Methods for production and purification of polypeptides

Abstract

The present invention provides a fusion polypeptide comprising a polypeptide of interest and a self-aggregating polypeptide, and methods for producing and purifying a polypeptide of interest by expressing the fusion polypeptide.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering. More specifically, this invention relates to fusion polypeptides comprising a target polypeptide moiety and a self-aggregating peptide moiety, and methods for producing and purifying the target polypeptide by expressing said fusion polypeptide. Background Technology

[0002] Currently, research and development on the application of peptides in medicine has broadly covered various aspects, including anti-tumor drugs, cardiovascular drugs, vaccines and antiviral drugs, as well as diagnostic kits (Leader et al., 2008). Compared with the rapidly growing market demand, the production methods of peptides have limited their development to some extent. Conventional chemical solid-phase synthesis methods, when producing medium- to long peptides with more than 30 amino acids, experience a significant increase in cost and difficulty as the peptide length increases (Bray et al., 2003).

[0003] Another effective method is to generate peptides within host cells using recombinant methods. In recombinant peptide production, the purification step is crucial. It has been reported that the separation and purification cost of recombinant peptides accounts for approximately 60%-80% of their total production cost (Chen et al., 2002). Purification methods for recombinant peptides include traditional ion exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. Ion exchange chromatography and hydrophobic interaction chromatography have certain requirements for sample starting conditions, making them less versatile and efficient than affinity chromatography. Affinity purification typically achieves yields exceeding 90%, making it the most commonly used method for recombinant protein purification. Commonly used affinity purification techniques include the fusion expression of histidine tags (his-tag) or glutathione transferase tags (GST-tag) with the target peptide, providing a universal purification method for the production of different target peptides. However, expensive purification columns make affinity purification costly, hindering its industrial application.

[0004] Human growth hormone (hGH) is a protein hormone secreted by the anterior pituitary gland. Its mature form is a non-glycosylated hydrophilic globulin with the signal peptide removed, composed of 191 amino acids and two disulfide bonds, with a relative molecular weight of approximately 22 kDa. hGH can reach various organs and tissues throughout the body via the bloodstream, and its receptors are distributed across various cells, allowing it to act on almost all tissues and cells. hGH plays many important functions in the human body, such as maintaining positive nitrogen balance, initiating protein synthesis in muscle cells, increasing amino acid uptake in skeletal muscle, regulating longitudinal bone growth, and protecting cardiomyocytes and lymphocytes from apoptosis (Levarski et al., 2014; Zamani et al., 2015). Therefore, hGH has been widely used to treat various diseases, and there are 11 FDA-approved indications for growth hormone. In my country, the approved indications for growth hormone mainly include six types: childhood growth hormone deficiency, growth hormone deficiency caused by burn symptoms and hypothalamic-pituitary disorders, Turner syndrome, adult growth hormone deficiency, and chronic renal insufficiency. Currently, the global sales of growth hormone exceed US$3 billion.

[0005] There are two main sources of human growth hormone (hGH) used clinically: direct extraction and traditional genetic engineering. Direct extraction requires extraction from the pituitary gland, resulting in low yields, high costs, and an inability to meet large-scale medical needs. Furthermore, it has been banned due to significant safety risks. Traditional genetic engineering, because hGH is glycosylated, utilizes prokaryotic expression systems, primarily recombinant *E. coli*. However, when directly expressed intracellularly in *E. coli*, hGH exists as inactive inclusion bodies, requiring subsequent renaturation to obtain biologically active hGH. Currently, fusion tags are mainly used for lysis promotion (e.g., glutathione fragments, TNFα, etc.) (Levarski et al., 2014; Nguyen et al., 2014) or periplasmic spatial expression (MBP tag) (Wang et al., 2018). These techniques require complex purification steps and various column chromatography techniques, such as affinity chromatography and gel size exclusion chromatography, resulting in low yields and high costs, leading to high prices for hGH products.

[0006] Human interferon-α2a belongs to type I interferon and is a multifunctional and highly active inducible protein produced by leukocytes and lymphocytes. It consists of 165 amino acids, contains two pairs of intramolecular disulfide bonds, and has a relative molecular weight of approximately 19.2 kDa. Recombinant human interferon-α2a has broad-spectrum antiviral activity. Its antiviral mechanism mainly involves the binding of interferon to interferon receptors on the surface of target cells, inducing various antiviral proteins in target cells, such as 2-5(A) synthase, protein kinase PKR, and MX protein, thereby preventing viral protein synthesis and inhibiting viral nucleic acid replication and transcription (Sen GC et al., 1992; Markus H. Heim et al., 1999). Interferon also has multiple immunomodulatory effects, such as enhancing the phagocytic activity of macrophages and strengthening the specific cytotoxicity of lymphocytes against target cells, promoting and maintaining the body's immune surveillance, immune protection, and immune homeostasis. Recombinant human interferon preparations are currently internationally recognized as effective treatments for hepatitis B and hepatitis C. According to statistics from the National Health and Family Planning Commission, there are approximately 350 million hepatitis B virus carriers worldwide. In addition, recombinant human interferon has been approved in China for the treatment of diseases such as chronic myeloid leukemia, hairy cell leukemia, renal cell carcinoma, and melanoma.

[0007] Early interferon was extracted from human leukocytes using purification techniques. This method was not only difficult to obtain and complex, but also resulted in low yields, high prices, and the potential for blood-borne viral contamination. It wasn't until the mid-1970s, with the development of biomedicine and the emergence of gene recombination technology, that interferon was gradually produced using a fermentation process with genetically engineered E. coli. However, this primarily yielded inactive inclusion bodies. Subsequently, a renaturation process was used to obtain biologically active interferon, and the interferon obtained through this method always retained a methionine residue at its N-terminus.

[0008] Recent studies have shown that the fusion expression of the target protein, inteptide, and self-assembled short peptide can induce the fusion protein to form active protein aggregates. The aggregates release the target protein into the supernatant through the self-cleavage of the inteptide (Wu et al., 2011; Xing et al., 2011; Zhou et al., 2012). Although this method for protein isolation and purification is low-cost and simple to operate, and has good application prospects in industrial production, existing technologies have reported that this method is only suitable for producing proteins without disulfide bonds. However, many important peptide drugs, such as human growth hormone and interferon α2a, have two disulfide bonds (the structural information of human growth hormone can be found in the UniProt database with accession number P01241, https: / / www.uniprot.org / uniprot / P01241; the structural information of interferon α2a can be found in the UniProt database with accession number P01563, https: / / www.uniprot.org / uniprot / P01563). To solve the problems caused by disulfide bonds, it is necessary to further attach a solubilizing tag to one end of the target protein, such as the TrxA tag (Zhao et al., 2016; Chinese Patent CN104755502 B), the SUMO tag (Regina L. Bis et al., 2014), or to use a complex renaturation method (Y. Mohammed et al., 2012).

[0009] Therefore, there is still a need in the field for low-cost, simple, and efficient methods for the production and purification of target peptides such as human growth hormone and interferon α2a. Summary of the Invention

[0010] This invention provides a low-cost, simple, and efficient method for the production and purification of disulfide-bonded peptides based on self-aggregating peptides and cleavage tags.

[0011] In one aspect, the present invention provides a fusion polypeptide comprising a target polypeptide moiety and a self-aggregating peptide moiety, wherein the target polypeptide is human growth hormone, the target polypeptide moiety is linked to the self-aggregating peptide moiety via a spacer, and wherein the cleavage tag includes a cleavage site. In some embodiments, the fusion polypeptide, after expression in host cells, can form active aggregates via the self-aggregating peptide moiety. In some embodiments, the target polypeptide moiety in the fusion polypeptide of the present invention is located at the N-terminus of the fusion polypeptide. In other embodiments, the target polypeptide moiety in the fusion polypeptide of the present invention is located at the C-terminus of the fusion polypeptide.

[0012] In some embodiments, the self-aggregating peptide moiety in the fusion polypeptide of the present invention comprises an amphiphilic self-assembling short peptide. In some embodiments, the self-aggregating peptide moiety comprises one or more tandemly repeated amphiphilic self-assembling short peptides.

[0013] In some embodiments, the amphiphilic self-assembling short peptide in the fusion polypeptide of the present invention is selected from amphiphilic β-sheet short peptides, amphiphilic α-helical short peptides, and surfactant-like short peptides. In some embodiments, surfactant-like short peptides are preferred.

[0014] In some embodiments, the surfactant-like short peptide has 7-30 amino acid residues, having an amino acid sequence represented by the following general formula from the N-terminus to the C-terminus:

[0015] AB or BA

[0016] Wherein, A is a peptide composed of hydrophilic amino acid residues, which may be the same or different, and are selected from Lys, Asp, Arg, Glu, His, Ser, Thr, Asn, and Gln; B is a peptide composed of hydrophobic amino acid residues, which may be the same or different, and are selected from Leu, Gly, Ala, Val, Ile, Phe, and Trp; A and B are linked by peptide bonds; and wherein the proportion of hydrophobic amino acid residues in the surfactant-like short peptide is 55%-95%. In some embodiments, the surfactant-like short peptide has 8 amino acid residues, wherein the proportion of hydrophobic amino acid residues in the surfactant-like short peptide is 75%. In some embodiments, the surfactant-like short peptide is selected from L6KD, L6KK, L6DD, L6DK, L6K2, L7KD, and DKL6. In some embodiments, the surfactant-like short peptide in the fusion polypeptide of the present invention is L6KD, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0017] In some embodiments, the amphiphilic β-sheet peptide has a length of 4-30 amino acid residues; and the content of hydrophobic amino acid residues is 40%-80%. In some embodiments, the amphiphilic β-sheet peptide in the fusion polypeptide of the present invention is EFK8, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0018] In some embodiments, the amphiphilic self-assembled short peptide is an amphiphilic α-helical short peptide with a length of 4-30 amino acid residues; and wherein the content of hydrophobic amino acid residues is 40%-80%. In some embodiments, the amphiphilic α-helical short peptide in the fusion polypeptide of the present invention is α3-peptide, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0019] In some embodiments, the target polypeptide in the fusion polypeptide of the present invention is human growth hormone. In some embodiments, the human growth hormone moiety comprises an amino acid sequence as shown in SEQ ID NO:5.

[0020] In some embodiments, the spacer in the fusion peptide of the present invention is directly linked to the target peptide portion and / or the self-aggregating peptide portion. In other embodiments, the spacer further includes a connector at its N-terminus and / or C-terminus, which is linked to the target peptide portion and / or the self-aggregating peptide portion via the connector.

[0021] In some embodiments, the cleavage site in the fusion polypeptide of the present invention is selected from temperature-dependent cleavage sites, pH-dependent cleavage sites, ion-dependent cleavage sites, enzyme cleavage sites, or self-cleavage sites. In some embodiments, the cleavage site is a self-cleavage site. In some specific embodiments, the spacer is an intein containing a self-cleavage site. In some embodiments, the intein is linked to the N-terminus or C-terminus of the human growth hormone moiety. In some embodiments, the intein is Mxe GyrA, having the sequence shown in SEQ ID NO: 4. In some optional embodiments, Mxe GyrA is linked to the C-terminus of the human growth hormone moiety.

[0022] In some embodiments, the linker in the spacer of the present invention is a GS-type linker, the amino acid sequence of which is shown in SEQ ID NO:6. In other embodiments, the linker is a PT-type linker, the amino acid sequence of which is shown in SEQ ID NO:7.

[0023] In another aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the fusion polypeptide of the present invention or its complementary sequence.

[0024] In another aspect, the present invention provides an expression construct comprising the polynucleotides of the present invention.

[0025] In another aspect, the present invention provides a host cell comprising the polynucleotide of the present invention or transformed from the expression construct of the present invention, wherein the host cell is capable of expressing the fusion polypeptide.

[0026] In some embodiments, the host cell is selected from prokaryotes, yeast, and higher eukaryotic cells. In some specific embodiments, the prokaryotes include Escherichia coli (Escherichia spp.). Escherichia ), Bacillus spp. Bacillus Salmonella ( Salmonella ) and Pseudomonas spp. ( Pseudomonas ) and Streptomyces ( Streptomyces More specifically, the prokaryotes are Escherichia coli, preferably Escherichia coli. E. coli ).

[0027] In another aspect, the present invention provides a method for producing and purifying human growth hormone, the method comprising the following steps:

[0028] (a) Culturing the host cells of the present invention to express the fusion polypeptide of the present invention;

[0029] (b) Lyse the host cells, then remove the soluble portion of the cell lysate and recover the insoluble portion;

[0030] (c) Releasing soluble human growth hormone from the insoluble portion by cleaving the cleavage site; and

[0031] (d) Remove the insoluble portion from step (c) and recover the soluble portion containing the human growth hormone.

[0032] In some embodiments, the lysis is carried out by ultrasound, homogenization, high pressure, hypotonicity, lysin, organic solvents, or combinations thereof. In other embodiments, the lysis is carried out under weakly alkaline pH conditions. In some specific embodiments, the cleavage is dithiothreitol (DTT)-mediated autocleavage.

[0033] In another aspect, the present invention provides a fusion polypeptide comprising a target polypeptide moiety and a self-aggregating peptide moiety, wherein the target polypeptide moiety is linked to the self-aggregating peptide moiety via a spacer, and wherein the cleavage tag includes a cleavage site. In some embodiments, the fusion polypeptide, after expression in a host cell, can form active aggregates via the self-aggregating peptide moiety. In some embodiments, the target polypeptide in the fusion polypeptide of the present invention is human growth hormone or human interferon α2a. In some embodiments, the target polypeptide moiety in the fusion polypeptide of the present invention is located at the N-terminus of the fusion polypeptide. In other embodiments, the target polypeptide moiety in the fusion polypeptide of the present invention is located at the C-terminus of the fusion polypeptide.

[0034] In some embodiments, the self-aggregating peptide moiety in the fusion polypeptide of the present invention comprises an amphiphilic self-assembling short peptide. In some embodiments, the self-aggregating peptide moiety comprises one or more tandemly repeated amphiphilic self-assembling short peptides.

[0035] In some embodiments, the amphiphilic self-assembling short peptide in the fusion polypeptide of the present invention is selected from amphiphilic β-sheet short peptides, amphiphilic α-helical short peptides, and surfactant-like short peptides. In some embodiments, surfactant-like short peptides are preferred.

[0036] In some embodiments, the surfactant-like short peptide has 7-30 amino acid residues, having an amino acid sequence represented by the following general formula from the N-terminus to the C-terminus:

[0037] AB or BA

[0038] Wherein, A is a peptide composed of hydrophilic amino acid residues, which may be the same or different, and are selected from Lys, Asp, Arg, Glu, His, Ser, Thr, Asn, and Gln; B is a peptide composed of hydrophobic amino acid residues, which may be the same or different, and are selected from Leu, Gly, Ala, Val, Ile, Phe, and Trp; A and B are linked by peptide bonds; and wherein the proportion of hydrophobic amino acid residues in the surfactant-like short peptide is 55%-95%. In some embodiments, the surfactant-like short peptide has 8 amino acid residues, wherein the proportion of hydrophobic amino acid residues in the surfactant-like short peptide is 75%. In some embodiments, the surfactant-like short peptide is selected from L6KD, L6KK, L6DD, L6DK, L6K2, L7KD, and DKL6. In some embodiments, the surfactant-like short peptide in the fusion polypeptide of the present invention is L6KD, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0039] In some embodiments, the amphiphilic β-sheet peptide has a length of 4-30 amino acid residues; and the content of hydrophobic amino acid residues is 40%-80%. In some embodiments, the amphiphilic β-sheet peptide in the fusion polypeptide of the present invention is EFK8, the amino acid sequence of which is shown in SEQ ID NO: 2.

[0040] In some embodiments, the amphiphilic self-assembled short peptide is an amphiphilic α-helical short peptide with a length of 4-30 amino acid residues; and wherein the content of hydrophobic amino acid residues is 40%-80%. In some embodiments, the amphiphilic α-helical short peptide in the fusion polypeptide of the present invention is α3-peptide, the amino acid sequence of which is shown in SEQ ID NO: 3.

[0041] In some embodiments, the amphiphilic self-assembling short peptide is an α-trihelical peptide. In some embodiments, the α-trihelical peptide in the fusion polypeptide of the present invention is TZ1H, the amino acid sequence of which is shown in SEQ ID NO: 36.

[0042] In some embodiments, the target polypeptide in the fusion polypeptide of the present invention contains at least two thiol groups, such as two, three, four, or more thiol groups, and disulfide bonds can be formed between the thiol groups. In some embodiments, the target polypeptide in the fusion polypeptide of the present invention contains one or more disulfide bonds. In some embodiments, the target polypeptide in the fusion polypeptide of the present invention contains one or more intramolecular disulfide bonds, such as one, two, or more disulfide bonds.

[0043] In some embodiments, the target peptide in the fusion peptide of the present invention has a length of 20-400 amino acids, such as 30-300 amino acids, 35-250 amino acids, or 40-200 amino acids.

[0044] In some embodiments, the target polypeptide in the fusion polypeptide of the present invention is human growth hormone. In some embodiments, the human growth hormone moiety comprises an amino acid sequence as shown in SEQ ID NO:5.

[0045] In some embodiments, the target polypeptide in the fusion polypeptide of the present invention is human interferon α2a. In some embodiments, the human interferon α2a moiety comprises an amino acid sequence as shown in SEQ ID NO:26.

[0046] In some embodiments, the spacer in the fusion peptide of the present invention is directly linked to the target peptide portion and / or the self-aggregating peptide portion. In other embodiments, the spacer further includes a connector at its N-terminus and / or C-terminus, which is linked to the target peptide portion and / or the self-aggregating peptide portion via the connector.

[0047] In some embodiments, the cleavage site in the fusion polypeptide of the present invention is selected from temperature-dependent cleavage sites, pH-dependent cleavage sites, ion-dependent cleavage sites, enzyme cleavage sites, or self-cleavage sites. In some embodiments, the cleavage site is a self-cleavage site. In some specific embodiments, the spacer is an intein containing a self-cleavage site. In some embodiments, the intein is attached to the N-terminus or C-terminus of the target polypeptide moiety. In some embodiments, the intein is attached to the C-terminus of the target polypeptide moiety. In some embodiments, the intein is Mxe GyrA, having the sequence shown in SEQ ID NO: 4. In some optional embodiments, the Mxe GyrA is attached to the C-terminus of the human growth hormone moiety.

[0048] In some embodiments, the cleavage site in the fusion polypeptide of the present invention is selected from temperature-dependent cleavage sites, pH-dependent cleavage sites, ion-dependent cleavage sites, enzyme cleavage sites, or self-cleavage sites. In some embodiments, the cleavage site is a pH-dependent cleavage site. In some specific embodiments, the spacer is an inteptide containing a pH-dependent cleavage site. In some embodiments, the inteptide is linked to the N-terminus or C-terminus of the target polypeptide moiety. In some embodiments, the inteptide is linked to the N-terminus of the target polypeptide moiety. In some embodiments, the inteptide is Mtu ΔI-CM, having the sequence shown in SEQ ID NO: 27. In some optional embodiments, the Mtu ΔI-CM is linked to the N-terminus of the human growth hormone moiety. In some optional embodiments, the Mtu ΔI-CM is linked to the N-terminus of the human interferon α2a moiety.

[0049] In some embodiments, the Mtu ΔI-CM includes a pH-dependent cleavage site that is cleaved under acidic conditions, preferably under weakly acidic conditions, such as at pH 6.0-6.5, and more preferably at pH 6.2. In some embodiments, the pH-dependent cleavage site is not cleaved under alkaline conditions.

[0050] In some embodiments, the integrin is a mutant of Mtu ΔI-CM. In some embodiments, the Mtu ΔI-CM has a mutation at position 73 and / or position 430. In some embodiments, the mutant of Mtu ΔI-CM has a mutation at position 73 of H73Y or H73V. In some embodiments, the mutant of Mtu ΔI-CM has a mutation at position 430 of T430V, T430S, or T430C. In some specific embodiments, the amino acid sequence of the mutant Mtu ΔI-CM having H73Y and T430V is shown in SEQ ID NO: 28. In some specific embodiments, the amino acid sequence of the mutant Mtu ΔI-CM having H73V and T430S is shown in SEQ ID NO: 29. In some specific embodiments, the amino acid sequence of the mutant Mtu ΔI-CM having H73V and T430C is shown in SEQ ID NO: 30.

[0051] In some embodiments, the linker in the spacer of the present invention is a GS-type linker, the amino acid sequence of which is shown in SEQ ID NO:6. In other embodiments, the linker is a PT-type linker, the amino acid sequence of which is shown in SEQ ID NO:7.

[0052] In another aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the fusion polypeptide of the present invention or its complementary sequence.

[0053] In another aspect, the present invention provides an expression construct comprising the polynucleotides of the present invention.

[0054] In another aspect, the present invention provides a host cell comprising the polynucleotides of the present invention or transformed from the expression constructs of the present invention, wherein the host cell is capable of expressing the fusion polypeptide.

[0055] In some embodiments, the host cell is selected from prokaryotes, yeast, and higher eukaryotic cells. In some specific embodiments, the prokaryotes include Escherichia coli (Escherichia spp.). Escherichia ), Bacillus spp. Bacillus Salmonella ( Salmonella ) and Pseudomonas spp. ( Pseudomonas ) and Streptomyces ( Streptomyces More specifically, the prokaryotes are Escherichia coli, preferably Escherichia coli. E. coli ).

[0056] In another aspect, the present invention provides a method for producing and purifying a target polypeptide, the method comprising the following steps:

[0057] (a) Culturing the host cells of the present invention to express the fusion polypeptide of the present invention;

[0058] (b) Lyse the host cells, then remove the soluble portion of the cell lysate and recover the insoluble portion;

[0059] (c) releasing a soluble target polypeptide from the insoluble portion by cleaving the cleavage site; and

[0060] (d) Remove the insoluble portion from step (c) and recover the soluble portion containing the target polypeptide.

[0061] In some embodiments, the lysis is carried out by ultrasound, homogenization, high pressure, hypotonicity, lysin, organic solvents, or combinations thereof. In other embodiments, the lysis is carried out under weakly alkaline pH conditions. In some specific embodiments, the cleavage is pH-dependent, for example, cleavage under acidic conditions, preferably under weakly acidic conditions, such as at pH 6.0-6.5, and preferably at pH 6.2. Attached Figure Description

[0062] Figure 1The expression and purification strategy for human growth hormone (hGH) based on self-aggregating peptides and the structural diagrams of the expression vectors used are shown. A: Expression and purification strategy; B: Vector structural diagrams of pET30-hGH-Mxe-L6KD, pET30-hGH-Mxe-EFK8, and pET30-hGH-Mxe-α3.

[0063] Figure 2 The image shows the SDS-PAGE analysis results of the expression and purification of human growth hormone (hGH) fusion protein. A: L6KD-based self-aggregating peptide; B: EFK8-based self-aggregating peptide; C: α3-peptide-based self-aggregating peptide.

[0064] Figure 3 The mass spectrometry analysis of human growth hormone (hGH) is shown.

[0065] Figure 4 The diagram shows the bioactivity analysis of human growth hormone (hGH).

[0066] Figure 5 The expression and purification strategies for human growth hormone (hGH) and human interferon α2a based on self-aggregating peptides are shown, along with the structural diagram of the expression vector used. A: Expression and purification strategies; B: pET32-L6KD-Mtu ΔI-CM-hGH, pET32-L6KD-Mtu ΔI-CM mutant strain 1-hGH, pET32-L6KD-Mtu ΔI-CM mutant strain 2-hGH, pET32-L6KD-Mtu ΔI-CM mutant strain 3-hGH, pET32-ELK16-Mtu ΔI-CM mutant strain 2-hGH, pET32-EFK8-Mtu ΔI-CM mutant strain 2-hGH, pET32-α3-Mtu ΔI-CM mutant strain 2-hGH, pET32-TZ1H-Mtu ΔI-CM mutant strain 2-hGH, pET32-L6KD-Mtu ΔI-CM-IFNα2a, pET32-L6KD-Mtu Vector structure diagrams of ΔI-CM mutant strains 1-IFNα2a, pET32-L6KD-Mtu, ΔI-CM mutant strains 2-IFNα2a, pET32-L6KD-Mtu, and ΔI-CM mutant strain 3-IFNα2a.

[0067] Figure 6 The image shows the SDS-PAGE analysis results of the expression and purification of human growth hormone (hGH) fusion protein. A: Expression and purification results of different MtuΔI-CM mutant strains in LB medium; B: Expression and purification results of different MtuΔI-CM mutant strains in fermentation medium; C: Supernatant after cleavage of fusion proteins with different self-aggregating peptides expressed in LB medium.

[0068] Figure 7The image shows the SDS-PAGE analysis results of column-purified human growth hormone (hGH).

[0069] Figure 8 The RP-HPLC chromatogram of human growth hormone (hGH) is shown.

[0070] Figure 9 The MS analysis chromatogram of human growth hormone (hGH) is shown.

[0071] Figure 10 The image shows a native-pAGE analysis of human growth hormone (hGH).

[0072] Figure 11 The CD (circular dichroism) analysis chromatogram of human growth hormone (hGH) is shown.

[0073] Figure 12 The image shows the SDS-PAGE analysis results of the expression and purification of human interferon α2a fusion protein. A: Mtu ΔI-CM; B: Mtu ΔI-CM mutants 1 and 2; C: Mtu ΔI-CM mutant 3; D: Expression and purification results using Mtu ΔI-CM mutant 2 in fermentation medium. Detailed Implementation

[0074] This invention is not limited to the specific methods, schemes, reagents, etc., described herein, as these can vary. The terminology used herein is for the purpose of describing particular embodiments only and not for limiting the scope of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0075] In one aspect, the present invention provides a fusion polypeptide comprising a target polypeptide moiety and a self-aggregating peptide moiety, wherein the target polypeptide moiety is human growth hormone, wherein the target polypeptide moiety is linked to the self-aggregating peptide moiety by a spacer, and wherein the cleavage tag comprises a cleavage site.

[0076] In another aspect, the present invention provides a fusion polypeptide comprising a target polypeptide portion and a self-aggregating peptide portion, wherein the target polypeptide portion is linked to the self-aggregating peptide portion by a spacer, and wherein the cleavage tag comprises a cleavage site.

[0077] In another aspect, the present invention provides a fusion polypeptide comprising a target polypeptide moiety and a self-aggregating peptide moiety, wherein the target polypeptide is human interferon α2a, wherein the target polypeptide moiety is linked to the self-aggregating peptide moiety by a spacer, and wherein the cleavage tag comprises a cleavage site.

[0078] In another aspect, the present invention provides an isolated polynucleotide comprising a nucleotide sequence encoding the fusion polypeptide of the present invention or its complementary sequence.

[0079] In another aspect, the present invention provides an expression construct comprising the polynucleotides of the present invention.

[0080] In another aspect, the present invention provides a host cell comprising the polynucleotide of the present invention or transformed from the expression construct of the present invention, wherein the host cell is capable of expressing the fusion polypeptide.

[0081] In another aspect, the present invention provides a method for producing and purifying human growth hormone, the method comprising the steps of: (a) culturing host cells of the present invention to express the fusion human polypeptide of the present invention; (b) lysing the host cells and then removing the soluble portion of the cell lysate and recovering the insoluble portion; (c) releasing soluble human growth hormone from the insoluble portion by cleaving the cleavage site; and (d) removing the insoluble portion in step (c) and recovering the soluble portion containing the human growth hormone.

[0082] In another aspect, the present invention provides a method for producing and purifying a target polypeptide, the method comprising the steps of: (a) culturing a host cell of the present invention to express the fusion polypeptide of the present invention; (b) lysing the host cell and then removing the soluble portion of the cell lysate and recovering the insoluble portion; (c) releasing the soluble target polypeptide from the insoluble portion by cleaving the cleavage site; and (d) removing the insoluble portion in step (c) and recovering the soluble portion containing the target polypeptide.

[0083] As used herein, the terms “peptide,” “polypeptide,” and “protein” are used interchangeably and are defined as biomolecules composed of amino acid residues linked together by peptide bonds.

[0084] As used herein, the amino acid sequence of the "target polypeptide" of the present invention contains at least two cysteine ​​residues, such as two, three, four, or more cysteine ​​residues, which can form intramolecular disulfide bonds, such as one, two, or more intramolecular disulfide bonds. The "target polypeptide" of the present invention contains at least two thiol groups, such as two, three, four, or more thiol groups, which can form disulfide bonds between themselves, such as one, two, or more intramolecular disulfide bonds. The length of the target polypeptide can be 20-400 amino acids, such as 30-300 amino acids, 35-250 amino acids, or 40-200 amino acids.

[0085] As used herein, "human growth hormone" and "target polypeptide" are used interchangeably. The term refers to a protein hormone secreted by the anterior pituitary gland. Its mature form is a non-glycosylated hydrophilic globulin with the signal peptide removed. It consists of 191 amino acids, has two disulfide bonds, and has a relative molecular weight of approximately 22 kDa. The human growth hormone portion of the fusion polypeptide of this invention contains the amino acid sequence shown in SEQ ID NO:5.

[0086] As used herein, “human interferon α2a” and “target polypeptide” are interchangeable. It is a class of multifunctional and highly active inducible proteins produced by leukocytes and lymphocytes, composed of 165 amino acids, containing two pairs of intramolecular disulfide bonds, with a relative molecular weight of approximately 19.2 kDa. The human interferon α2a portion of the fusion polypeptide of the present invention contains the amino acid sequence shown in SEQ ID NO:26.

[0087] In some specific embodiments, the "target polypeptide" of the present invention has a structure similar to that of "human growth hormone". In some specific embodiments, the "target polypeptide" of the present invention has a structure similar to that of "human interferon α2a".

[0088] In some embodiments, the fusion polypeptide, after expression in host cells, can form active aggregates via the self-aggregating peptide moiety. In some embodiments, the target polypeptide moiety in the fusion polypeptide of the present invention is located at the N-terminus of the fusion polypeptide. In other embodiments, the target polypeptide moiety in the fusion polypeptide of the present invention is located at the C-terminus of the fusion polypeptide.

[0089] As used herein, a "self-aggregating peptide" refers to a peptide that, after fusing with a target polypeptide moiety and being expressed in a host cell, mediates the formation of an insoluble, active aggregate of the fusion protein within the cell. As used herein, an "active aggregate" refers to a human growth hormone moiety that remains correctly folded and active, or to an aggregate in which the human growth hormone moiety remains soluble after separation from the self-aggregating peptide.

[0090] Unintentionally limited by any theory, it is known in the art that some amphipathic peptides, due to their separated hydrophilic and hydrophobic regions, can spontaneously form specific self-assembling structures under the influence of hydrophobic interactions and other driving forces (Zhao et al., 2008). The inventors have surprisingly discovered that some amphipathic short peptides with self-assembling capabilities can induce the formation of intracellular active aggregates. The amphipathic self-assembling short peptides used as the self-aggregating peptides of the present invention can be selected from amphipathic β-sheet short peptides, amphipathic α-helical short peptides, and surfactant-like short peptides. The amphipathic self-assembling short peptides used as the self-aggregating peptides of the present invention can also be selected from α-trihelical peptides.

[0091] As used herein, "surfactant-like peptide" is a class of amphiphilic polypeptides that can be used as self-aggregating peptides in this invention. It typically consists of 7-30 amino acid residues, has an elongation of approximately 2-5 nm, and a structure similar to lipids, consisting of a hydrophobic amino acid tail and a hydrophilic amino acid head. The properties of surfactant-like structures are similar to those of surfactants; in aqueous solutions, they can form assembled structures such as micelles and nanotubes. Suitable surfactant-like short peptides for use as self-aggregating peptides in this invention can be 7-30 amino acid residues in length, comprising an amino acid sequence represented by the following general formula from the N-terminus to the C-terminus:

[0092] AB or BA,

[0093] A and B are linked by peptide bonds. A is a hydrophilic head composed of hydrophilic amino acid residues, which can be the same or different polar amino acids and are selected from Lys, Asp, Arg, Glu, His, Ser, Thr, Asn, and Gln. Examples of A include KD, KK, or DK. B is a hydrophobic tail composed of hydrophobic amino acid residues, which can be the same or different nonpolar amino acids and are selected from Leu, Gly, Ala, Val, Ile, Phe, and Trp. Examples of B include LLLLLLL (L6), L7, or GAVIL. In the surfactant-like short peptide of the present invention, the proportion of hydrophobic amino acids is higher than the proportion of hydrophilic amino acids, and the proportion of hydrophobic amino acids in the surfactant-like short peptide can be 55-95%, 60-95%, 65-95%, 70-95%, 75-95%, 80-95%, 85-95%, or 90-95%. In some embodiments, the surfactant-like short peptide has 8 amino acid residues, of which 75% are hydrophobic amino acids. In aqueous solution, the surfactant-like peptide undergoes self-assembly, causing the hydrophobic tail to aggregate internally while the hydrophilic head is exposed to the solution, interacting with the aqueous solution and preventing the hydrophobic region from contacting the aqueous solution. Specific examples of surfactant-like short peptides suitable for the self-aggregating peptides of the present invention include L6KD, L6KK, L6DD, L6DK, L6K2, L7KD, and DKL6. The fusion polypeptide of the present invention utilizes L6KD, the amino acid sequence of which is shown in SEQ ID NO: 1.

[0094] Furthermore, those skilled in the art know that surfactant-like peptides with the above-mentioned structures (such as L6KD, L6K2, L6D2, etc.) have similar activities and can all mediate the formation of insoluble active aggregates of fusion proteins within cells (Zhou et al., 2012).

[0095] As used herein, "amphiphilic β-sheet short peptide" refers to a short peptide having 4-30 amino acid residues, composed of alternating hydrophobic and charged hydrophilic amino acids, where, when forming a β-sheet, one side consists of hydrophobic amino acid residues, and the other side consists of alternating positively and negatively charged hydrophilic amino acid residues. These short peptides can self-assemble under hydrophobic interactions, electrostatic interactions, and hydrogen bonding. Generally, the longer the amphiphilic β-sheet structure or the stronger its hydrophobicity, the easier self-assembly occurs, and the stronger the mechanical strength of the resulting self-aggregate. To ensure sufficient self-assembly capability, the amphiphilic β-sheet short peptide of the present invention should contain a certain amount of hydrophobic amino acids. The amphiphilic β-sheet short peptide of the present invention comprises 40-80%, 45-70%, 50-60%, for example, approximately 50% hydrophobic amino acid residues. A specific example of an amphiphilic β-sheet short peptide that can be used as a self-aggregating peptide of the present invention is EFK8, whose amino acid sequence is shown in SEQ ID NO:2.

[0096] An α-helix is ​​a protein secondary structure in which the peptide backbone extends helically around an axis. As used herein, a "short amphiphilic α-helical peptide" refers to a short peptide with 4-30 amino acid residues, exhibiting a unique hydrophilic-hydrophobic amino acid arrangement compared to a regular α-helix, such that one side of the formed α-helix structure is predominantly composed of hydrophilic amino acids, while the other side is predominantly composed of hydrophobic amino acids. It is hypothesized that amphiphilic α-helices self-assemble in aqueous solution by forming coiled-coils, in which two α-helices bind through hydrophobic interactions, and this binding is further stabilized by electrostatic interactions of charged amino acids. The amphiphilic α-helical peptides of this invention comprise 40-80%, 45-70%, 50-60%, for example, approximately 50% hydrophobic amino acid residues. A specific example of an amphiphilic α-helical peptide that can be used as a self-aggregating peptide of this invention is α3-peptide, whose amino acid sequence is shown in SEQ ID NO:3. As used herein, an “α-trihelical peptide” consists of six heptapeptide repeat sequences, with three histidine residues at the d position of the first, third, and fifth heptapeptide repeat sequences. A specific example of an α-trihelical peptide that can be used as a self-aggregating peptide of the present invention is TZ1H, whose amino acid sequence is shown in SEQ ID NO: 36 (Lou et al., 2019).

[0097] There are existing reports in this field of polypeptides with self-aggregating properties formed by the tandem repetition of multiple repeating units, such as elastin-like peptide-1 (ELP), which consists of 110 VPGXG repeating units, and whose aggregation properties are related to the number of repeating units (Banki, et al., 2005; MacEwan and Chilkoti, 2010). There are also reports showing that the self-aggregating tendency of amphiphilic β-sheets composed of multiple repeating units increases with the number of repeating units (Zhang et al., 1992). It can be expected that polypeptides composed of multiple of the aforementioned "amphiphilic self-assembling short peptides" tandemly can retain or even acquire enhanced self-assembly capabilities.

[0098] Therefore, the self-aggregating peptide portion of the present invention may include one or more tandemly linked amphiphilic self-assembled short peptides. The self-aggregating peptide portion of the present invention may contain 1-150, 1-130, 1-110, 1-90, 1-70, 1-50, 1-30, 1-10, or 1-5 of the amphiphilic self-assembled short peptides, for example, 1, 2, 3, 4, or 5. Two or more amphiphilic self-assembled short peptides in the self-aggregating peptide portion may form tandem repeats. To facilitate recombination operations and considering production costs, it is desirable to use fewer repeats. Therefore, in some embodiments, the "self-aggregating peptide portion" contains only one of the amphiphilic self-assembled short peptides.

[0099] In addition, it has been reported that some protein domains, such as β-amyloid peptide, VP1, MalE31, and CBD, have been involved. clos Such domains can also induce fusion proteins to form aggregates, and this invention anticipates that such domains can also be used as the "self-aggregating peptides" of this invention. However, the structures of these domains are relatively complex and the mechanism by which they induce aggregation remains unclear (Mitraki, 2010). In this invention, it is preferable to use short, amphiphilic self-assembling peptides with relatively simple structures and short lengths.

[0100] Existing research has found that when self-aggregating peptides (such as amphiphilic self-assembling peptides) capable of inducing the formation of active aggregates are expressed as fusion proteins with target peptides in host cells, the expressed fusion proteins can form insoluble aggregates. Aggregate formation avoids degradation of the fusion protein by intracellular proteases, thus increasing the yield of the target peptide. After cell lysis, the insoluble aggregates can be easily collected from the cell lysate by centrifugation or filtration, removing soluble impurities and achieving preliminary purification of the fusion protein. Subsequently, by cleaving the cleavage site located at the linker between the self-aggregating peptide and the target peptide, the soluble portion containing the target peptide is released from the insoluble portion (precipitate) and distributed in the supernatant. Insoluble impurities can then be removed again by simple centrifugation or filtration, yielding the soluble target peptide. This method of producing peptides based on self-aggregating peptides simplifies the separation and purification steps, avoids the use of expensive purification columns, and significantly reduces production costs.

[0101] Existing technologies also report that the above methods are only suitable for producing a class of proteins that do not contain disulfide bonds, such as Bacillus subtilis (…). Bacillus subtilis Lipase A (LipA) (Van Pouderoyen et al., 2001), Aspergillus fumigatus ( Aspergillus fumigatus Type II ketoamine oxidase (AMA) (Collard et al., 2008), Bacillus pumilus ( Bacillus pumilus Xylosidase (XynB) (its structural information can be found in the Protein Database PDB with accession number 1YIF, https: / / www.rcsb.org / structure / 1YIF), etc. Target proteins with disulfide bonds (such as CCL5 (2 disulfide bonds), SDF-1α (3 disulfide bonds), and leptin (1 disulfide bond) tend to aggregate after intein-mediated cleavage and cannot be released into the supernatant; the reason these cleaved target proteins remain aggregated may be due to exposed hydrophobic sequences or the difficulty in forming the correct disulfide bonds in the periplasmic space of *E. coli* (Zhao et al., 2016). To address the problem caused by disulfide bonds, current research has found that adding a solubilizing tag to one end of the target protein can effectively produce proteins with disulfide bonds (Zhao et al., 2016; Chinese Patent CN 104755502 B), such as the TrxA tag (Zhao et al., 2016) and the SUMO tag (Regina L. Bis et al., 2014).

[0102] However, surprisingly, the inventors discovered that although human growth hormone has two disulfide bonds, it can be effectively produced using the self-aggregating peptide method described above, even without the addition of a solubilizing tag. Furthermore, the inventors also discovered that human interferon-α2a, which has a similar structure to human growth hormone and also has two disulfide bonds, can also be produced using the self-aggregating peptide method described above.

[0103] As used herein, a "spacer" refers to a polypeptide of a certain length composed of amino acids, including sequences necessary for cleavage, such as protease recognition sequences for enzyme cleavage, integrin sequences for self-cleavage, etc., to connect the various parts of the fusion protein without affecting the structure and activity of each part. Therefore, the spacer of the present invention contains a "cleavage site." In the fusion polypeptide of the present invention, the spacer is directly linked to the target polypeptide part and / or the self-aggregating peptide part. In other embodiments, the spacer further includes a linker at its N-terminus and / or C-terminus, which links to the target polypeptide part and / or the self-aggregating peptide part via the linker.

[0104] In some embodiments, the spacer is an integrity containing a self-cleaving site. In some embodiments, the integrity is linked to the N-terminus or C-terminus of the human growth hormone moiety. It should be understood that those skilled in the art can select appropriate integrities and suitable linking sites as needed.

[0105] The cleavage sites of this invention for releasing the soluble target polypeptide moiety from the insoluble moiety (precipitate) include those selected from temperature-dependent cleavage sites, pH-dependent cleavage sites, ion-dependent cleavage sites, enzyme-dependent cleavage sites, or self-cleavage sites, or any other cleavage sites known to those skilled in the art. Preferred cleavage sites in this invention are self-cleavable, for example, containing the amino acid sequence of a self-cleavable integrin. This is because integrity-based cleavage methods do not require the addition of enzymes or the use of harmful substances such as hydrogen bromide used in chemical methods; cleavage can be easily induced simply by altering the buffer environment of the aggregate (Wu et al., 1998; TELENTI et al., 1997). Various self-cleavable integrins are known in the art, such as a series of integrins from NEB with different self-cleavage properties. In some embodiments, the cleavage site may also be a pH-dependent cleavage site.

[0106] In some specific embodiments of the invention, the integrin is Mxe GyrA, having the sequence shown in SEQ ID NO: 4. In some optional embodiments, Mxe GyrA is attached to the C-terminus of the human growth hormone moiety. In one specific embodiment, the integrin Mxe GyrA can be induced to self-cleave at its amino terminus by adding an appropriate amount of dithiothreitol (DTT) to a buffer system. Those skilled in the art can determine the DTT concentration and reaction time as needed. Optionally, DTT can be removed in subsequent operations.

[0107] In some specific embodiments of the present invention, the inteptide is Mtu ΔI-CM, having the sequence shown in SEQ ID NO:27. In some optional embodiments, Mtu ΔI-CM is attached to the N-terminus of the human growth hormone moiety. In some optional embodiments, Mtu ΔI-CM is attached to the N-terminus of the human interferon α2a moiety. In one specific embodiment, the inteptide Mtu ΔI-CM can be induced to self-cleave at its carboxyl terminus in a buffer system at pH 6.2.

[0108] As used in this article, “Mtu ΔI-CM” is derived from the wild-type Mtu recA intein. It is obtained by deleting the endonuclease domain of the Mtu recA extra-large intein, retaining 110 amino acids at the N-terminus and 58 amino acids at the C-terminus, and then introducing four mutations: C1A, V67L, D24G, and D422G (Wood et al., 1999).

[0109] This invention also provides mutants of Mtu ΔI-CM, which can also be used as integrins of this invention. In some specific embodiments, because Mtu ΔI-CM contains pH-dependent cleavage sites, autocleavage may occur during in vivo expression due to insufficient pH control before the final in vitro cleavage step, resulting in the loss of part of the target peptide, i.e., premature autocleavage during in vivo maturation. To reduce the proportion of premature autocleavage during in vivo maturation, the inventors introduced mutations at positions 73 and / or 430 of Mtu ΔI-CM. Optionally, the mutation at position 73 is selected from H73Y and H73V, and the mutation at position 430 is selected from T430V, T430S, and T430C. Preferably, the mutant has a mutation combination selected from the following: H73Y / T430V (SEQ ID NO: 28), H73V / T430S (SEQ ID NO: 29) and H73V / T430C (SEQ ID NO: 30); more preferably, the mutant has a mutation combination selected from the following: H73V / T430S (SEQ ID NO: 29) and H73V / T430C (SEQ ID NO: 30).

[0110] Furthermore, since the activity of Mtu ΔI-CM is temperature-sensitive, premature in vivo autocleavage can be inhibited by lowering the temperature. For example, lowering the temperature to 18°C ​​when expressing the fusion protein can help. o C, and the bacterial cells were thoroughly cooled before IPTG was added to induce recombinant protein expression in order to reduce the proportion of autocleavage in vivo.

[0111] Those skilled in the art will understand that, in order to reduce mutual interference between different parts of the fusion protein of the present invention, the different parts of the fusion protein can be connected by a linker. As used herein, a linker is a polypeptide of a certain length composed of amino acids with low hydrophobicity and low charge effect, which, when used in a fusion protein, allows the connected parts to fully unfold and fold into their respective native conformations without interference.

[0112] Commonly used linkers in this art include, for example, flexible GS-type linkers rich in glycine (G) and serine (S); and rigid PT-type linkers rich in proline (P) and threonine (T). In some embodiments, the amino acid sequence of the GS-type linker used in this invention is shown in SEQ ID NO:6. In other embodiments, the amino acid sequence of the PT-type linker used in this invention is shown in SEQ ID NO:7.

[0113] In the production of peptide drugs, it is often necessary for the recombinant peptide to have a sequence identical to the target peptide, i.e., without any additional amino acid residues at both ends, so that the produced peptide has pharmacokinetics consistent with naturally occurring peptides. In this invention, this can be achieved by selecting appropriate cleavage sites and their connection methods with the target peptide. Those skilled in the art will understand how to make such selections based on the characteristics of the cleavage sites. For example, in one embodiment, the Mxe GyrA cleavage site can be directly linked to the C-terminus of the target peptide moiety, so that there are no additional amino acid residues between it and the human growth hormone moiety. In other embodiments, the "target peptide" and the "spacer" of this invention can contain a short sequence, such as "MRM," to improve cleavage efficiency without affecting the final activity of the target peptide. In still other embodiments, the amino acid sequence of the target peptide obtained by self-cleavage of the carboxyl terminus of Mtu ΔI-CM will be completely identical to the target sequence, which is of significant importance for peptide drugs, both from a drug approval perspective and from a biological action perspective. Those skilled in the art will understand that by selecting spacers with different cleavage sites, it is possible to cleave target peptides without extra amino acid residues at the C-terminus and / or N-terminus.

[0114] As described above, the present invention also relates to polynucleotides comprising a nucleotide sequence encoding the fusion polypeptide of the present invention or its complementary sequence. As used herein, a "polynucleotide" refers to a macromolecule consisting of multiple nucleotides linked by 3'-5'-phosphodiester bonds, wherein said nucleotides include ribonucleotides and deoxyribonucleotides. The sequences of the polynucleotides of the present invention can be codon-optimized for different host cells (such as Escherichia coli) to improve the expression of the fusion protein. Methods for codon optimization are known in the art.

[0115] As described above, the present invention also relates to expression constructs comprising the polynucleotides described above. In the expression constructs of the present invention, the sequence of the polynucleotide encoding the fusion protein is operatively linked to an expression control sequence to perform desired transcription and ultimately produce the fusion polypeptide in a host cell. Suitable expression control sequences include, but are not limited to, promoters, enhancers, ribosome-acting sites such as ribosome binding sites, polyadenylation sites, transcription splicing sequences, transcription termination sequences, and sequences stabilizing mRNA, etc.

[0116] Vectors used for the expression constructs of this invention include those that replicate autonomously in host cells, such as plasmid vectors; and also include vectors capable of integrating into and replicating with the host cell DNA. Many commercially available vectors suitable for this invention are readily available. In one specific embodiment, the expression construct of this invention is derived from Novagen's pET30a(+).

[0117] This invention also relates to a host cell containing the polynucleotides of this invention or transformed from the expression constructs of this invention, wherein said host cell is capable of expressing the fusion polypeptide of this invention. Host cells for expressing the fusion polypeptide of this invention include prokaryotes, yeast, and higher eukaryotic cells. Exemplary prokaryotic hosts include Escherichia coli (…). Escherichia ), Bacillus spp. Bacillus Salmonella ( Salmonella ) and Pseudomonas spp. ( Pseudomonas ) and Streptomyces ( Streptomyces The host cell is a bacterium. In a preferred embodiment, the host cell is an Escherichia coli cell, preferably an Escherichia coli. In a specific embodiment of the invention, the host cell used is an Escherichia coli BL21(DE3) strain cell (Novagen).

[0118] The recombinant expression construct of the present invention can be introduced into host cells using one of many well-known techniques, including but not limited to: heat shock conversion, electroporation, DEAE-glucan transfection, microinjection, liposome-mediated transfection, calcium phosphate precipitation, protoplasmic fusion, microparticle bombardment, viral transformation, and similar techniques.

[0119] The present invention also relates to a method for producing and purifying human growth hormone, the method comprising the steps of: (a) culturing host cells of the present invention to express the fusion polypeptide of the present invention; (b) lysing the host cells, then removing the soluble portion of the cell lysate and recovering the insoluble portion; (c) releasing soluble human growth hormone from the insoluble portion by cleaving the cleavage site; and (d) removing the insoluble portion in step (c) and recovering the soluble portion containing the human growth hormone. A schematic diagram of the method of the present invention can be seen in [reference needed]. Figure 1 A.

[0120] This invention also provides a method for producing and purifying a target polypeptide, the method comprising the following steps: (a) culturing host cells of the invention to express the fusion polypeptide of the invention; (b) lysing the host cells, then removing the soluble portion of the cell lysate and recovering the insoluble portion; (c) releasing the soluble target polypeptide from the insoluble portion by cleaving the cleavage site; and (d) removing the insoluble portion in step (c) and recovering the soluble portion containing the target polypeptide. A schematic diagram of the method of the invention can be seen in [reference needed]. Figure 5 A.

[0121] In this invention, the method for lysing host cells is selected from commonly used processing methods in the art, such as ultrasound, homogenization, high pressure (e.g., in a Freund's crusher), osmolysis, detergents, lysing enzymes, organic solvents, or combinations thereof, and the lysis is carried out under weakly alkaline pH conditions (e.g., pH 7.5-8.5), thereby causing the cell membrane of the host cells to rupture, releasing the active aggregates from the cell, but still keeping them in an insoluble state.

[0122] The released aggregates are recovered directly as precipitates, eliminating the need to change environmental conditions (such as temperature, ion concentration, pH) to obtain fused proteins in a precipitated state, and also avoiding the impact of drastic changes in environmental conditions on protein stability and activity.

[0123] In traditional growth hormone production, because human growth hormone contains disulfide bonds, it is necessary to use a tag to secrete the growth hormone into the periplasmic space of E. coli to overcome the expression problems caused by the disulfide bonds. This protein expression method, which involves secretion into the periplasmic space, is generally considered to have a yield of 0.1-10 mg / L, mostly around 1 mg / L. Furthermore, the purification process mainly employs two methods: using a particularly expensive antibody against growth hormone (antibody-specific purification is used, but the antibody is very expensive, and the number of batches used is limited, requiring replacement after a few batches) packed into a column for purification (Chang et al., 1986); or using an affinity tag, followed by a series of complex steps including: 1) purifying the fusion protein with the affinity tag, 2) changing the buffer, 3) adding an external protease to cleave the tag, 4) purifying the affinity tag to remove the protease and tag, and 5) changing the buffer again, before finally purifying the growth hormone through molecular sieves (Nguyen et al., 2014; Moony et al., 2014).

[0124] Unlike existing techniques that address disulfide bond issues by adding solubilizing tags, the present invention, despite the presence of two disulfide bonds in its target polypeptide, human growth hormone, has surprisingly demonstrated that a fusion method based on self-aggregating peptides without adding solubilizing tags can successfully produce large quantities of active human growth hormone. The self-aggregating peptides used in this invention induce the fusion protein to form numerous active protein aggregates, preventing degradation of human growth hormone within the host and facilitating its proper folding in prokaryotic cells to form active human growth hormone. The human growth hormone obtained by this invention is a correctly folded soluble protein, requiring no cumbersome renaturation operations, and exhibits high yield and purity. The purification of the human growth hormone from this invention requires minimal equipment, eliminates the need for purification columns, and results in low production costs and simple operation.

[0125] As used in this article, "purity" refers to the purity of the target protein, that is, the proportion of the target polypeptide, such as human growth hormone, in the total protein in the purification solution. Since the target protein is expressed through cells, there are a large number of other proteins inside the cell (e.g., E. coli contains thousands of proteins). Purifying the target protein from such a diverse and abundant mixture of proteins has always been a key technical challenge. Through steps such as cell disruption, centrifugation, and separation after cleavage, the purification solution contains essentially only proteins and inorganic salts. Therefore, the higher the proportion of human growth hormone in the purification solution, the higher the purity of the product.

[0126] Example

[0127] To make the technical solutions and advantages of the present invention clearer, the embodiments of the present invention will be described in further detail below. It should be understood that the embodiments should not be construed as limiting, and those skilled in the art can make further adjustments to the embodiments based on the principles of the present invention.

[0128] Unless otherwise specified, all methods used in the following examples are conventional methods. For specific steps, please refer to, for example, Molecular Cloning: A Laboratory Manual (Sambrook, J., Russell, David W., Molecular Cloning: A Laboratory Manual, 3rd edition, 2001, NY, Cold SpringHarbor). All primers used were synthesized by Shanghai Sangon Biotech.

[0129] Example 1: Construction of a human growth hormone fusion protein expression construct containing the intima-peptide MxeGyrA

[0130] The construction process of the expression vectors pET30-hGH-Mxe-L6KD, pET30-hGH-Mxe-EFK8, and pET30-hGH-Mxe-α3 used in the embodiments of this application is similar. The following takes the construction of pET30-hGH-Mxe-L6KD as an example. The required primers were designed by oligo 6 and synthesized by Shanghai Sangon Biotech as shown in Table 1.

[0131] Table 1. Oligonucleotide primers used in this embodiment.

[0132]

[0133] a The underlined parts of the primers represent restriction endonucleases. Nde I, Xho I and Spe I is the identification site.

[0134] First, the polynucleotide sequence of human growth hormone (hGH) (NCBI number: AAA98618.1) was obtained from NCBI. Codon optimization for *E. coli* was performed using jcat software, and the gene fragment was synthesized by Shanghai Sangon Biotech. Using the synthesized gene as a template and hGH-F and hGH-R as primers, the hGH polynucleotide fragment was amplified by PCR. The PCR reaction used NEB's Q5 polymerase (New England Biolab (NEB)). The PCR conditions were: 98 ℃ for 30 sec, 98 ℃ for 10 sec, 60 ℃ for 30 sec, and 72 ℃ for 30 sec, for a final incubation at 72 ℃ for 2 min. After the reaction, the PCR amplification product was separated and recovered by 1% agarose gel electrophoresis.

[0135] Using pET30-lipA-Mxe-L6KD (Xing Lei et al., 2011) as a template and MxeL6KD-F and MxeL6KD-R as primers, the Mxe-L6KD polynucleotide fragment was amplified by PCR. The PCR reaction used NEB Q5 polymerase, and the PCR conditions were: 98 ℃ for 30 sec, 98 ℃ for 10 sec, 60 ℃ for 30 sec, 72 ℃ for 30 sec, for a final incubation at 72 ℃ for 2 min. After the reaction, the PCR amplification products were separated and recovered by 1% agarose gel electrophoresis. Then, the hGH and Mxe-L6KD fragments were subjected to an overlap PCR reaction: first, without primers, 98 ℃ for 30 sec, 98 ℃ for 10 sec, 68 ℃ for 30 sec, 72 ℃ for 25 sec, for a final incubation at 72 ℃ for 5 min. Then, primers hGH-F and MxeL6KD-R were added, and the reaction was incubated at 98 °C for 30 sec, 98 °C for 10 sec, 68 °C for 30 sec, and 72 °C for 25 sec for a total of 30 cycles; finally, the reaction was incubated at 72 °C for 5 min. After the reaction, the PCR amplification products were detected by electrophoresis, and the results showed that the PCR amplification produced the correct bands as expected, which were then separated and recovered. The overlapped PCR recovered products were treated with restriction endonucleases. Nde I and Xho After double digestion with enzyme I, the plasmid pET30(a) was ligated with T4 ligase. The ligation product was transformed into E. coli DH5α competent cells. The transformed cells were plated on LB plates supplemented with 50 μg / mL kanamycin to screen for positive clones. The plasmid was extracted and sequenced. The sequencing results showed that the cloned pET30-hGH-Mxe-L6KD sequence was correct.

[0136] The correctly sequenced plasmid was then transformed into *E. coli* BL21(DE3)(Novagen) competent cells. Transformed cells were plated on LB agar plates supplemented with 50 μg / mL kanamycin to screen for positive clones for subsequent expression and purification. The pET30-hGH-Mxe-EFK8 and pET30-hGH-Mxe-α3 plasmids and their expression strains were obtained using a similar method. For the construction of pET30-hGH-Mxe-EFK8, primer Mxe-EFK-R was used instead of Mxe-L6KD-R for cloning. For the construction of pET30-hGH-Mxe-α3, the hGH-Mxe nucleotide fragment was obtained from pET30-hGH-Mxe-L6KD using primers hGH-F and hGHalpha-R, and then inserted into the pET30-hGH-Mxe-α3 plasmid. Nde I and Spe The pET30-lipA-Mxe-α3 plasmid vector was double-digested with restriction endonucleases (Lin Zhanglin et al., 2018). The structures of the constructed pET30-hGH-Mxe-L6KD, pET30-hGH-Mxe-EFK8, and pET30-hGH-Mxe-α3 plasmids are shown below. Figure 1 As shown in B.

[0137] Example 2: Expression and purification of human growth hormone fusion protein

[0138] The strains constructed in Example 1 (containing plasmids pET30-hGH-Mxe-L6KD, pET30-hGH-Mxe-EFK8, and pET30-hGH-Mxe-α3) were inoculated into LB liquid medium containing 50 μg / mL kanamycin and cultured in a shaker at 37 °C until the logarithmic growth phase (OD50). 600 =0.4-0.6), add 0.2 mM IPTG, and induce at 18 ℃ for 18 hours and 30 ℃ for 6 hours. Harvest cells and measure bacterial concentration (OD). 600 (The following will use 1 mL of OD) 60 The number of cells with a value of 0 to 1 is called 1 OD.

[0139] The bacterial cells were resuspended in lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, and 0.37 g Na₂EDTA·2H₂O dissolved in 800 mL of water, pH adjusted to 8.5, and then diluted to 1 L with water) to a concentration of 20 OD / mL, and then sonicated (lysis conditions: 200 W power, 3 sec sonication time, 3 sec interval, 99 sonications). The cells were centrifuged at 12000 rpm for 20 min at 4 °C, and the supernatant and precipitate were collected separately. The precipitate was washed twice with lysis buffer and then thoroughly resuspended in cleavage buffer (20 mM Tris-HCl, 500 mM NaCl, 40 mM dithiothreitol, 1 mM EDTA, pH 8.5) and incubated at 4 °C overnight for 24 h to allow for complete self-cleavage of the integrins. The suspension was then centrifuged, and the supernatant and precipitate were analyzed by SDS-PAGE along with the uncut precipitate (the precipitate was resuspended in the same volume of lysis buffer as in the previous resuspension step). Results are as follows: Figure 2 As shown. Lanes a and d contain human growth hormone (hGH) expression and purification samples, respectively: a: cell lysate supernatant; b: cell lysate precipitate, showing clear aggregates of fusion protein expression; c: precipitate separated after cleavage; d: supernatant separated after cleavage, showing clear human growth hormone (hGH) bands. Lanes 1-4 contain protein quantification standards containing bovine serum albumin (BSA), with loading amounts of 4 μg, 2 μg, 1 μg, and 0.5 μg, respectively.

[0140] According to the protein quantification standards, the optical density of the target band was analyzed using Bio-Rad's Quantity ONE gel quantification analysis software. The aggregate yield of the fusion protein, the human growth hormone (hGH) yield released into the supernatant after peptide-mediated self-cleavage, the Mxe GyrA cleavage efficiency, the human growth hormone (hGH) recovery rate, and its purity in the supernatant were calculated. The results are shown in Table 2.

[0141] Table 2. Expression and purification of human growth hormone (hGH)

[0142]

[0143] a Protein aggregate production b Including peptide-mediated autocleavage of human growth hormone (hGH) production (in bacterial concentration OD) 600 When the value is 2, each liter of LB medium contains 2.66 mg of E. coli cells (calculated based on wet weight). c Inteptide-mediated autocleavage efficiency = 100% × (pre-cleavage aggregate expression level - post-cleavage aggregate remaining amount) / pre-cleavage aggregate yield.d Recovery rate = 100% × actual hGH yield / theoretical yield of human growth hormone (hGH) that can be produced by protein aggregates under complete cleavage.

[0144] The three fusion proteins used (hGH-Mxe-L6KD, hGH-Mxe-EFK8, and hGH-Mxe-α3) existed in precipitated form, with aggregate expression levels ranging from 44.9 to 150.0 μg / mg cell wet weight. The three fusion proteins were autocleaved by the intima-peptide MxeGyrA, separating hGH from the Mxe-L6KD / EFK8 / α3-peptide. The cleavage efficiency was 52.8%–64.2%, and the yield of human growth hormone (hGH) released into the supernatant after cleavage was 2.8–21.4 μg / mg cell wet weight. The purity of the recovered hGH after cleavage was 31.4%–88.2%. Among them, the hGH-Mxe-L6KD fusion protein had the highest yield and purity of human growth hormone (hGH). Specifically, the human growth hormone (hGH) yield was 21.4 μg / mg cell wet weight and the purity was 88.2% obtained by one-step purification using this purification technology based on self-aggregating peptides and self-cleaving tags.

[0145] Example 3: Determination of the molecular weight of human growth hormone (hGH)

[0146] Taking the human growth hormone (hGH) sample obtained from L6KD self-aggregating peptides in Experimental Example 2 as an example, molecular weight determination was performed. The human growth hormone (hGH) sample was dialyzed with the mobile phase (solution A:solution B = 1:1) to prepare a 2 mg / mL hGH sample, which was then analyzed by HPLC-MS. Instruments: Agilent 1260 HPLC connected to a Waters SYNAPT G2-S time-of-flight mass spectrometry system; Column: Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm particlesize, 130 Å, Waters, USA); Mobile phase: Solution A was an aqueous solution of 0.1% (v / v) formic acid, and Solution B was an acetonitrile solution of 0.1% (v / v) formic acid, with the gradient shown in Table 2; Injection volume: 10 μL; Flow rate: 0.4 mL / min; Temperature: 60 ℃.

[0147] Table 3. Setting parameters for mobile phase gradient change

[0148]

[0149] from Figure 3 The obtained molecular weight is 22678.0 Daltons, which is basically consistent with the calculated molecular weight of 22678.8 Daltons. The difference of 0.8 Daltons is within the range of machine measurement error, proving that the obtained hGH sequence is correct.

[0150] Example 4: Bioactivity assay of human growth hormone

[0151] Taking the human growth hormone (hGH) sample obtained from the L6KD self-aggregating peptide in Experiment 2 as an example, bioactivity was tested. The NB2-11 cell line (European Center for Cell Lines / Microbial Collections (ECACC)) was used as the standard human growth hormone proliferation test cell line. Well-grown NB2-11 cells were digested with trypsin and counted. Cells were resuspended in serum-free medium to prepare a cell suspension, and 5000 cells were seeded into each well of a 96-well cell culture plate for 24 hours of serum starvation. Each sample was diluted to the set concentration and added to the corresponding cell culture wells, and cultured in an incubator for 24 hours. Proliferation was detected using the CCK8 assay kit (Shanghai Beyotime Biotechnology Co., Ltd.). 20 μL of CCK8 solution was added to each well; the culture plate was incubated in an incubator for 2 hours; and the absorbance at 450 nm was measured using a microplate reader. The samples tested included bovine serum albumin (BSA), human growth hormone (hGH) obtained from L6KD self-aggregating peptides in Experiment 2, and commercial human growth hormone (hGH) (proteintech, USA), with sample concentrations of 1, 5, 10, 20, 30, 40, and 50 ng / mL.

[0152] like Figure 4 As shown, the human growth hormone (hGH) purified by this method effectively promoted the proliferation of NB2-11 cells, with the effect increasing with increasing concentration from 1 to 50 ng / mL, a trend consistent with that of commercial hGH samples. Under the condition of adding 50 ng / mL hGH, the proliferative activity of the human growth hormone (hGH) purified by this method on NB2-11 cells was 88.5% that of the commercial hGH sample. Considering that the purity of the tested hGH sample was 88.2%, the bioactivity of the obtained human growth hormone (hGH) sample is comparable to that of commercial human growth hormone (hGH).

[0153] Example 5: Construction of a human growth hormone fusion protein expression vector containing the intima-peptide Mtu ΔI-CM

[0154] The construction processes of the expression vectors pET32-L6KD-Mtu ΔI-CM-hGH, pET32-L6KD-Mtu ΔI-CM mutant strain 1-hGH, pET32-L6KD-Mtu ΔI-CM mutant strain 2-hGH, pET32-L6KD-Mtu ΔI-CM mutant strain 3-hGH, pET32-ELK16-Mtu ΔI-CM mutant strain 2-hGH, pET32-EFK8-Mtu ΔI-CM mutant strain 2-hGH, pET32-α3-Mtu ΔI-CM mutant strain 2-hGH, and pET32-TZ1H-Mtu ΔI-CM mutant strain 2-hGH used in this application embodiment are similar. The following uses the construction of pET32-L6KD-Mtu ΔI-CM-hGH as an example. The required primers are obtained through oligo... 6. Oligonucleotide primers as shown in Table 1 were designed and synthesized by Shanghai Sangon Biotech.

[0155] Table 4. Oligonucleotide primers used in this example.

[0156]

[0157] The growth hormone hGH polynucleotide fragment was amplified by PCR using primers J19040-hGH-F and J19041-hGH-R (PCR instrument (Bio-rad / C1000Touch)). The PCR reaction was performed using NEB Q5 polymerase (New England Biolab (NEB)). The PCR conditions were: 98 ℃ for 30 sec, 98 ℃ for 10 sec, 60 ℃ for 30 sec, and 72 ℃ for 30 sec, for a total of 30 cycles; the final incubation was at 72 ℃ for 2 min. After the reaction, the PCR amplification products were subjected to 1% agarose gel electrophoresis and then recovered using an ultrathin DNA gel product recovery kit (Magen, D2110-03).

[0158] The L6KD-Mtu ΔI-CM nucleotide fragment was amplified from pET30a-L6KD-Mtu ΔI-CM-AMA (Zhou B. et al., 2012) using primers J20001-Mtu-F and J19042-Mtu-R via PCR. The PCR reaction was performed using NEB Q5 polymerase (New England Biolab (NEB)) under the following conditions: 98 ℃ for 30 sec, 98 ℃ for 10 sec, 72 ℃ for 30 sec, 72 ℃ for 1 min, for a total of 30 cycles; the final cycle was 72 ℃ for 2 min. After the reaction, the PCR amplification product was separated and recovered by 1% agarose gel electrophoresis.

[0159] Overlap PCR was performed on the growth hormone (hGH) polynucleotide fragment and the L6KD-Mtu ΔI-CM nucleotide fragment. The PCR reaction used NEB Q5 polymerase, and the PCR conditions were: 98 °C for 30 sec, 98 °C for 10 sec, 72 °C for 30 sec, 72 °C for 2 min, for a total of 30 cycles; the final cycle was 72 °C for 2 min. The amplified fragments were subjected to 1% agarose gel electrophoresis and then recovered using an ultrathin DNA gel product recovery kit (Magen, D2110-03). The purified fragments and pET32a plasmid (Novagen) were processed using restriction endonucleases. Eco RI and Xho The DNA fragments were double-digested with enzyme I, then purified. After purification, they were ligated with T4 DNA ligase, and the ligation product was transformed into E. coli DH5α competent cells. The transformed cells were plated on LB plates supplemented with 100 μg / mL carbenicillin to screen for positive clones. Plasmids were extracted using a plasmid extraction kit and sequenced.

[0160] The correctly sequenced plasmid was then transformed into E. coli BL21(DE3)(Novagen) competent cells. The transformed cells were plated on LB plates supplemented with 100 μg / mL carbenicillin to screen for positive clones for subsequent expression and purification.

[0161] Using a similar method, plasmids and their expression strains of the following mutant strains were obtained: pET32-L6KD-Mtu ΔI-CM 1-hGH, pET32-L6KD-Mtu ΔI-CM 2-hGH, pET32-L6KD-Mtu ΔI-CM 3-hGH, pET32-ELK16-Mtu ΔI-CM 2-hGH, pET32-EFK8-Mtu ΔI-CM 2-hGH, pET32-α3-Mtu ΔI-CM 2-hGH, and pET32-TZ1H-Mtu ΔI-CM 2-hGH. The structure of the constructed pET32-L6KD-Mtu ΔI-CM-hGH plasmid is shown below. Figure 5 As shown in B.

[0162] Example 6: Expression and purification of human growth hormone fusion protein in LB medium

[0163] The strains constructed in Example 5 (containing the plasmids described above) were inoculated into LB liquid medium containing 100 μg / mL carbenicillin and cultured in a shaker at 37 °C until the logarithmic growth phase (OD600 = 0.4-0.6). IPTG was added to a final concentration of 0.2 mM, and the cells were induced at 18 °C for 24 hours. Cells were then harvested, and the bacterial concentration (OD600) was measured. Hereinafter, 1 mL of cells with an OD600 of 1 is referred to as 1 OD.

[0164] The bacterial cells were resuspended in lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, and 0.37 g Na₂EDTA·2H₂O dissolved in 800 mL of water, pH adjusted to 8.5, and then diluted to 1 L with water) to a concentration of 20 OD / mL, and then sonicated (lysis conditions: 200 W power, 3 sec sonication time, 3 sec interval, 99 sonications). The cells were centrifuged at 15000 g for 20 min at 4 °C, and the supernatant and precipitate were collected separately. The precipitate was washed twice with an equal volume of lysis buffer, and then resuspended thoroughly in an equal volume of cleavage buffer (PBS supplemented with 40 mM Bis-Tris, pH 6.2, 2 mM EDTA) and incubated at 25 °C for 24 h to allow for complete autocleavage of the integrins. The sample was then centrifuged at 4 °C and 15000 g for 20 min. The precipitate was resuspended in an equal volume of lysis buffer. The resulting supernatant and precipitate were then analyzed by SDS-PAGE along with the supernatant and precipitate before cleavage. Results are as follows: Figure 6 As shown in Figure A, lanes ES, EP, CP, and CS represent human growth hormone (hGH) expression and purification samples. ES: cell lysate supernatant; EP: cell lysate precipitate, showing clearly detectable aggregates of the fusion protein; CP: precipitate separated after cleavage; CS: supernatant separated after cleavage, showing a clear hGH band. Lanes 1-5 represent MtuΔI-CM (without 18℃ cooling), Mtu ΔI-CM (after 18℃ cooling), Mtu ΔI-CM mutant strain 1, Mtu ΔI-CM mutant strain 2, and Mtu ΔI-CM mutant strain 3, respectively. Lanes I-IV contain bovine serum albumin (BSA) protein quantification standards, with loading amounts of 2.5 μg, 1.25 μg, 0.625 μg, and 0.3125 μg, respectively. SDS-PAGE analysis results for different aggregated peptides are shown below. Figure 6 As shown in C, the supernatant separated after lane CS was cut showed clear human growth hormone (hGH) bands. Lanes 1-5 were L6KD, ELK16, EFK8, α3, and TZ1H.

[0165] According to the protein quantification standards, the optical density of the target band was analyzed using ImageJ gel quantification software. The aggregate yield of the fusion protein, the human growth hormone (hGH) yield released into the supernatant after peptide-mediated self-cleavage, the Mtu ΔI-CM cleavage efficiency, the human growth hormone (hGH) recovery rate and its purity in the supernatant were calculated. The results are shown in Table 5.

[0166] Table 5. Expression and purification of human growth hormone (hGH)

[0167]

[0168] a Protein aggregate production b Intended peptide-mediated autocleavage of human growth hormone (hGH) production (calculated as the amount of protein produced by E. coli cells per liter of LB medium). c Inteptide-mediated autocleavage efficiency = 100% × (pre-cleavage aggregate expression level - post-cleavage aggregate remaining amount) / pre-cleavage aggregate yield. d Recovery rate = 100% × actual hGH yield / theoretical yield of human growth hormone (hGH) that can be produced by protein aggregates under complete cleavage.

[0169] The four different Mtu ΔI-CM mutant fusion proteins (L6KD-Mtu-hGH, L6KD-Mtu(1)-hGH, L6KD-Mtu(2)-hGH, L6KD-Mtu(3)-hGH) and four different aggregate peptide fusion proteins ELK16-Mtu ΔI-CM mutant 2-hGH, EFK8-Mtu ΔI-CM mutant 2-hGH, α3-Mtu ΔI-CM mutant 2-hGH, and TZ1H-Mtu ΔI-CM mutant 2-hGH were all present in precipitate form. The expression levels of the aggregates of the four different Mtu ΔI-CM mutant 2 (Mtu(2)) were 446~536 mg / L LB medium. Four different Mtu ΔI-CM mutant fusion proteins were autocleaved by the inner peptide Mtu ΔI-CM, separating hGH from L6KD-Mtu. The cleavage efficiency ranged from 31% to 72%, and the yield of human growth hormone (hGH) released into the supernatant after cleavage was 8–72 mg / L LB culture medium. The purity of the recovered hGH was 49–82%. Among them, the L6KD-Mtu-hGH fusion protein had the highest yield and purity of hGH. Specifically, after cooling at 18 °C, the hGH yield was 72 mg / L LB culture medium wet weight with a purity of 82% after one-step purification using this purification technique based on self-aggregating peptides and self-cleavage tags. The expression levels of four different aggregated peptides ranged from 4 to 303 mg / L LB culture medium. The four different aggregated peptides were autocleaved by the inteptide Mtu ΔI-CM, and hGH was separated from L6KD-Mtu. The cleavage efficiency was 22% to 46%. The yield of human growth hormone (hGH) released into the supernatant after cleavage was 1% to 33 mg / L LB culture medium, and the purity of the recovered hGH after cleavage was 17% to 98%.

[0170] Example 7: Expression and purification of human growth hormone fusion protein using fermentation medium

[0171] The strain constructed in Example 5 was inoculated into fermentation medium containing 100 μg / mL carbenicillin (Shao-Yang Hu et al., 2004) and cultured in a shaker at 37 °C until the logarithmic growth phase (OD600 = 0.4-0.6). A final concentration of 0.2 mMIPTG was added, and the cells were induced at 18 °C for 24 hours. Cells were then harvested, and the bacterial concentration (OD600) was measured. Hereinafter, 1 mL of cells with an OD600 of 1 is referred to as 1 OD. The components of the fermentation medium used are shown in Table 6.

[0172] Table 6 Fermentation medium components

[0173]

[0174] Glucose was sterilized separately from other components at 121 °C for 20 min. The trace element solution was sterilized by filtration through a 0.22 μm filter in a laminar flow hood. Carbenicillin was added to a final concentration of 100 mg / L before use of the prepared culture medium.

[0175] The bacterial cells were resuspended in lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, and 0.37 g Na₂EDTA·2H₂O dissolved in 800 mL of water, pH adjusted to 8.5, and then diluted to 1 L with water) to a concentration of 20 OD / mL, and then sonicated (lysis conditions: 200 W power, 3 sec sonication time, 3 sec interval, 99 sonications). The cells were centrifuged at 15000 g for 20 min at 4 °C, and the supernatant and precipitate were collected separately. The precipitate was washed twice with an equal volume of lysis buffer, and then resuspended thoroughly in an equal volume of cleavage buffer (PBS supplemented with 40 mM Bis-Tris, pH 6.2, 2 mM EDTA) and incubated at 25 °C for 24 h to allow for complete autocleavage of the integrins. The sample was then centrifuged at 4 °C and 15000 g for 20 min. The precipitate was resuspended in an equal volume of lysis buffer. The resulting supernatant and precipitate were then analyzed by SDS-PAGE along with the supernatant and precipitate before cleavage. Results are as follows: Figure 6 As shown in B. Lanes ES, EP, CP, and CS contain human growth hormone (hGH) expression and purification samples, respectively: ES: cell lysate supernatant; EP: cell lysate precipitate, showing clearly detectable aggregates of fusion protein expression; CP: precipitate separated after cleavage; CS: supernatant separated after cleavage, showing clearly detectable human growth hormone (hGH) bands; Lanes 1-5 contain Mtu ΔI-CM (without 18℃ cooling), Mtu ΔI-CM (after 18℃ cooling), Mtu ΔI-CM mutant strain 1, Mtu ΔI-CM mutant strain 2, and Mtu ΔI-CM mutant strain 3. Lanes I-IV contain protein quantification standards containing bovine serum albumin (BSA), with loading amounts of 2.5 μg, 1.25 μg, 0.625 μg, and 0.3125 μg, respectively.

[0176] According to the protein quantification standards, the optical density of the target band was analyzed using ImageJ gel quantification software. The aggregate yield of the fusion protein, the human growth hormone (hGH) yield released into the supernatant after peptide-mediated self-cleavage, the Mtu ΔI-CM cleavage efficiency, the recovery rate of human growth hormone (hGH) and its purity in the supernatant were calculated. The results are shown in Table 7.

[0177] Table 7. Expression and purification of human growth hormone (hGH) in fermentation medium.

[0178]

[0179] a Protein aggregate production b The yield of human growth hormone (hGH) mediated by peptide-mediated autocleavage (calculated as the amount of protein produced by E. coli cells per liter of fermentation medium). c Inteptide-mediated autocleavage efficiency = 100% × (pre-cleavage aggregate expression level - post-cleavage aggregate remaining amount) / pre-cleavage aggregate yield. d Recovery rate = 100% × actual hGH yield / theoretical yield of human growth hormone (hGH) that can be produced by protein aggregates under complete cleavage.

[0180] The four different Mtu ΔI-CM fusion proteins (L6KD-Mtu-hGH, L6KD-Mtu(1)-hGH, L6KD-Mtu(2)-hGH, and L6KD-Mtu(3)-hGH) used were all present in precipitate form, and the expression levels of the four different Mtu ΔI-CM aggregates were 1696~2983 mg / L of fermentation broth. After the four different Mtu ΔI-CM fusion proteins were self-cleaved by the intrinating peptide Mtu ΔI-CM, hGH was separated from L6KD-Mtu, with a cleavage efficiency of 29~63%. The yield of human growth hormone hGH released into the supernatant after cleavage was 69~362 mg / L of fermentation broth, and the purity of the recovered hGH after cleavage was 56~88%.

[0181] Example 8: Purification of Human Growth Hormone Fusion Protein

[0182] Taking the human growth hormone (hGH) sample obtained from L6KD self-aggregating peptides in Example 6 as an example, approximately 12 mg of the hGH sample was finely purified using an anion exchange column (Capto HiRes Q 5 / 50) and a molecular sieve column (Sephacryl S200HR (16 / 60)). During ion exchange column purification, after loading the sample, unbound protein was washed away with binding buffer (20 mM Tris-HCl, pH 8.0), followed by linear elution with 20 CV and 50% Elution buffer (20 mM Tris-HCl, 1.0 M NaCl, pH 8.0), collecting the peak eluted with approximately 34% Elution buffer. The protein purified by the ion exchange column was further purified using a molecular sieve column, eluted with buffer (20 mM NaCl, 20 mM Tris-HCl, pH 7.5) for 120 CV, collecting the peak after approximately 90 min. The collected elution peaks were detected by SDS-PAGE, and the results are shown below. Figure 7As shown in the diagram. Lane 1 contains hGH purified by cSAT; lane 2 contains hGH purified by ion exchange column; and lane 3 contains hGH purified by molecular sieve. Recombinant human growth hormone (hGH) protein with a purity greater than 99% can be obtained through two-step purification using ion exchange column and molecular sieve.

[0183] Example 9: RP-HPLC determination of human growth hormone (hGH)

[0184] Taking the human growth hormone (hGH) sample purified by ion exchange column and molecular sieve in Example 8 as an example, RP-HPLC determination was performed. The standard and the purified human growth hormone (hGH) sample were prepared into a 0.1 mg / mL hGH sample using sterile water, and analyzed by RP-HPLC. The results are as follows. Figure 8 As shown in Table 8. Instrument: Agilent 1260; Column: YMC-Pack ODS-A; Mobile phase: Solution A was 0.1% (v / v) trifluoroacetic acid in acetonitrile, Solution B was 0.1% (v / v) 0.1% (v / v) trifluoroacetic acid in aqueous solution, and the gradient used was as shown in Table 8; Injection volume was 99 μL, flow rate was 1 mL / min, and temperature was 30 ℃.

[0185] Table 8. Parameters for setting the gradient change of the mobile phase

[0186]

[0187] Example 10: Determination of the molecular weight of human growth hormone (hGH)

[0188] Taking the human growth hormone (hGH) sample obtained from L6KD self-aggregating peptides in Experiment 6 as an example, molecular weight determination was performed. The human growth hormone (hGH) sample was dialyzed with the mobile phase (solution A:solution B = 1:1) to prepare a 2 mg / mL hGH sample, which was then analyzed by HPLC-MS. Instruments: Agilent 1260 HPLC connected to a Waters SYNAPT G2-S time-of-flight mass spectrometry system; Column: Acquity UPLC BEH C18 column (2.1 mm × 100 mm, 1.7 μm particlesize, 130 Å, Waters, USA); Mobile phase: Solution A was an aqueous solution of 0.1% (v / v) formic acid, and Solution B was an acetonitrile solution of 0.1% (v / v) formic acid, with the gradient shown in Table 9; Injection volume: 10 μL; Flow rate: 0.4 mL / min; Temperature: 60 ℃.

[0189] Table 9. Setting parameters for mobile phase gradient change

[0190]

[0191] from Figure 9 The obtained molecular weight is 22,123.8 Daltons, which is consistent with the molecular weight of 22,123.8 Daltons determined by the medical hGH standard (Jintropin), proving that the obtained hGH sequence is correct.

[0192] Example 11: Native-PAGE assay of human growth hormone (hGH)

[0193] Taking the human growth hormone (hGH) sample purified by ion exchange column and molecular sieve in Example 8 as an example, secondary structure determination was performed. Standard and purified human growth hormone (hGH) sample were prepared into a 0.1 mg / mL hGH sample using sterile water for electrophoresis. The entire electrophoresis process was performed on ice at 80V. Coomassie brilliant blue staining results are shown below. Figure 10 As shown. From Figure 10 It can be seen that the hGH obtained by cSAT purification has a structure that is basically consistent with the medical hGH standard.

[0194] Example 12: Determination of the secondary structure of human growth hormone (hGH)

[0195] Taking the human growth hormone (hGH) sample purified by ion exchange column and molecular sieve in Example 8 as an example, secondary structure determination was performed. Standard and purified human growth hormone (hGH) sample were prepared into a 0.1 mg / mL hGH sample using sterile water. The secondary structure of the hGH sample was determined using far-ultraviolet circular dichroism (CBD). Instrument: Chirascan™ circular dichroism spectrometer. Before protein sample detection, 200 μL of distilled water was added to the sample cell, and CBD was performed in the far-ultraviolet region (190 nm-260 nm). The obtained chromatographic signal was subtracted as background signal. The scanning parameters used are shown in Table 10.

[0196] Table 10 Circular Dichroism Analysis Scan Settings

[0197]

[0198] from Figure 11 It can be seen that the obtained secondary structure analysis spectrum is basically consistent with the spectrum of medical hGH standard, proving that the obtained hGH secondary structure is correct.

[0199] Example 13: Construction of human interferon α2a fusion protein expression vector

[0200] The construction processes of the expression vectors pET32-L6KD-Mtu ΔI-CM-IFNα2a, pET32-L6KD-Mtu ΔI-CM mutant 1-IFNα2a, pET32-L6KD-Mtu ΔI-CM mutant 2-IFNα2a, and pET32-L6KD-Mtu ΔI-CM mutant 3-IFNα2a used in the embodiments of this application are as follows. Taking the construction of pET32-L6KD-Mtu ΔI-CM-IFNα2a as an example, the required primers were designed using oligo 6 and synthesized by Shanghai Sangon Biotech as shown in Table 11.

[0201] Table 11 Oligonucleotide primers used in this embodiment

[0202]

[0203] First, using pET32-L6KD-Mtu ΔI-CM-hGH as a template and J20016-PT-F and J20017-Mtu-R as primers, the L6KD-Mtu ΔI-CM polynucleotide fragment was amplified by PCR. The PCR reaction used NEB's Q5 polymerase, and the PCR conditions were: 98 ℃ for 30 sec, 98 ℃ for 10 sec, 72 ℃ for 30 sec, 72 ℃ for 1 min, for a total of 30 cycles; and a final incubation at 72 ℃ for 2 min. After the reaction, the PCR amplification product was separated and recovered by 1% agarose gel electrophoresis. The human interferon α2a (NCBI number: NM_000605.4) polynucleotide sequence was obtained from NCBI, and codon optimization and synthesis were performed by Shanghai Sangon Biotech. Using the synthesized gene as a template and J20018-IFN-F and J20019-IFN-R as primers, the human interferon α2a polynucleotide fragment was amplified by PCR. The PCR reaction was performed using Q5 polymerase from New England Biolab (NEB). The PCR conditions were: 98 ℃ for 30 sec, 98 ℃ for 10 sec, 72 ℃ for 30 sec, 72 ℃ for 1 min, for a total of 30 cycles; the final temperature was 72 ℃ for 2 min. After the reaction, the PCR amplification products were separated and recovered by 1% agarose gel electrophoresis.

[0204] Overlap PCR reactions were performed on IFNα2a and L6KD-Mtu ΔI-CM fragments using primers J20016-PT-F and J20019-IFN-R: 30 cycles of 98 ℃ for 30 sec, 98 ℃ for 10 sec, 72 ℃ for 1 min, and 72 ℃ for 2 min; the final cycle was 72 ℃ for 2 min. After the reaction, the PCR amplification products were detected by electrophoresis, and the PCR amplification showed the correct bands as expected, which were then recovered by gel extraction.

[0205] Using pET32-L6KD-Mtu ΔI-CM-hGH as a template and J20020-Term-F and J20003-Ori-R as primers, the f1ori-AmpR-ori polynucleotide fragment was amplified by PCR. The rop-lacI-T7 promoter-RBS polynucleotide fragment was amplified by PCR using J20004-Bom-F and J20015-RBS-R as primers. PCR was performed using NEB Q5 polymerase under the following conditions: 98 ℃ for 30 sec, 98 ℃ for 10 sec, 72 ℃ for 1 sec, 72 ℃ for 3 min, for a total of 30 cycles; the final temperature was 72 ℃ for 4 min. After the reaction, the PCR products were separated and recovered by 1% agarose gel electrophoresis.

[0206] The recovered product from the overlap PCR and the two amplified polynucleotide fragments were assembled using Gibson at 50 °C for 1 h. The ligation product was then transformed into E. coli DH5α competent cells. The transformed cells were plated on LB plates supplemented with 100 μg / mL carbenicillin to screen for positive clones. The plasmid was extracted and sequenced. The sequencing results showed that the constructed pET32-L6KD-Mtu ΔI-CM-IFNα2a plasmid was correct.

[0207] The correctly sequenced plasmid was then transformed into *E. coli* BL21(DE3)(Novagen) competent cells. The transformed cells were plated on LB agar plates supplemented with 100 μg / mL carbenicillin to screen for positive clones for subsequent expression and purification. Using a similar method, plasmids and their expression strains for the pET32-L6KD-Mtu ΔI-CM mutant strains 1-IFNα2a, 2-IFNα2a, and 3-IFNα2a were obtained. The structure of the constructed pET32-L6KD-Mtu ΔI-CM-IFNα2a plasmid is shown below. Figure 5 As shown in B.

[0208] Example 14: Expression and purification of human interferon α2a fusion protein in LB liquid medium

[0209] The strains constructed in Example 13 (containing plasmids pET32-L6KD-Mtu ΔI-CM-IFNα2a, pET32-L6KD-Mtu ΔI-CM mutant 1-IFNα2a, pET32-L6KD-Mtu ΔI-CM mutant 2-IFNα2a, and pET32-L6KD-Mtu ΔI-CM mutant 3-IFNα2a) were inoculated into LB liquid medium containing 100 μg / mL carbenicillin and cultured in a shaker at 37°C until the logarithmic growth phase (OD600 = 0.4-0.6). IPTG was added to a final concentration of 0.2 mM, and the cells were induced at 18°C ​​for 24 hours. Cells were then harvested, and the bacterial concentration (OD600) was measured. (Hereinafter, the amount of cells with an OD600 of 1 mL is referred to as 1 OD).

[0210] The bacterial cells were resuspended in lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, and 0.37 g Na₂EDTA·2H₂O dissolved in 800 mL of water, pH adjusted to 8.5, and then diluted to 1 L with water) to a concentration of 20 OD / mL, and then sonicated (lysis conditions: 200 W power, 3 sec sonication time, 3 sec interval, 99 sonications). The cells were centrifuged at 15000 g for 20 min at 4 °C, and the supernatant and precipitate were collected separately. The precipitate was washed twice with an equal volume of lysis buffer, and then resuspended thoroughly in an equal volume of cleavage buffer (PBS supplemented with 40 mM Bis-Tris, pH 6.2, 2 mM EDTA) and incubated at 25 °C for 24 h to allow for complete cleavage of the integrins. The sample was then centrifuged at 4 °C and 15000 g for 20 min. The precipitate was resuspended in an equal volume of lysis buffer. The resulting supernatant and precipitate were then analyzed by SDS-PAGE along with the supernatant and precipitate before cleavage. Results are as follows: Figure 12 As shown in A-12B, lanes ES, EP, CP, and CS contain human interferon α2a expression and purification samples. ES: cell lysate supernatant; EP: cell lysate precipitate, showing clearly detectable aggregates of the fusion protein; CP: precipitate separated after cleavage; CS: supernatant separated after cleavage, showing a clear human interferon α2a band. Lanes I-IV contain protein quantification standards containing bovine serum albumin (BSA), with loading amounts of 2.5 μg, 1.25 μg, 0.625 μg, and 0.3125 μg, respectively.

[0211] According to the protein quantification standards, the optical density of the target band was analyzed using ImageJ gel quantification software. The aggregate yield of the fusion protein, the yield of human interferon α2a released into the supernatant after peptide-mediated self-cleavage, the Mtu ΔI-CM cleavage efficiency, the recovery rate of human interferon α2a and its purity in the supernatant were calculated. The results are shown in Table 9.

[0212] Table 12 Expression and purification of human interferon α2a

[0213]

[0214] a Protein aggregate production b The yield of human interferon α2a mediated by peptide self-cleavage (calculated as the amount of protein produced by E. coli cells per liter of LB medium). c Inteptide-mediated autocleavage efficiency = 100% × (pre-cleavage aggregate expression level - post-cleavage aggregate remaining amount) / pre-cleavage aggregate yield. d Recovery rate = 100% × Actual yield of IFN α2a / Theoretical yield of human interferon α2a that can be produced by protein aggregates under complete cleavage.

[0215] The four fusion proteins used (L6KD-Mtu-IFNα2a, L6KD-Mtu(1)-IFNα2a, L6KD-Mtu(2)-IFNα2a, and L6KD-Mtu(3)-IFNα2a) all existed in precipitate form, with aggregate expression levels of 446–536 mg / L LB culture medium. The four fusion proteins were autocleaved by the intrinating peptide Mtu ΔI-CM, separating IFNα2a from L6KD-Mtu. The cleavage efficiency was 31–72%, and the yield of human interferon α2a released into the supernatant after cleavage was 3–25 mg / L LB culture medium. The purity of the recovered IFNα2a after cleavage was 25–68%. Among them, the L6KD-Mtu(3)-IFNα2a fusion protein had the highest IFNα2a yield and purity. That is, the human interferon α2a yield was 25 mg / L LB culture medium wet weight and the purity was 68% by one-step purification technology based on self-aggregating peptide and self-cleaving tag.

[0216] Example 15: Expression and purification of human interferon IFNα2a fusion protein using fermentation medium

[0217] The strain constructed in Example 12 was inoculated into fermentation medium containing 100 μg / mL carbenicillin and cultured in a shaker at 37 °C until the logarithmic growth phase (OD600 = 0.4-0.6). IPTG was added to a final concentration of 0.2 mM, and the cells were induced at 18 °C for 24 hours. Cells were then harvested, and the bacterial concentration (OD600) was measured. (Hereinafter, the amount of cells with an OD600 of 1 mL is referred to as 1 OD). The components of the fermentation medium used are shown in Table 3.

[0218] The bacterial cells were resuspended in lysis buffer B1 (2.4 g Tris, 29.22 g NaCl, and 0.37 g Na₂EDTA·2H₂O dissolved in 800 mL of water, pH adjusted to 8.5, and then diluted to 1 L with water) to a concentration of 20 OD / mL, and then sonicated (lysis conditions: 200 W power, 3 sec sonication time, 3 sec interval, 99 sonications). The cells were centrifuged at 15000 g for 20 min at 4 °C, and the supernatant and precipitate were collected separately. The precipitate was washed twice with an equal volume of lysis buffer, and then resuspended thoroughly in an equal volume of cleavage buffer (PBS supplemented with 40 mM Bis-Tris, pH 6.2, 2 mM EDTA) and incubated at 25 °C for 24 h to allow for complete cleavage of the integrins. The sample was then centrifuged at 4 °C and 15000 g for 20 min. The precipitate was resuspended in an equal volume of lysis buffer. The resulting supernatant and precipitate were then analyzed by SDS-PAGE along with the supernatant and precipitate before cleavage. Results are as follows: Figure 12 As shown in D. Lanes ES, EP, CP, and CS are for human interferon α2a expression and purification samples, respectively: ES: cell lysate supernatant; EP: cell lysate precipitate, with clearly detectable aggregates of fusion protein expression; CP: precipitate separated after cleavage; CS: supernatant separated after cleavage, with clearly detectable human interferon α2a bands; Lanes I-IV are protein quantification standards containing bovine serum albumin (BSA), with loading amounts of 2.5 μg, 1.25 μg, 0.625 μg, and 0.3125 μg, respectively.

[0219] According to the protein quantification standards, the optical density of the target band was analyzed using ImageJ gel quantification software. The aggregate yield of the fusion protein, the yield of human interferon α2a released into the supernatant after peptide-mediated self-cleavage, the Mtu ΔI-CM cleavage efficiency, the recovery rate of human interferon α2a and its purity in the supernatant were calculated. The results are shown in Table 13.

[0220] Table 13 Expression and purification of human interferon α2a in fermentation medium

[0221]

[0222] a Protein aggregate production b The yield of human interferon α2a mediated by peptide self-cleavage (calculated as the amount of protein produced by E. coli cells per liter of fermentation medium). c Inteptide-mediated autocleavage efficiency = 100% × (pre-cleavage aggregate expression level - post-cleavage aggregate remaining amount) / pre-cleavage aggregate yield. d Recovery rate = 100% × Actual yield of IFN α2a / Theoretical yield of human interferon α2a that can be produced by protein aggregates under complete cleavage.

[0223] The fusion protein L6KD-Mtu(2)-IFNα2a was present in precipitate form, with an aggregate expression level of 1098 mg / L fermentation broth. The fusion protein was self-cleaved by the inner peptide Mtu ΔI-CM, separating IFNα2a from L6KD-Mtu with a cleavage efficiency of 88%. The yield of human interferon α2a released into the supernatant after cleavage was 90 mg / L fermentation broth, and the purity of the recovered IFNα2a was 50%. In other words, this purification technique based on self-aggregating peptides and self-cleavage tags can purify human interferon α2a in one step to a yield of 90 mg / L fermentation broth wet weight with a purity of 50%.

[0224] References

[0225] [1] Levarski Z., et al., High-level expression and purification ofrecombinant human growth hormone produced in soluble form in Escherichia coli .Protein Expression and Purification. 2014. 100, 40–47.

[0226] [2] Zamani M., et al., Cloning, expression, and purification of asynthetic human growth hormone in Escherichia coli using response surfacemethodology. Molecular Biotechnology. 2015. 57, 241–250.

[0227] [3] Nguyen MT, et al., Prokaryotic soluble overexpression and purification of bioactive human growth hormone by fusion to thioredoxin, maltose binding protein, and protein disulfide isomerase. PLoS One. 2014. 9, e89038.

[0228] [4] Wang Kuqiang et al., A method for preparing an engineered bacterium that efficiently expresses growth hormone and its application, CN201811442625.8

[0229] [5] Sen GC, Lengyel P., The interferon system. A bird's eye view ofits biochemistry. Journal of Biological Chemistry. 1992. 267, 5017–5020.

[0230] [6] Heim MH, Moradpour D., Blum HEC, Expression of HepatitisC Virus Proteins Inhibits Signal Transduction through the Jak-STAT Pathway. Journal of Virology. 1999. 73, 8469-8475.

[0231] [7] Wu W., et al., Active protein aggregates induced by terminallyattached self-assembling peptide ELK16 in Escherichia coli. Microbial CellFactories, 2011. 10, 9

[0232] [8] Xing L., et al., Streamlined protein expression and purificationusing cleavable self-aggregating tags. Microbial Cell Factories, 2011. 10, 42

[0233] [9] Zhou B., et al., Small surfactant-like peptides can drive solubleproteins into active aggregates. Microbial Cell Factories, 2012. 11, 10

[0234]

[10] Lin Z., et al., α-helical self-assembling short peptides and their applications in protein purification, CN201811557416.8

[0235]

[11] Zhao Q., et al., Recombinant production of medium-to large-sizedpeptides in Escherichia coli using a cleavable self-aggregating tag. MicrobialCell Factories, 2016. 15:136

[0236]

[12] van Pouderoyen G., et al., The crystal structure of Bacillus subtilis lipase: a minimal α / β hydrolase fold enzyme. Journal of MolecularBiology, 2001. 309, 215-226

[0237]

[13] Collard F., et al., Crystal structure of the deglycating enzymefructosamine oxidase (Amadoriase II). The Journal of Biological Chemistry,2008. 283:40

[0238]

[14] Bray B.L., Large-scale manufacture of peptide therapeutics by chemical synthesis. Nature Reviews Drug Discovery, 2003, 2(7):587-593.

[0239]

[15] Leader B., et al. Protein therapeutics: a summary and pharmacological classification. Nature Reviews Drug Discovery, 2008, 7(1):21-39

[0240]

[16] Chen Hao, et al., Chinese Journal of Biotechnology, 2002, 22(5): p. 87-92.

[0241]

[17] Chang C., et al. High-level secretion of human growth hormone by Escherichia coli . Gene, 1987, 55(23): 189-196.

[0242]

[18] Mooney J.T., et al. Purification of a recombinant human growth hormone by an integrated IMAC procedure. Protein Expression and Purification, 2014: 85-94.

[0243]

[19] Wood D.W., et al. A genetic system yields self-cleaving inteins for bioseparations. Nature Biotechnology, 1999, 17: 889-892.

[0244]

[20] Lou S., et al. Peptide Tectonics: Encoded StructuralComplementarity Dictates Programmable Self-Assembly. Advanced Science, 2019,6:1802043. sequence list <110> South China University of Technology <120> Methods for the production and purification of peptides <130> P2020TC1338 <150> CN201911053169.2 <151> 2019-10-31 <160> 36 <170> PatentIn version 3.5 <210> 1 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> L6KD <400> 1 Leu Leu Leu Leu Leu Leu Lys Asp 1 5 <210> 2 <211> 8 <212> PRT <213> Artificial Sequence <220> <223> EFK8 <400> 2 Phe Glu Phe Glu Phe Lys Phe Lys 1 5 <210> 3 <211> twenty one <212> PRT <213> Artificial Sequence <220> <223> α3-peptide <400> 3 Leu Glu Thr Leu Ala Lys Ala Leu Glu Thr Leu Ala Lys Ala Leu Glu 1 5 10 15 Thr Leu Ala Lys Ala 20 <210> 4 <211> 198 <212> PRT <213> Artificial Sequence <220> <223> Mxe GyrA intein <400> 4 Cys Ile Thr Gly Asp Ala Leu Val Ala Leu Pro Glu Gly Glu Ser Val 1 5 10 15 Arg Ile Ala Asp Ile Val Pro Gly Ala Arg Pro Asn Ser Asp Asn Ala 20 25 30 Ile Asp Leu Lys Val Leu Asp Arg His Gly Asn Pro Val Leu Ala Asp 35 40 45 Arg Leu Phe His Ser Gly Glu His Pro Val Tyr Thr Val Arg Thr Val 50 55 60 Glu Gly Leu Arg Val Thr Gly Thr Ala Asn His Pro Leu Leu Cys Leu 65 70 75 80 Val Asp Val Ala Gly Val Pro Thr Leu Leu Trp Lys Leu Ile Asp Glu 85 90 95 Ile Lys Pro Gly Asp Tyr Ala Val Ile Gln Arg Ser Ala Phe Ser Val 100 105 110 Asp Cys Ala Gly Phe Ala Arg Gly Lys Pro Glu Phe Ala Pro Thr Thr 115 120 125 Tyr Thr Val Gly Val Pro Gly Leu Val Arg Phe Leu Glu Ala His His 130 135 140 Arg Asp Pro Asp Ala Gln Ala Ile Ala Asp Glu Leu Thr Asp Gly Arg 145 150 155 160 Phe Tyr Tyr Ala Lys Val Ala Ser Val Thr Asp Ala Gly Val Gln Pro 165 170 175 Val Tyr Ser Leu Arg Val Asp Thr Ala Asp His Ala Phe Ile Thr Asn 180 185 190 Gly Phe Val Ser His Ala 195 <210> 5 <211> 191 <212> PRT <213> Artificial Sequence <220> <223> hGH <400> 5 Phe Pro Thr Ile Pro Leu Ser Arg Leu Phe Asp Asn Ala Met Leu Arg 1 5 10 15 Ala His Arg Leu His Gln Leu Ala Phe Asp Thr Tyr Gln Glu Phe Glu 20 25 30 Glu Ala Tyr Ile Pro Lys Glu Gln Lys Tyr Ser Phe Leu Gln Asn Pro 35 40 45 Gln Thr Ser Leu Cys Phe Ser Glu Ser Ile Pro Thr Pro Ser Asn Arg 50 55 60 Glu Glu Thr Gln Gln Lys Ser Asn Leu Glu Leu Leu Arg Ile Ser Leu 65 70 75 80 Leu Leu Ile Gln Ser Trp Leu Glu Pro Val Gln Phe Leu Arg Ser Val 85 90 95 Phe Ala Asn Ser Leu Val Tyr Gly Ala Ser Asp Ser Asn Val Tyr Asp 100 105 110 Leu Leu Lys Asp Leu Glu Glu Gly Ile Gln Thr Leu Met Gly Arg Leu 115 120 125 Glu Asp Gly Ser Pro Arg Thr Gly Gln Ile Phe Lys Gln Thr Tyr Ser 130 135 140 Lys Phe Asp Thr Asn Ser His Asn Asp Asp Ala Leu Leu Lys Asn Tyr 145 150 155 160 Gly Leu Leu Tyr Cys Phe Arg Lys Asp Met Asp Lys Val Glu Thr Phe 165 170 175 Leu Arg Ile Val Gln Cys Arg Ser Val Glu Gly Ser Cys Gly Phe 180 185 190 <210> 6 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> GS linker <400> 6 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5 10 15 <210> 7 <211> 17 <212> PRT <213> Artificial Sequence <220> <223> PT linker <400> 7 Pro Thr Pro Pro Thr Thr Pro Thr Pro Pro Thr Thr Pro Thr Pro Thr 1 5 10 15 Pro <210> 8 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> hGH‑F primer <400> 8 cgccatatgt tcccgaccat cccgctg 27 <210> 9 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> hGH‑R primer <400> 9 gatgcacatt cgcatgaaac cgcaag 26 <210> 10 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Primer Mxe‐L6KD‐F <400> 10 cctaatgttt catgcgaatg tgcatcacg 29 <210> 11 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> Primer Mxe‐L6KD‐R <400> 11 tgctcgagtc aatctttcag cagcagcagc agcagcggcg tcggggttgg 50 <210> 12 <211> 47 <212> DNA <213> Artificial Sequence <220> <223> Primer Mxe‐EFK8‐R <400> 12 tgctcgagtc acttgaactc gaattcgaac tcgaacggcg tcggggt 47 <210> 13 <211> 42 <212> DNA <213> Artificial Sequence <220> <223> Primer hGHalpha‐R <400> 13 cggactagtg catctcccgt gatgcacatt cgcatgaaac cg 42 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> J20001‐Mtu‐F <400> 14 ctgctgctga aagatccaac ccc 23 <210> 15 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> J19042‐Mtu‐R <400> 15 atggtcggga agttatgaac cacaacgcct t 31 <210> 16 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> J19040‐hGH‐F <400> 16 ttgtggttca taacttcccg accatcccgc tgtctcgt 38 <210> 17 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> J19041‐hGH‐R <400> 17 ttagcagccg gatctcagtg gt 22 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> J20016‐PT‐F <400> 18 ctgctgctga aagatccaac ccc 23 <210> 19 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> J20017‑Mtu‑R <400> 19 gcaggtcgca gttatgaacc acaacgcctt ccgca 35 <210> 20 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> J20018‑IFN‑F <400> 20 tgtggttcat aactgcgacc tgccgcagac 30 <210> 21 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> J20019‑IFN‑R <400> 21 ttagcagccg gatctcagtg gt 22 <210> 22 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> J20020‑Term‑F <400> 22 accactgaga tccggctgct aacaaag 27 <210> 23 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> J20003‑Ori‑R <400> 23 gcggtatcag ctcactcaaa ggcggtaata cgg 33 <210> 24 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> J20004‑Bom‑F <400> 24 cctttgagtg agctgatacc gctcgccgca gccgaac 37 <210> 25 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> J20015‑RBS‑R <400> 25 gggttggatc tttcagcagc agcagcagca gcatatgt 38 <210> 26 <211> 165 <212> PRT <213> Artificial Sequence <220> <223> hIFN‑alpha2a <400> 26 Cys Asp Leu Pro Gln Thr His Ser Leu Gly Ser Arg Arg Thr Leu Met 1 5 10 15 Leu Leu Ala Gln Met Arg Lys Ile Ser Leu Phe Ser Cys Leu Lys Asp 20 25 30 Arg His Asp Phe Gly Phe Pro Gln Glu Glu Phe Gly Asn Gln Phe Gln 35 40 45 Lys Ala Glu Thr Ile Pro Val Leu His Glu Met Ile Gln Gln Ile Phe 50 55 60 Asn Leu Phe Ser Thr Lys Asp Ser Ser Ala Ala Trp Asp Glu Thr Leu 65 70 75 80 Leu Asp Lys Phe Tyr Thr Glu Leu Tyr Gln Gln Leu Asn Asp Leu Glu 85 90 95 Ala Cys Val Ile Gln Gly Val Gly Val Thr Glu Thr Pro Leu Met Lys 100 105 110 Glu Asp Ser Ile Leu Ala Val Arg Lys Tyr Phe Gln Arg Ile Thr Leu 115 120 125 Tyr Leu Lys Glu Lys Lys Tyr Ser Pro Cys Ala Trp Glu Val Val Arg 130 135 140 Ala Glu Ile Met Arg Ser Phe Ser Leu Ser Thr Asn Leu Gln Glu Ser 145 150 155 160 Leu Arg Ser Lys Glu 165 <210> 27 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> Mtu ΔI‑CM <400> 27 Ala Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr 1 5 10 15 His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val 20 25 30 Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp 35 40 45 Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly 50 55 60 Ala Ile Leu Trp Ala Thr Pro Asp His Lys Val Leu Thr Glu Tyr Gly 65 70 75 80 Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Gln Pro 85 90 95 Arg Arg Phe Asp Gly Phe Gly Asp Ser Ala Pro Ile Pro Ala Arg Val 100 105 110 Gln Ala Leu Ala Asp Ala Leu Asp Asp Lys Phe Leu His Asp Met Leu 115 120 125 Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu Pro Thr Arg 130 135 140 Arg Ala Arg Thr Phe Gly Leu Glu Val Glu Glu Leu His Thr Leu Val 145 150 155 160 Ala Glu Gly Val Val Val His Asn 165 <210> 28 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> Mtu ΔI‑CM Mut 1 <400> 28 Ala Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr 1 5 10 15 His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val 20 25 30 Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp 35 40 45 Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly 50 55 60 Ala Ile Leu Trp Ala Thr Pro Asp Tyr Lys Val Leu Thr Glu Tyr Gly 65 70 75 80 Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Gln Pro 85 90 95 Arg Arg Phe Asp Gly Phe Gly Asp Ser Ala Pro Ile Pro Ala Arg Val 100 105 110 Gln Ala Leu Ala Asp Ala Leu Asp Asp Lys Phe Leu His Asp Met Leu 115 120 125 Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu Pro Thr Arg 130 135 140 Arg Ala Arg Thr Phe Gly Leu Glu Val Glu Glu Leu His Val Leu Val 145 150 155 160 Ala Glu Gly Val Val Val His Asn 165 <210> 29 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> Mtu ΔI‑CM Mut 2 <400> 29 Ala Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr 1 5 10 15 His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val 20 25 30 Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp 35 40 45 Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly 50 55 60 Ala Ile Leu Trp Ala Thr Pro Asp Val Lys Val Leu Thr Glu Tyr Gly 65 70 75 80 Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Gln Pro 85 90 95 Arg Arg Phe Asp Gly Phe Gly Asp Ser Ala Pro Ile Pro Ala Arg Val 100 105 110 Gln Ala Leu Ala Asp Ala Leu Asp Asp Lys Phe Leu His Asp Met Leu 115 120 125 Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu Pro Thr Arg 130 135 140 Arg Ala Arg Thr Phe Gly Leu Glu Val Glu Glu Leu His Ser Leu Val 145 150 155 160 Ala Glu Gly Val Val Val His Asn 165 <210> 30 <211> 168 <212> PRT <213> Artificial Sequence <220> <223> Mtu ΔI‑CM Mut 3 <400> 30 Ala Leu Ala Glu Gly Thr Arg Ile Phe Asp Pro Val Thr Gly Thr Thr 1 5 10 15 His Arg Ile Glu Asp Val Val Asp Gly Arg Lys Pro Ile His Val Val 20 25 30 Ala Ala Ala Lys Asp Gly Thr Leu His Ala Arg Pro Val Val Ser Trp 35 40 45 Phe Asp Gln Gly Thr Arg Asp Val Ile Gly Leu Arg Ile Ala Gly Gly 50 55 60 Ala Ile Leu Trp Ala Thr Pro Asp Val Lys Val Leu Thr Glu Tyr Gly 65 70 75 80 Trp Arg Ala Ala Gly Glu Leu Arg Lys Gly Asp Arg Val Ala Gln Pro 85 90 95 Arg Arg Phe Asp Gly Phe Gly Asp Ser Ala Pro Ile Pro Ala Arg Val 100 105 110 Gln Ala Leu Ala Asp Ala Leu Asp Asp Lys Phe Leu His Asp Met Leu 115 120 125 Ala Glu Glu Leu Arg Tyr Ser Val Ile Arg Glu Val Leu Pro Thr Arg 130 135 140 Arg Ala Arg Thr Phe Gly Leu Glu Val Glu Glu Leu His Cys Leu Val 145 150 155 160 Ala Glu Gly Val Val Val His Asn 165 <210> 31 <211> 495 <212> DNA <213> Artificial Sequence <220> <223> hIFN‑alpha2a <400> 31 tgcgacctgc cgcagaccca ctctctgggt tctcgtcgta ccctgatgct gctggctcag 60 atgcgtaaga tctctctgtt ctcttgcctg aaagaccgtc acgacttcgg tttcccgcag 120 gaagaattcg gtaaccagtt ccagaaagct gaaaccatcc cggttctgca cgaaatgatc 180 cagcagatct tcaacctgtt ctctaccaaa gactcttctg ctgcttggga cgaaaccctg 240 ctggacaaat tctacaccga actgtaccag cagctgaacg acctggaagc gtgcgttatc 300 cagggtgttg gtgttaccga aaccccgctg atgaaagaag actctatcct ggctgttcgt 360 aaatacttcc agcgtatcac cctgtacctg aaagagaaga aatactctcc gtgcgcttgg 420 gaagttgttc gtgctgaaat catgcgttct ttctctctgt ctaccaacct gcaggaatct 480 ctgcgttcta aagaa 495 <210> 32 <211> 504 <212> DNA <213> Artificial Sequence <220> <223> Mtu ΔI‑CM <400> 32 gcgctggctg aaggcacgcg catttttgat ccggtcacgg gcacgacgca ccgcattgaa 60 gatgttgttg atggccgcaa gccgattcat gtggttgcgg ccgcaaaaga tggcaccctg 120 cacgcccgtc cggtcgtgag ttggtttgat cagggtacgc gtgacgtcat tggtctgcgt 180 atcgcgggcg gtgcaattct gtgggcaacc ccggatcata aagtgctgac ggaatatggc 240 tggcgtgctg cgggtgaact gcgtaagggt gaccgtgttg cacagccgcg tcgctttgat 300 ggcttcggtg acagcgcacc gattccggct cgcgttcaag ccctggcaga tgctctggat 360 gacaagttcc tgcacgacat gctggcggaa gaactgcgtt actctgttat ccgcgaagtc 420 ctgccgaccc gtcgcgcccg cacgtttggt ctggaagtgg aagaactgca taccctggtt 480 gcggaaggcg ttgtggttca taac 504 <210> 33 <211> 504 <212> DNA <213> Artificial Sequence <220> <223> Mtu ΔI‑CM Mut 1 <400> 33 gcgctggctg aaggcacgcg catttttgat ccggtcacgg gcacgacgca ccgcattgaa 60 gatgttgttg atggccgcaa gccgattcat gtggttgcgg ccgcaaaaga tggcaccctg 120 cacgcccgtc cggtcgtgag ttggtttgat cagggtacgc gtgacgtcat tggtctgcgt 180 atcgcgggcg gtgcaattct gtgggcaacc ccggattata aagtgctgac ggaatatggc 240 tggcgtgctg cgggtgaact gcgtaagggt gaccgtgttg cacagccgcg tcgctttgat 300 ggcttcggtg acagcgcacc gattccggct cgcgttcaag ccctggcaga tgctctggat 360 gacaagttcc tgcacgacat gctggcggaa gaactgcgtt actctgttat ccgcgaagtc 420 ctgccgaccc gtcgcgcccg cacgtttggt ctggaagtgg aagaactgca tgttctggtt 480 gcggaaggcg ttgtggttca taac 504 <210> 34 <211> 504 <212> DNA <213> Artificial Sequence <220> <223> Mtu ΔI‑CM Mut 2 <400> 34 gcgctggctg aaggcacgcg cattttgat ccggtcacgg gcacgacgca ccgcattgaa 60 gatgttgttg atggccgcaa gccgattcat gtggttgcgg ccgcaaaaga tggcaccctg 120 cacgcccgtc cggtcgtgag ttggtttgat cagggtacgc gtgacgtcat tggtctgcgt 180 atcgcgggcg gtgcaattct gtgggcaacc ccggatgtta aagtgctgac ggaatatggc 240 tggcgtgctg cgggtgaact gcgtaagggt gaccgtgttg cacagccgcg tcgctttgat 300 ggcttcggtg acagcgcacc gattccggct cgcgttcaag ccctggcaga tgctctggat 360 gacaagttcc tgcacgacat gctggcggaa gaactgcgtt actctgttat ccgcgaagtc 420 ctgccgaccc gtcgcgcccg cacgtttggt ctggaagtgg aagaactgca tagtctggtt 480 gcggaaggcg ttgtggttca taac 504 <210> 35 <211> 504 <212> DNA <213> Artificial Sequence <220> <223> Mut ΔI‑CM Mut 3 <400> 35 gcgctggctg aaggcacgcg cattttgat ccggtcacgg gcacgacgca ccgcattgaa 60 gatgttgttg atggccgcaa gccgattcat gtggttgcgg ccgcaaaaga tggcaccctg 120 cacgcccgtc cggtcgtgag ttggtttgat cagggtacgc gtgacgtcat tggtctgcgt 180 atcgcgggcg gtgcaattct gtgggcaacc ccggatgtta aagtgctgac ggaatatggc 240 tggcgtgctg cgggtgaact gcgtaagggt gaccgtgttg cacagccgcg tcgctttgat 300 ggcttcggtg acagcgcacc gattccggct cgcgttcaag ccctggcaga tgctctggat 360 gacaagttcc tgcacgacat gctggcggaa gaactgcgtt actctgttat ccgcgaagtc 420 ctgccgaccc gtcgcgcccg cacgtttggt ctggaagtgg aagaactgca ttgtctggtt 480 gcggaaggcg ttgtggttca taac 504 <210> 36 <211> 41 <212> PRT <213> Artificial Sequence <220> <223> TZ1H <400> 36 Glu Ile Ala Gln His Glu Lys Glu Ile Gln Ala Ile Glu Lys Lys Ile 1 5 10 15 Ala Gln His Glu Tyr Lys Ile Gln Ala Ile Glu Glu Lys Ile Ala Gln 20 25 30 His Lys Glu Lys and Gln Ala and Lys 35 40

Claims

1. A separated fusion polypeptide comprising a target polypeptide moiety and a self-aggregating peptide moiety, wherein the target polypeptide moiety is linked to the self-aggregating peptide moiety by a spacer, wherein the spacer is an intima-peptide Mxe GyrA, the sequence of which is shown in SEQ ID NO: 4, wherein the target polypeptide is human growth hormone as shown in SEQ ID NO: 5, and wherein the self-aggregating peptide is L6KD, the amino acid sequence of which is shown in SEQ ID NO:

1.

2. The fusion polypeptide of claim 1, wherein the target polypeptide portion is located at the N-terminus of the fusion polypeptide.

3. The fusion polypeptide of claim 1, wherein the target polypeptide portion is located at the C-terminus of the fusion polypeptide.

4. The fusion polypeptide of any one of claims 1-3, wherein the spacer is directly connected to the target polypeptide portion and / or the self-aggregating peptide portion; or wherein the spacer further comprises a connector at its N-terminus and / or C-terminus, which is connected to the target polypeptide portion and / or the self-aggregating peptide portion via the connector.

5. The fusion polypeptide of claim 1, wherein the Mxe GyrA is linked to the N-terminus or C-terminus of the human growth hormone moiety.

6. The fusion polypeptide of claim 4, wherein the linker is a GS type linker, the amino acid sequence of which is shown in SEQ ID NO:6; or wherein the linker is a PT type linker, the amino acid sequence of which is shown in SEQ ID NO:

7.

7. An isolated polynucleotide comprising a nucleotide sequence encoding a fusion polypeptide of any one of claims 1-6.

8. An expression construct comprising the polynucleotide of claim 7.

9. A host cell comprising the polynucleotide of claim 7 or transformed from the expression construct of claim 8, wherein the host cell is capable of expressing the fusion polypeptide.

10. The host cell of claim 9, wherein the host cell is selected from prokaryotes, yeast and higher eukaryotic cells.

11. The host cell of claim 10, wherein the prokaryote includes Escherichia coli (Escherichia coli). Escherichia ), Bacillus spp. Bacillus Salmonella ( Salmonella ) and Pseudomonas spp. ( Pseudomonas ) and Streptomyces ( Streptomyces ) bacteria.

12. The host cell of claim 10, wherein the prokaryote is Escherichia coli.

13. The host cell of claim 11, wherein the prokaryote is *Escherichia coli* (…). E. coli ).

14. A method for producing and purifying a target polypeptide, the method comprising the following steps: (a) Culturing host cells according to any one of claims 9-13 to express the fusion polypeptide; (b) Lyse the host cells, then remove the soluble portion of the cell lysate and recover the insoluble portion; (c) Release of a soluble target polypeptide from the insoluble portion by cleaving the cleavage site; and (d) Remove the insoluble portion from step (c) and recover the soluble portion containing the target polypeptide.

15. The method of claim 14, wherein the lysis is performed by ultrasound, homogenization, high pressure, hypotonicity, lysin, organic solvent, or a combination thereof.

16. The method of claim 14, wherein the cleavage is carried out under weakly alkaline pH conditions.

17. The method of claim 14, wherein the cleavage is a dithiothreitol (DTT)-mediated self-cleavage.

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