide having any 5 amino acid residues.

[0009] According to some embodiments of the present application, the fusion polypeptide comprises the structure of Formula II,

[0010] Z2-SerX1HisX n X2Arg-Z1-SerX1HisX n X2Arg(II),

[0011] Wherein, Z2 and Z1 are independently selected from polypeptides or amino acid residues,

[0012] X1 is any amino acid residue,

[0013] X n Does not exist or X n is a peptide having 1 to 5 amino acid residues,

[0014] X2 is Lys or Arg,

[0015] And wherein Ser is connected to the N-terminus of Z1 or Z2, and Arg is connected to the C-terminus of Z1.

[0016] According to some embodiments of the present application, Formula I is selected from Z2-SerGluHisGluArgArg-Z1, Z2-SerAlaHisGlyGlyAlaArgArg-Z1 and Z2-SerLysHisHisGlyAlaLysArg-Z1.

[0017] According to some embodiments of the present application, Z2 is the same as Z1.

[0018] According to some embodiments of the present application, Z2 is different from Z1.

[0019] According to some embodiments of the present application, at least one of Z2 and Z1 is a target polypeptide, and in particular, both are target polypeptides.

[0020] According to some embodiments of the present application, Z2 is a target polypeptide, and Z1 is not a target polypeptide.

[0021] According to some embodiments of the present application, Z1 is a target polypeptide, and Z2 is not a target polypeptide.

[0022] According to some embodiments of the present application, when X n When not present, the structure of Formula I can also be described as Z2-SerX1HisX2Arg-Z1, and the structure of Formula II can also be described as Z2-SerX1HisX2Arg-Z1-SerX1HisX2Arg.

[0023] According to some embodiments of the present application, the length of the sequence of the target polypeptide is 10 to 100 amino acids.

[0024] According to some embodiments of the present application, the fusion polypeptide includes at least 3 target polypeptide sequences connected in series, for example, the fusion polypeptide includes 3 to 25 target polypeptide sequences connected in series, for example, 3, 5, 10, 15, 20, 25 or any interval thereof.

[0025] According to some embodiments of the present application, at least one of Z2 and Z1 comprises a tag sequence, and the tag sequence comprises at least one of a His tag and a TRX fusion tag.

[0026] According to some embodiments of the present application, at least one of Z2 and Z1 does not contain the structure of Formula I in its internal sequence, and in particular, neither Z2 nor Z1 contains the structure of Formula I in its internal sequence.

[0027] According to some embodiments of the present application, at least one of Z2 and Z1 does not contain SerX1His, in particular, neither Z2 nor Z1 contains SerX1His. According to some embodiments of the present application, at least one of Z2 and Z1 does not contain X2Arg, in particular, neither Z2 nor Z1 contains X2Arg.

[0028] According to a preferred embodiment of the present application, both Z2 and Z1 contain neither SerX1His nor X2Arg.

[0029] According to a preferred embodiment of the present application, at least one of Z2 and Z1 comprises a partial or complete sequence of at least one of semaglutide, liraglutide, linaclotide, and teriparatide.

[0030] According to some embodiments of the present application, the fusion polypeptide includes two or more structures of Formula I, for example, at least 3, at least 5, at least 10, at least 20 or at least 50.

[0031] According to some embodiments of the present application, the fusion polypeptide includes two or more structures of Formula II, for example, at least 3, at least 5, at least 10, at least 20 or at least 50.

[0032] In a second aspect of the present application, a method for preparing a target polypeptide by fusion polypeptide is provided, the method comprising the following steps:

[0033] (i) providing a fusion polypeptide comprising the structure of Formula I,

[0034] Z2-SerX1HisX n X2Arg-Z1(I)

[0035] Wherein, Z2 and Z1 are independently selected from polypeptides or amino acid residues,

[0036] X1 is any amino acid residue,

[0037] X n Does not exist or X n is a peptide having 1 to 5 amino acid residues,

[0038] X2 is Lys or Arg,

[0039] Among them, Ser is connected to the N-terminus of Z2, and Arg is connected to the C-terminus of Z1.

[0040] Furthermore, at least one of Z2 and Z1 is a target polypeptide;

[0041] (ii) subjecting the fusion polypeptide to nickel ion catalyzed cleavage to separate Z2;

[0042] (iii) subjecting the fusion polypeptide to cleavage by a protease, in particular Kex2, to separate Z1;

[0043] (iv) collecting the target polypeptide.

[0044] In the method of the present invention, the interior of the polypeptide sequences of Z2 and Z1 will not be cleaved, that is, the protease or nickel ions used in the method of the present invention cannot cleave the peptide bonds within the polypeptide sequences of Z2 and Z1.

[0045] According to some embodiments of the method of the present application, the method comprises the following steps:

[0046] (i) providing a fusion polypeptide comprising the structure of II,

[0047] Z2-SerX1HisX n X2Arg-Z1-SerX1HisX n X2Arg(II),

[0048] Wherein, Z2 and Z1 are independently selected from polypeptides or amino acid residues,

[0049] X1 is any amino acid residue,

[0050] X n Does not exist or X n is a peptide having 1 to 5 amino acid residues,

[0051] X2 is Lys or Arg,

[0052] wherein Ser is connected to the N-terminus of Z1 or Z2, and Arg is connected to the C-terminus of Z1,

[0053] Furthermore, at least one of Z2 and Z1 is a target polypeptide;

[0054] (ii) subjecting the fusion polypeptide to nickel ion catalytic cleavage to disconnect Ser from the N-terminus of Z1 or Z2;

[0055] (iii) subjecting the fusion polypeptide to cleavage by a protease, particularly Kex2, to separate Arg from the C-terminus of Z1;

[0056] (iv) collecting the target polypeptide.

[0057] In the third aspect of the present application, a DNA molecule is provided, which encodes the fusion polypeptide according to the present application. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 The electrophoresis SDS-PAGE diagram of the fermentation expression of the fusion polypeptide concatemer of Example 1 is schematically shown.

[0059] Figure 2 The HPLC detection results of the fusion polypeptide concatemer of Example 1 after dissolution are schematically shown.

[0060] Figure 3 The HPLC detection results of the ZL02 monomer after the fusion polypeptide concatemer of Example 1 was dissolved and cleaved by nickel ions are schematically shown.

[0061] Figure 4The HPLC detection results of the ZL03 monomer obtained by cleaving the ZL02 monomer in Example 1 by KEX2 enzyme are schematically shown.

[0062] Figure 5 The mass spectrometry molecular weight spectrum of the ZL02 monomer of Example 1 is schematically shown.

[0063] Figure 6 The mass spectrometry molecular weight spectrum of the ZL03 monomer of Example 1 is schematically shown.

[0064] Figure 7 The HPLC test results of semaglutide (ZL06) prepared in Example 1 are schematically shown.

[0065] Figure 8 The mass spectrometry molecular weight spectrum of semaglutide (ZL06) prepared in Example 1 is schematically shown.

[0066] Fig. 9 The electrophoresis SDS-PAGE diagram of the fermentation expression of the fusion polypeptide concatemer of Example 2 is schematically shown.

[0067] Fig.10 The HPLC detection results of the fusion polypeptide concatemer of Example 2 after dissolution are schematically shown.

[0068] Fig.11 The HPLC detection results of the KL02 monomer after the fusion polypeptide concatemer of Example 2 was dissolved and cleaved by nickel ions are schematically shown.

[0069] Fig.12 The HPLC detection results of the KL03 monomer obtained by cleaving the KL02 monomer of Example 2 by KEX2 enzyme are schematically shown.

[0070] Fig.13 The mass spectrometry molecular weight spectrum of the KL02 monomer of Example 2 is schematically shown.

[0071] Fig.14 The mass spectrometry molecular weight spectrum of the KL03 monomer of Example 2 is schematically shown.

[0072] Fig.15 The HPLC test results of semaglutide (KL06) prepared in Example 2 are schematically shown.

[0073] Fig.16 The mass spectrometry molecular weight spectrum of semaglutide (KL06) prepared in Example 2 is schematically shown.

[0074] Fig.17 The electrophoresis SDS-PAGE diagram of the fermentation expression of the fusion polypeptide concatemer of Example 3 is schematically shown.

[0075] Fig.18 The HPLC detection results after dissolution and purification of the fusion polypeptide concatemer of Example 3 are schematically shown.

[0076] Fig.19 The HPLC test results of the ZL02 monomer after the fusion polypeptide concatemer of Example 3 was dissolved, coupled, and then purified by nickel ion cutting are schematically shown.

[0077] Fig. 20 The HPLC detection results of the AM03 monomer obtained by cleavage and purification of the AM02 monomer in Example 3 by KEX2 enzyme are schematically shown.

[0078] Fig.21 The mass spectrometry molecular weight spectrum of the AM02 monomer of Example 3 is schematically shown.

[0079] Fig. 22 The mass spectrometry molecular weight spectrum of the AM03 monomer of Example 3 is schematically shown. DETAILED DESCRIPTION

[0080] In order to make the purpose, technical scheme and advantages of the present application clearer, the technical scheme of the present application will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. The relevant embodiments described herein are illustrative and are used to provide a basic understanding of the present application. The embodiments of the present application should not be interpreted as limiting the present application.

[0081] For simplicity, only some numerical ranges are specifically disclosed herein. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, and any upper limit can be combined with any other upper limit to form an unspecified range. In addition, each separately disclosed point or single value can itself be combined as a lower limit or upper limit with any other point or single value or with other lower limits or upper limits to form an unspecified range.

[0082] In the description herein, unless otherwise specified, “above” and “below” include the number.

[0083] Unless otherwise specified, the terms used in this application have the commonly known meanings generally understood by those skilled in the art. Unless otherwise specified, the numerical values ​​of the various parameters mentioned in this application can be measured using various measurement methods commonly used in the art (for example, they can be tested according to the methods given in the examples of this application).

[0084] A list of items connected by the terms "at least one of," "at least one of," "at least one of," or other similar terms may mean any combination of the listed items. For example, if items A and B are listed, the phrase "at least one of A and B" means only A; only B; or A and B. In another example, if items A, B, and C are listed, the phrase "at least one of A, B, and C" means only A; or only B; only C; A and B (excluding C); A and C (excluding B); B and C (excluding A); or all of A, B, and C. Item A may contain a single component or multiple components. Item B may contain a single component or multiple components. Item C may contain a single component or multiple components.

[0085] In the context of the present invention, the terms "protein", "polypeptide" and "peptide" can be used interchangeably to refer to polypeptides. It should be understood that the specific terms used do not limit the size of the molecule (unless directly specified in a specific case).

[0086] In the context of the present invention, the term "fusion polypeptide" is intended to mean a polypeptide comprising two or more polypeptides fused together, for example, to form a non-naturally occurring polypeptide. The size of the fused polypeptide can vary and depends on the purpose of the fusion polypeptide. In order to improve expression, fusion polypeptides are often used in the recombinant expression process of proteins to promote the maintenance of soluble expression products, to promote the discharge of fusion polypeptides or parts thereof to the extracellular medium, to protect polypeptides from being unintentionally processed by proteases or peptidases, etc. In such a fusion polypeptide, one of at least two constituent polypeptides is usually designated as a "target polypeptide", that is, a polypeptide to be prepared by a recombinant expression process.

[0087] In the context of the present invention, "additional residues" refer to other amino acid residues besides those in the target protein sequence.

[0088] In order to achieve the above-mentioned purpose and enable technicians in this technical field to understand the scheme of the present invention, the specific implementation scheme adopted by the present invention is exemplified as follows. It should be stated that the described embodiments are only partial embodiments of the present invention, rather than all embodiments.

[0089] Example 1

[0090] 1. Construction of pET-29a-1 recombinant plasmid and engineered bacteria

[0091] The artificially synthesized nucleotide sequence SEQ ID NO: 1 was obtained from a gene company, and the amino acid sequence expressed by the sequence is shown in SEQ ID NO: 2.

[0092] (SEQ ID NO: 1).

[0093] GSEHERREGTFTSDVSSYLEGQAAKEFIAWLVRGRGSEHERREGTFTSDVSSYLEGQAAKEFIAWLVRGRGSEHERREGTFTSDVSSYLEGQAAKEFIAWLVRGRGSEHERREGTFTSDVSSYLEGQAAKEFIAWLVRGRGSEHERREGTFTSDVSSYLEGQAAKEFIAWLVRGRG (SEQ ID NO: 2).

[0094] The nucleotide sequence SEQ ID NO: 1 was connected to the pET29a vector by double restriction enzymes xho1 and nde1 to obtain the pET-29a-1 recombinant plasmid. After DNA sequencing confirmed that the DNA sequence in the recombinant plasmid was correct, the recombinant plasmid was introduced into the competent cells of the Escherichia coli expression host bacteria BL21 (DE3) by the heat shock method, and the agar plate was incubated and cultured with LB medium and cultured at 37°C overnight. A single bacterial cell was picked from the kanamycin sulfate LB plate, and IPTG was induced after shaking culture at 37°C and 220rpm for 6 hours. After electrophoresis detection, the strains that can express normally were screened as engineering bacteria.

[0095] 2. Fermentation culture

[0096] Transfer the engineered bacteria to a 500ml Erlenmeyer flask containing 100ml LB culture medium at a ratio of 1% and culture at 37°C overnight to form the seed solution for the upper tank.

[0097] Inoculate 3% of the seed liquid from the upper tank into a 5L fermenter containing YT culture medium, and culture at 37°C. Maintain the dissolved oxygen above 25% by adjusting the rotation speed, air volume, and pure oxygen volume. Use ammonia water to adjust the pH and maintain it at 7.0. When the OD600 of the bacterial solution reaches 30-40, add isopropyl-β-D-thiogalactoside at a final concentration of 1.0mM. Continue to culture for 8 hours, then stop the fermentation, collect the bacterial solution, centrifuge at 10,000rpm for 10 minutes, discard the supernatant, collect the bacteria, and store in a -20°C refrigerator for later use. SDS-PAGE of the bacterial solution after the recombinant engineered bacteria induced expression is as follows Figure 1 As shown, the size of the obtained fusion polypeptides was between 17 kDa and 25 kDa.

[0098] 3. Acquisition of target peptides

[0099] 3.1 The fermented cells of the recombinant engineering bacteria were resuspended in a crushing buffer, homogenized twice under high pressure (pressure 600 bar to 800 bar), centrifuged at room temperature with stirring, and the precipitate was collected;

[0100] 3.2 Resuspend the precipitate with washing solution according to the mass volume ratio; stir at room temperature for 30 minutes, and collect the precipitate by centrifugation (pretreatment of fusion polypeptide);

[0101] 3.3 The precipitate was dissolved in urea according to the mass volume ratio, and the HPLC test was performed as follows: Figure 2 The protein content was calculated according to the peak area, the pH was adjusted to 9.5-10.5, 0.1M nickel chloride solution was added at 5 ml / g, stirred at 37°C to 42°C, reacted for 3-5 hours, and HPLC detection was performed as shown. Figure 3 As shown, the content was determined and the supernatant was collected by centrifugation (cutting 1: cutting by nickel ions);

[0102] 3.4 Routine cleaning of the cationic chromatography column, equilibration with equilibration solution for 2CV, chemical cutting sample adjusted to pH 2.5-3.2, filtration and mounting on the chromatography column (conductivity less than 5ms / cm), re-equilibration with equilibration solution for 2CV, elution with eluent in one step, and collection of the target peak;

[0103] 3.5 Add KEX2 enzyme to the eluent at a certain mass-volume ratio, stir at 30℃ for 1-2 hours, and detect by HPLC. Figure 4 As shown, the content was determined and the supernatant was collected by centrifugation (cutting two: cutting by KEX2 enzyme);

[0104] 3.6C8 reverse column loading, equilibration, gradient elution to collect target polypeptide, the purity of the obtained target polypeptide is not less than 95%.

[0105] Characterization of the products obtained by cleavage

[0106] The mass spectrometry molecular weight spectrum of the obtained product is as follows Figure 5 The theoretical molecular weight of the product ZL02 obtained after the cleavage is completed is 3970. Figure 5 It can be seen that the mass spectrometry molecular weight detection of ZL02 is a multi-charged molecular weight: under the M+H condition, the molecular weight detected with 4 charges is 993.4, the molecular weight detected with 5 charges is 794.9, the molecular weight detected with 6 charges is 662.6, and the molecular weight detected with 7 charges is 568.1, which is consistent with the theoretical molecular weight of 3969. The molecular weights of Zl02 were tested by mass spectrometer: 3973.6, 3974.5, 3975.6, 3976.7.

[0107] (Due to isotope differences) Characterization confirmed that ZL02 was the desired product.

[0108] The amino acid sequence of ZL02 is SEHERREGTFTSDVSSYLEGQAAKEFIAWLVRGRG.

[0109] Characterization of the products obtained by cleavage II

[0110] The mass spectrometry molecular weight spectrum of the obtained product is as follows Figure 6 After the second cleavage is completed, ZL03 is obtained theoretically. The theoretical molecular weight of ZL03 is: 3175. Figure 6 It can be seen that the mass spectrometry molecular weight detection of ZL02 is a multi-charged molecular weight: under the M+H condition, the molecular weight detected with 4 charges is 794.7, and the molecular weight detected with 3 charges is 1059.3, which is consistent with the theoretical molecular weight of ZL03, which is 3175. Through characterization, it can be determined that ZL03 is the desired product. The amino acid sequence of ZL03 is EGTFTSDVSSYLEGQAAKEFIAWLVRGRG.

[0111] Preparation of semaglutide by target peptide

[0112] An equivalent of semaglutide side chain (Ste-Glu-AEEA-AEEA-OSU CAS: 1169630-40-3, purchased from Chengdu Pukang) was weighed at a ratio of 1:1.1, dissolved and added dropwise to the alkaline aqueous solution of the target polypeptide, the pH was adjusted to precipitate the product, the precipitate was centrifuged and washed with water and then redissolved; 4 equivalents of linker (Fmoc-His-Aib-OH CAS: 1446013-08-6, purchased from Chengdu Pukang) were weighed, dissolved and activated and added dropwise to the redissolved aqueous solution, after the reaction was completed, 20% piperidine was added to react for 20 minutes, and ethyl acetate was added for extraction to obtain an aqueous solution of semaglutide. Reverse preparation and purification was performed until the purity of semaglutide reached 99%.

[0113] The HPLC detection spectrum of semaglutide obtained in Example 1 is as follows Figure 7 shown.

[0114] The mass spectrometry molecular weight spectrum of semaglutide obtained in Example 1 is as follows Figure 8 The theoretical molecular weight of semaglutide is 4113.6. Figure 8 It can be seen that the mass spectrometry molecular weight detection of ZL06 is a multi-charged molecular weight: under the M+H condition, the molecular weight detected with three charges is 1372.05, which is consistent with the theoretical molecular weight of 4113. The sequence information of ZL06 is EGTFTSDVSSYLEGQAAK(-AEEA-AEEA-Glu-Ste)EFIAWLVRGRG, and its structure is shown below:

[0115]

[0116] It can be confirmed through characterization that semaglutide was successfully prepared using the target polypeptide obtained in Example 1.

[0117] Example 2

[0118] 1. Construction of pET-29a-1 recombinant plasmid and engineered bacteria

[0119] The artificially synthesized nucleotide sequence SEQ ID NO: 3 was obtained from a gene company, and the amino acid sequence expressed by the sequence is shown in SEQ ID NO: 4.

[0120] GGCAGCGAACATGAAAAACGCGAAGGCACCTTTACCAGCGATGTGAGCAGCTATCTGGAAGGCCAGGCGGCGAAAGAATTTATTGCGTGGCTGGTGCGCGGCCGCGGCAGCGAACATGAAAAACGCGAAGGCACCTTTACCAGCGATGTGAGCAGCTATCTGGAAGGCCAGGCGGCGAAAGAATTTATTGCGTGGCTGGTGCGCGGCCGCGGCAGCGAACATGAAAAACGCGAAGGCACCTTTACCAGCGATGTGAGCAGCTATCTGGAAGGCCAGGCGGCGAAAGAATTTATTGCGTGGCTGGTGCGCGGCCGCGGCAGCGAACATGAAAAACGCGAAGGCACCTTTACCAGCGATGTGAGCAGCTATCTGGAAGGCCAGGCGGCGAAAGAATTTATTGCGTGGCTGGTGCGCGGCCGCGGCAGCGAACATGAAAAACGCGAAGGCACCTTTACCAGCGATGTGAGCAGCTATCTGGAAGGCCAGGCGGCGAAAGAATTTATTGCGTGGCTGGTGCGCGGCCGCGGC

[0121] (SEQ ID NO:3)。

[0122] GSEHEKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGSEHEKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGSEHEKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGSEHEKREGTFTSDVSSYLEGQAAKEFIAWLVRGRGSEHEKREGTFTSDVSSYLEGQAAKEFIAWLVRGRG(SEQ ID NO:4)。

[0123] The nucleotide sequence SEQ ID NO:3 was connected to the pET29a vector by double digestion with xho1 and nde1 to obtain the pET-29a-2 recombinant plasmid. After DNA sequencing confirmed that the DNA sequence in the recombinant plasmid was correct, the recombinant plasmid was introduced into the competent cells of the Escherichia coli expression host bacteria BL21 (DE3) by the heat shock method, and the agar plate was incubated and cultured with LB medium and cultured at 37°C overnight. A single bacterial cell was picked from the kanamycin sulfate LB plate, and IPTG was induced after shaking culture at 37°C and 220rpm for 6 hours. The strain that can express normally was screened as the engineering bacteria.

[0124] 2. Fermentation culture

[0125] Transfer the engineered bacteria to a 500ml Erlenmeyer flask containing 100ml LB culture medium at a ratio of 1% and culture at 37°C overnight to form the seed solution for the upper tank.

[0126] Inoculate 3% of the seed liquid from the upper tank into a 5L fermenter containing YT culture medium, and culture at 37°C. Maintain the dissolved oxygen above 25% by adjusting the rotation speed, air flow, and pure oxygen flow. Use ammonia water to adjust the pH and maintain it at 7.0. When the OD600 of the bacterial solution reaches about 40, add isopropyl-β-D-thiogalactoside at a final concentration of 1.0 mM. Continue to culture for 12 hours, then stop the fermentation, collect the bacterial solution, centrifuge at 10,000 rpm for 5 minutes, discard the supernatant, collect the bacteria, and store in a -20°C refrigerator for later use. SDS-PAGE of the bacterial solution after the recombinant engineered bacteria induced expression is shown in Figure 2. Fig. 9 As shown, the size of the obtained fusion polypeptides was between 17 kDa and 25 kDa.

[0127] 3. Acquisition of target peptides

[0128] 3.1 The fermented cells of the recombinant engineering bacteria were resuspended in a crushing buffer, homogenized twice under high pressure (pressure 600 bar to 800 bar), centrifuged at room temperature with stirring, and the precipitate was collected;

[0129] 3.2 Resuspend the precipitate with washing solution according to the mass volume ratio; stir at room temperature for 30 minutes, and collect the precipitate by centrifugation (pretreatment of fusion polypeptide);

[0130] 3.3 The precipitate was dissolved in 6M urea and the HPLC test was performed as follows: Fig.10 The protein content was calculated according to the peak area, the pH was adjusted to 9.5-10.5, 0.1 M nickel chloride solution was added at 5 ml / g, and the mixture was stirred at 37°C to 42°C until the HPLC detection was as shown. Fig.11 As shown, the content was determined and the supernatant was collected by centrifugation (cutting 1: cutting by nickel ions);

[0131] 3.4 Routine cleaning of the cationic chromatography column, equilibration with equilibration solution for 2CV, chemical cutting sample adjusted to pH 2.8-3.0, re-equilibration with equilibration solution for 2CV, elution with eluent in one step, and collection of target peaks;

[0132] 3.5 Add KEX2 enzyme to the eluent at a certain mass-volume ratio, stir at 37℃ for 1-2 hours, and detect by HPLC. Fig.12 As shown, the content was determined and the supernatant was collected by centrifugation (cutting two: cutting by KEX2 enzyme);

[0133] 3.6 Reverse column loading, equilibration, and gradient elution to collect the target polypeptide, and the purity of the obtained target polypeptide is not less than 95%.

[0134] Characterization of the products obtained by cleavage

[0135] The mass spectrometry molecular weight spectrum of the obtained product is as follows Fig.13 The theoretical molecular weight of the product KL02 obtained after the cleavage is completed is 3942. Fig.13 It can be seen that the mass spectrometry molecular weight detection of KL02 is a multi-charged molecular weight: under the M+H condition, the molecular weight detected with 4 charges is 986.3, the molecular weight detected with 5 charges is 789.3, the molecular weight detected with 6 charges is 658.0, and the molecular weight detected with 7 charges is 564.1, which is consistent with the theoretical molecular weight. The molecular weights of Kl02 were tested by mass spectrometer: 3941.2, 3941.5, 3942, 3941.7.

[0136] (Due to isotopic differences) Characterization confirmed that KL02 was the desired product.

[0137] The amino acid sequence of KL02 is SEHEKREGTFTSDVSSYLEGQAAKEFIAWLVRGRG.

[0138] Characterization of the products obtained by cleavage II

[0139] The mass spectrometry molecular weight spectrum of the obtained product is as follows Fig.14 After the second cleavage is completed, KL03 is obtained theoretically. The theoretical molecular weight of KL03 is: 3175. Fig.14 It can be seen that the mass spectrometry molecular weight detection of KL03 is a multi-charged molecular weight: under the M+H condition, the molecular weight detected with 4 charges is 794.7, the molecular weight detected with 3 charges is 1059.2, and the molecular weight detected with 2 charges is 1588.3, ​​which is consistent with the theoretical molecular weight of KL03 of 3175. Kl03 was tested by mass spectrometer and the molecular weights were: 3174.8, 3174, 3174.6. (Isotope differences) It can be determined through characterization that KL03 is the desired product.

[0140] The amino acid sequence of KL03 is EGTFTSDVSSYLEGQAAKEFIAWLVRGRG.

[0141] 4. Preparation of semaglutide via target peptide

[0142] An equivalent amount of semaglutide side chain (Ste-Glu-AEEA-AEEA-OSU CAS: 1169630-40-3) was weighed at a ratio of 1:1.1, dissolved and then added dropwise to the alkaline aqueous solution of the target polypeptide, the pH was adjusted to precipitate the product, the precipitate was centrifuged and washed with water and then redissolved; a certain equivalent amount of linker (Fmoc-His-Aib-OH CAS: 1446013-08-6) was weighed, dissolved and activated and then added dropwise to the redissolved aqueous solution, after the reaction was completed, 20% piperidine was added to react for 20 minutes, and ethyl acetate was added for extraction to obtain an aqueous solution of semaglutide. Reverse preparation and purification was performed until the purity of semaglutide reached 99%.

[0143] The HPLC detection spectrum of semaglutide obtained in Example 2 is as follows Fig.15 shown.

[0144] The mass spectrometry molecular weight spectrum of semaglutide obtained in Example 2 is as follows Fig.16 The theoretical molecular weight of semaglutide is 4113.6. Fig.16 It can be seen that the mass spectrometry molecular weight detection of KL06 is a multi-charged molecular weight: under the M+H condition, the molecular weight detected with 5 charges is 823.6, the molecular weight detected with 4 charges is 1029.2, and the molecular weight detected with 3 charges is 1372, which is consistent with the theoretical molecular weight of 4113. KL06 was tested by mass spectrometer and the molecular weights were: 4113, 4112.8, 4113. The sequence information of KL06 is EGTFTSDVSSYLEGQAAK(-AEEA-AEEA-Glu-Ste)EFIAWLVRGRG, and its structural schematic diagram is shown below:

[0145]

[0146] It can be confirmed through characterization that semaglutide was successfully prepared using the target polypeptide obtained in Example 2.

[0147] Example 3

[0148] The artificially synthesized nucleotide sequence SEQ ID NO: 5 was obtained from a gene company, and the amino acid sequence expressed by the sequence is shown in SEQ ID NO: 6.

[0149]

[0150] GSEHERREGTFTSDVSSYLEEQAAREFIAWLVRGRKGGGGEASELSTAALGRLSAELHEL

[0151] ATLPRTETGSGSPGSEHERREGTFTSDVSSYLEEQAAREFIAWLVRGRKGGGGEASELST

[0152] AALGRLSAELHELATLPRTETGSGSPGSEHERREGTFTSDVSSYLEEQAAREFIAWLVRG

[0153] RKGGGGEASELSTAALGRLSAELHELATLPRTETGSGSPGSEHERREGTFTSDVSSYLEE

[0154] QAAREFIAWLVRGRKGGGGEASELSTAALGRLSAELHELATLPRTETGSGSPGSEHERRE

[0155] GTFTSDVSSYLEEQAAREFIAWLVRGRKGGGGEASELSTAALGRLSAELHELATLPRTETGSGSPG (SEQ ID NO: 6).

[0156] 1. Construction of recombinant plasmids and engineered bacteria

[0157] The nucleotide sequence SEQ ID NO:5 was connected to the pET29a vector by double digestion with xho1 and nde1 to obtain the pET-29a-3 recombinant plasmid. After DNA sequencing confirmed that the DNA sequence in the recombinant plasmid was correct, the recombinant plasmid was introduced into the competent cells of the Escherichia coli expression host bacteria BL21 (DE3) by heat shock method, LB medium was incubated and cultured on agar plates, cultured at 37°C overnight, single bacteria were picked from the kanamycin sulfate LB plate, shaken and cultured at 37°C, 220rpm for 12 hours, and electrophoresis was performed to screen the strains that can express normally as engineering bacteria.

[0158] 2. Fermentation culture

[0159] Transfer the engineered bacteria to a 500ml Erlenmeyer flask containing 100ml LB culture medium at a ratio of 1% and culture at 37°C overnight to form the seed solution for the upper tank.

[0160] Inoculate 3% of the seed liquid from the upper tank into a 5L fermenter containing YT culture medium, and culture at 37°C. Maintain the dissolved oxygen above 25% by adjusting the rotation speed, air volume, and pure oxygen volume. Use ammonia water to adjust the pH and maintain it at 7.0. When the OD600 of the bacterial solution reaches 30-40, add isopropyl-β-D-thiogalactoside at a final concentration of 1.0mM. Continue to culture for 8 hours, then stop the fermentation, collect the bacterial solution, centrifuge at 10,000rpm for 10 minutes, discard the supernatant, collect the bacteria, and store in a -20°C refrigerator for later use. SDS-PAGE of the bacterial solution after the recombinant engineered bacteria induced expression is as follows Fig.17 As shown, the size of the obtained fusion polypeptide protein is about 41 kDa.

[0161] 3. Acquisition of target peptides

[0162] 3.1 The fermented cells of the recombinant engineering bacteria were resuspended in a crushing buffer, homogenized twice under high pressure, centrifuged at room temperature, and the precipitate was collected;

[0163] 3.2 Resuspend the precipitate with washing solution according to the mass volume ratio; stir at room temperature for 30 minutes, and collect the precipitate by centrifugation (pretreatment of fusion polypeptide);

[0164] 3.3 The precipitate was dissolved with 6M urea, and the target peptide was collected by reverse column loading, equilibrium, and gradient elution. The target peptide was detected by HPLC as follows: Fig.18 shown.

[0165] 3.4 Dissolve the C18 side chain (Ste-Glu-AEEA-AEEA-OSU) at a ratio of 1:0.5 and add it dropwise to the alkaline aqueous solution of the target peptide.

[0166] 3.5 Adjust the pH to 10.0-10.4, add 0.1M nickel chloride solution at 5 ml / g, stir at 25°C for 24 hours, load on the reverse column, balance, and gradient elute to collect the target polypeptide. The purity of the obtained target polypeptide is not less than 90%. HPLC detection is as follows: Fig.19 As shown, the supernatant was collected (cut 1: cut by nickel ions);

[0167] 3.6 After the eluate is treated, KEX2 enzyme is added according to the volume ratio, and the reaction is stirred at 30°C for 1-2 hours. The target polypeptide is collected by reverse column loading, equilibrium, and gradient elution. The purity of the obtained target polypeptide is not less than 90%. HPLC detection is as follows: Fig. 20 As shown, (cutting two: cutting by KEX2 enzyme);

[0168] Characterization of the products obtained by cleavage

[0169] The mass spectrometry molecular weight spectrum of the obtained product is as follows Fig.21 The theoretical molecular weight of the product AM02 obtained after the cleavage is completed is 8476. Fig.21 It can be seen that the AM02 mass spectrometry molecular weight detection is a multi-charged molecular weight: under the M+H conditions, the molecular weight detected with 7 charges is 1211.9, the molecular weight detected with 8 charges is 1060.5, the molecular weight detected with 9 charges is 942.7, and the molecular weight detected with 10 charges is 848.6, which is consistent with the theoretical molecular weight of 8476.

[0170] AM02 was tested by mass spectrometer and the molecular weights were: 8476.3, 8476, 8475.3, 8476.

[0171] (Isotope differences lead to) Through characterization, it can be determined that AM02 is the desired product. The amino acid sequence of AM02 is SEHERREGTFTSDVSSYLEEQAAREFIAWLVRGRK(Ste-Glu-AEEA-AEEA-OSU)GGGGEASELSTAALGRLSAELHELATLPRTETGSGSPG

[0172] Characterization of the products obtained by cleavage II

[0173] The mass spectrometry molecular weight spectrum of the obtained product is as follows Fig. 22 After the second cleavage is completed, AM03 is obtained theoretically. The theoretical molecular weight of AM03 is: 7681. Fig. 22 It can be seen that the mass spectrometry molecular weight detection of AM03 is a multi-charged molecular weight: under the M+H condition, the detection molecular weight with 7 charges is 1098.3, and the detection molecular weight with 6 charges is 1281.3, which is consistent with the theoretical molecular weight of AM03 of 7681. AM03 was tested by mass spectrometer and the molecular weights were: 7681.1, 7681.8. (Isotope differences lead to) It can be determined by characterization that AM03 is the desired product. The amino acid sequence of AM03 is EGTFTSDVSSYLEEQAAREFIAWLVRGRK(Ste-Glu-AEEA-AEEA-OSU)GGGGEASELSTAALGRLSAELHELATLPRTETGSGSPG.

Claims

1. A fusion polypeptide, characterized in that: The fusion polypeptide comprises the structure of Formula I, Z2-SerX1HisXnX2Arg-Z1(I), Wherein, Z2 and Z1 are independently selected from polypeptides or amino acid residues, X1 is any amino acid residue, X n Does not exist or X n is a peptide having 1 to 5 amino acid residues, X2 is Lys or Arg, And wherein, Ser is connected to the N-terminus of Z2, and Arg is connected to the C-terminus of Z1.

2. The fusion polypeptide according to claim 1, characterized in that Z2 is the same as Z1.

3. The fusion polypeptide according to claim 1, characterized in that The fusion polypeptide comprises the structure of Formula II, Z2-SerX1HisXnX2Arg-Z1-SerX1HisXnX2Arg(II), Wherein, Z2 and Z1 are independently selected from polypeptides or amino acid residues, X1 is any amino acid residue, X n Does not exist or X n is a peptide having 1 to 5 amino acid residues, X2 is Lys or Arg, And wherein Ser is connected to the N-terminus of Z1 or Z2, and Arg is connected to the C-terminus of Z1.

4. The fusion polypeptide according to claim 1, characterized in that Z2 is the same as Z1.

5. The fusion polypeptide according to claim 1, characterized in that Formula I is selected from the group consisting of Z2-SerGluHisGluArgArg-Z1, Z2-SerAlaHisGlyGlyAlaArgArg-Z1 and Z2-SerLysHisHisGlyAlaLysArg-Z1.

6. The fusion polypeptide according to claim 1, characterized in that At least one of Z2 and Z1 comprises a tag sequence, wherein the tag sequence comprises at least one of a His tag and a TRX fusion tag.

7. The fusion polypeptide according to claim 1, characterized in that At least one of Z2 and Z1 comprises 10 to 100 amino acid residues.

8. The fusion polypeptide according to claim 1, characterized in that At least one of Z2 and Z1 does not contain SerX1His, and / or at least one of Z2 and Z1 does not contain X2Arg.

9. The fusion polypeptide according to claim 1, characterized in that: At least one of Z2 and Z1 comprises a partial or complete sequence of at least one of semaglutide, liraglutide, linaclotide, and teriparatide.

10. A method for preparing a target polypeptide by fusion polypeptide, characterized in that: The method comprises the following steps: (i) providing a fusion polypeptide comprising the structure of Formula I, Z2-SerX1HisXnX2Arg-Z1(I) Wherein, Z2 and Z1 are independently selected from polypeptides or amino acid residues, X1 is any amino acid residue, X n Does not exist or X n is a peptide having 1 to 5 amino acid residues, X2 is Lys or Arg, Among them, Ser is connected to the N-terminus of Z2, and Arg is connected to the C-terminus of Z1. Furthermore, at least one of Z2 and Z1 is a target polypeptide; (ii) subjecting the fusion polypeptide to nickel ion catalyzed cleavage to separate Z2; (iii) subjecting the fusion polypeptide to cleavage by a protease, in particular Kex2, to separate Z1; (iv) collecting the target polypeptide.

11. A method for preparing a target polypeptide by fusion polypeptide, characterized in that: The method comprises the following steps: (i) providing a fusion polypeptide comprising the structure of II, Z2-SerX1HisXnX2Arg-Z1-SerX1HisXnX2Arg(II), Wherein, Z2 and Z1 are independently selected from polypeptides or amino acid residues, X1 is any amino acid residue, X n Does not exist or X n is a peptide having 1 to 5 amino acid residues, X2 is Lys or Arg, wherein Ser is connected to the N-terminus of Z1 or Z2, and Arg is connected to the C-terminus of Z1, Furthermore, at least one of Z2 and Z1 is a target polypeptide; (ii) subjecting the fusion polypeptide to nickel ion catalytic cleavage to disconnect Ser from the N-terminus of Z1 or Z2; (iii) subjecting the fusion polypeptide to cleavage by a protease, particularly Kex2, to separate Arg from the C-terminus of Z1; (iv) collecting the target polypeptide.

12. A DNA molecule, characterized in that The DNA molecule encodes the fusion polypeptide according to claim 1.

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