Method for screening high-yield exoprotein or polypeptide strain based on antibacterial peptide

Abstract

The invention relates to a method for screening a high-yield exoprotein or polypeptide strain based on an antibacterial peptide. The method comprises the following steps: obtaining an eukaryotic expression strain of an exoprotein or polypeptide expression module integrated with an antibacterial peptide label; co-culturing the eukaryotic expression strain and antibacterial peptide sensitive bacteria on a solid plate, and observing the size of an inhibition zone of the exoprotein or polypeptide marked by the secreted antibacterial peptide in the growth process of the eukaryotic expression strain for inhibiting the growth of the antibacterial peptide sensitive bacteria; and screening according to the size of the inhibition zone to obtain the strain with high yield of the exosome protein or polypeptide. According to the method, the eukaryotic expression strain of high-performance protein or polypeptide exosome is visually displayed through the size of the inhibition zone, and the method has universality, can be suitable for detection of any protein, and is low in cost and easy in raw material obtaining.

Description

Technical Field

[0001] The present invention relates to the field of bioengineering technology, and in particular to a method for screening strains with high production of exocrine proteins or polypeptides based on antimicrobial peptides. Background Art

[0002] Pichia pastoris is a highly efficient heterologous protein expression system, offering advantages such as rapid growth, simple genetic manipulation, and large-scale fermentation. It is currently a commonly used host for recombinant protein expression. Compared to higher eukaryotes, yeast offers advantages such as cost-effectiveness, high biomass, high protein titers, and the absence of immunogenic compounds. Pichia pastoris contains less than 10% endogenous secreted proteins, which allows purification of recombinant proteins or peptides secreted into the culture medium without being affected by other proteins and contaminants, thereby reducing the cost of subsequent purification.

[0003] Furthermore, Pichia pastoris possesses eukaryotic protein processing and post-translational modification capabilities, such as glycosylation and disulfide bond formation. Compared to prokaryotic expression systems such as Escherichia coli and Bacillus subtilis, it is more suitable for the synthesis of recombinant proteins or peptides that require protein processing and post-translational modification. These advantages make Pichia pastoris a suitable platform for producing a variety of functionally active recombinant proteins or peptides.

[0004] Currently, expression modules for exogenous proteins or peptides are primarily integrated into the Pichia pastoris genome via random nonhomologous recombination. The integration site and copy number of the exogenous gene significantly influence protein secretion levels, leading to significant variability in recombinant protein or peptide expression between recombinant strains. Using traditional protein purification and quantitative methods to screen for recombinant strains with high secretion performance is time-consuming and labor-intensive, while specific methods for detecting exosomes are expensive and not applicable to all proteins. Currently, there is a lack of a universal, simple, and low-cost method for screening strains with high exosome secretion performance. Summary of the Invention

[0005] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. To this end, the present invention aims to provide a method for screening strains with high production of exosome proteins or polypeptides based on antimicrobial peptides.

[0006] To this end, according to an embodiment of the present invention, the present invention proposes a method for screening strains with high production of exocytic proteins or polypeptides based on antimicrobial peptides, which comprises:

[0007] Obtaining a eukaryotic expression strain integrating an antimicrobial peptide-tagged exoprotein or polypeptide expression module;

[0008] Co-culturing the eukaryotic expression strain and antimicrobial peptide-sensitive bacteria on a solid plate, and observing the size of the inhibition zone of the antimicrobial peptide-labeled exoprotein or polypeptide secreted by the eukaryotic expression strain during its growth to inhibit the growth of antimicrobial peptide-sensitive bacteria;

[0009] According to the size of the inhibition zone, a strain with high exoprotein or polypeptide production performance is screened and obtained.

[0010] According to an embodiment of the present invention, a method for screening strains with high exocytotic protein or polypeptide production based on antimicrobial peptides is provided. The method utilizes antimicrobial peptides to label exocytotic proteins or polypeptides without affecting the protein structure and function. A eukaryotic expression strain and antimicrobial peptide-sensitive bacteria are co-cultured on a solid plate. When the eukaryotic expression strain secretes the exocytotic protein or polypeptide labeled with the antimicrobial peptide, the growth of the antimicrobial peptide-sensitive bacteria is inhibited, resulting in the formation of an inhibition zone. The inhibition zone can thus be used to visually display the amount of exocytted protein, thereby demonstrating the high-performance protein exocytosis of the expression strain. The method is universal, applicable to the detection of any protein, and has low cost and readily available raw materials.

[0011] In addition, the method for screening strains with high exocrine protein or polypeptide production based on antimicrobial peptides according to the above embodiment of the present invention may also have the following additional technical features:

[0012] Optionally, the antimicrobial peptide is human defensin 5 or plectasin.

[0013] Optionally, the eukaryotic expression strain is a Pichia pastoris strain or a Saccharomyces lipolytica strain.

[0014] Optionally, the method of obtaining a eukaryotic expression strain for integrating an antimicrobial peptide-tagged exoprotein or polypeptide expression module comprises:

[0015] Amplifying the antimicrobial peptide fragment by PCR, and connecting the antimicrobial peptide fragment to the expression vector pGAPZ(alpha)A to obtain a first recombinant plasmid;

[0016] PCR amplification to obtain a gene fragment of the target protein or polypeptide, and ligating the target gene fragment to the first recombinant plasmid to obtain a second recombinant plasmid;

[0017] The second recombinant plasmid is electroporated into a eukaryotic expression strain, and positive colonies are screened.

[0018] Furthermore, the target gene fragment is a β-lactamase β-LAC fragment.

[0019] Furthermore, the second recombinant plasmid was linearized by BglⅡ cutting and transformed into Pichia pastoris GS115 competent cells by electroporation. The electroporated cells were spread on YPD+100 μg Ble plates to screen for positive colonies.

[0020] Optionally, co-culturing the eukaryotic expression strain and antimicrobial peptide-sensitive bacteria on a solid plate comprises:

[0021] After the E. coli bacterial solution is evenly spread on a YPD plate, the eukaryotic expression strain bacterial solution is then dropped onto the plate. After overnight culture, an inhibition zone appears.

[0022] Additional aspects and advantages of the present invention will be set forth in part in the description which follows and, in part, will be obvious from the description which follows, or may be learned by practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Schematic diagram of the principle of using antimicrobial peptides to screen high-exoprotein-producing strains according to an embodiment of the present invention;

[0024] Figure 2 This is a PCR verification diagram of the integration of the β-LAC-HD5 expression module into the Pichia pastoris genome according to an embodiment of the present invention;

[0025] Figure 3 This is a diagram showing an antibacterial experiment of exocytic proteins or polypeptides labeled with exocytic antimicrobial peptides expressed by a Pichia pastoris expression strain according to an embodiment of the present invention. DETAILED DESCRIPTION

[0026] The technical solution of the present invention is described below through specific examples. It should be understood that the one or more method steps mentioned in the present invention do not exclude the presence of other method steps before and after the combination step or the insertion of other method steps between these explicitly mentioned steps; it should also be understood that these embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. Moreover, unless otherwise specified, the numbering of each method step is only a convenient tool for identifying each method step, and is not intended to limit the order of arrangement of each method step or to define the scope of the present invention. Changes or adjustments in their relative relationships, without substantially changing the technical content, should also be regarded as the scope of the present invention.

[0027] In order to better understand the above technical solutions, exemplary embodiments of the present invention are described in more detail below. Although exemplary embodiments of the present invention are shown, it should be understood that the present invention can be implemented in various forms and should not be limited by the embodiments set forth herein. On the contrary, these embodiments are provided to enable a more thorough understanding of the present invention and to fully convey the scope of the present invention to those skilled in the art.

[0028] The test materials used in the present invention are all common commercial products and can be purchased on the market; unless otherwise specified, the experiments involved are all conventional experimental methods.

[0029] Sources of Materials: Pichia pastoris GS115 and TOP10 were commercially available; the TOP10 strain was used for vector construction and antibacterial experiments. The Pichia pastoris expression vector pGAPZ(alpha)A was commercially available. Phusion high-fidelity DNA polymerase and restriction endonucleases were purchased from Xiamen Lulong Biotechnology Development Co., Ltd. Plasmid extraction kits, DNA purification kits, gel recovery kits, and genomic DNA extraction kits were purchased from Hangzhou Bio-Tech. Zeocin was purchased from InvivoGen.

[0030] LB medium composition: 10 g·L -1 Peptone, 5g·L -1 Yeast extract, 10g·L -1 NaCl, make up to 1 L with double-distilled water, and sterilize at 0.1 MPa pressure and 121°C for 20 min.

[0031] The composition of YPD medium is: 10 g L -1 Yeast extract, 20g·L -1 Peptone, 20 g L -1 Glucose was added and made up to 1 L with double distilled water, and sterilized at 0.1 MPa pressure and 121°C for 20 min.

[0032] Table 1 Primers used for PCR amplification

[0033]

[0034]

[0035] The present invention is described below with reference to specific examples. It should be noted that these examples are merely illustrative and do not limit the present invention in any way.

[0036] Example 1 Obtaining Pichia pastoris expression vector

[0037] Using the primers HD5_XbaI_F (SEQ ID NO: 1) and HD5_SalI_R (SEQ ID NO: 2) as primers (Table 1), primer dimers were formed to obtain the HD5 fragment by PCR amplification. The target band was recovered using a DNA purification kit to obtain a recovered product. PCR amplification conditions were: 94°C for 2 min, 94°C for 15 s, 52°C for 15 s, and 72°C for 15 s, for 30 cycles. The recovered product was ligated with the expression vector pGAPZ(alpha)A and transformed into competent Escherichia coli Top10 cells. The pGAPZ-MCS-HD5 plasmid was isolated and sequenced to ensure complete consistency with the designed plasmid DNA sequence. Specifically, the HD5 sequence obtained by PCR was cut with XbaI-SalI and ligated into the XbaI-SalI sites of the pGAPZ(alpha)A vector to obtain the pGAPZ-MCS-HD5 plasmid.

[0038] Using primers β-LAC_EcoRI_F (SEQ ID NO:3) and β-LAC_XbaI_R (SEQ ID NO:4) as primers (Table 1), the pUC18 vector was used as a template to amplify the β-LAC fragment. The target band was recovered using a DNA purification kit to obtain the recovered product. PCR amplification conditions were: 94°C for 2 min, 94°C for 15 s, 52°C for 15 s, and 72°C for 30 s, for 30 cycles. The recovered product was ligated with the expression vector pGAPZ-MCS-HD5 and transformed into competent Escherichia coli Top10 cells. The pGAPZ-β-LAC-HD5 plasmid was isolated and sequenced to ensure complete consistency with the designed plasmid DNA sequence. Specifically, the β-LAC gene fragment obtained by PCR was cut with EcoRI-XbaI and ligated into the EcoRI-XbaI sites of the recombinant vector pGAPZ-MCS-HD5 to obtain the pGAPZ-β-LAC-HD5 plasmid.

[0039] Example 2 Obtaining a recombinant strain of Pichia pastoris expressing an exocytotic target protein

[0040] The pGAPZ-β-LAC-HD5 plasmid obtained in Example 1 was linearized by BglⅡ cutting and electroporated into Pichia pastoris GS115 competent cells. The electroporated cells were plated on YPD + 100 μg Ble plates to screen for positive colonies. PCR identification was performed using primers such as β-LAC_EcoRI_F shown in SEQ ID NO: 3 and HD5_SalI_R shown in SEQ ID NO: 2 to confirm that the Pichia pastoris recombinant strain had clearly integrated the β-LAC-HD5 expression module. The results are as follows: Figure 2 shown.

[0041] Example 3 Screening of high-yield exoprotein Pichia pastoris expression strains using inhibition zones

[0042] After the E. coli liquid was evenly spread on the YPD plate, the recombinant strain (positive colonies obtained in Example 2) liquid was dropped on the plate. After overnight culture, an inhibition zone appeared. The results are as follows. Figure 3 As shown, through Figure 3 The size of the inhibition zone displayed can be used to intuitively judge the protein secretion ability of the Pichia pastoris expression strain.

[0043] In summary, according to the technology for screening strains with high exosome protein or polypeptide production based on antimicrobial peptides in an embodiment of the present invention, antimicrobial peptides are small peptides with broad-spectrum antimicrobial activity against bacteria and fungi; first, the exosome protein or polypeptide of the expression strain is labeled with the antimicrobial peptide; then, the expression strain integrating the antimicrobial peptide-labeled exosome protein or polypeptide expression module and Escherichia coli are co-cultured on a solid plate; when the expression strain secretes the antimicrobial peptide-labeled exosome protein or polypeptide, the antimicrobial peptide secreted by the expression strain can inhibit the growth of Escherichia coli. Based on the size of the inhibition zone, it can be used to quickly screen the protein exocytosis performance of the expression strain (such as Pichia pastoris), thereby intuitively displaying the exocytosis of high-performance proteins or polypeptides through the size of the inhibition zone. In addition, the use of sensitive bacteria such as Escherichia coli for screening experiments is low-cost and readily available raw materials.

[0044] In the description of this specification, the reference terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" mean that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic expressions of the above terms should not be understood as necessarily referring to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art can combine and combine different embodiments or examples described in this specification.

[0045] Although the embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and are not to be construed as limitations on the present invention. A person skilled in the art may change, modify, replace and modify the above embodiments within the scope of the present invention.

Claims

1. A method for screening strains with high production of exocytic proteins or polypeptides based on antimicrobial peptides, characterized in that: include: Obtaining a eukaryotic expression strain integrating an antimicrobial peptide-tagged exoprotein or polypeptide expression module; Co-culturing the eukaryotic expression strain and antimicrobial peptide-sensitive bacteria on a solid plate, and observing the size of the inhibition zone of the antimicrobial peptide-labeled exoprotein or polypeptide secreted by the eukaryotic expression strain during its growth to inhibit the growth of antimicrobial peptide-sensitive bacteria; According to the size of the inhibition zone, a strain with high exoprotein or polypeptide production performance is screened and obtained.

2. The method according to claim 1, wherein The antimicrobial peptide is human defensin 5 or plectasin.

3. The method according to claim 1, wherein The eukaryotic expression strain is a Pichia pastoris strain or a Saccharomyces lipolytica strain.

4. The method according to claim 1, wherein The eukaryotic expression strain for obtaining an exoprotein or polypeptide expression module integrated with an antimicrobial peptide marker comprises: Amplifying the antimicrobial peptide fragment by PCR, and connecting the antimicrobial peptide fragment to the expression vector pGAPZ(alpha)A to obtain a first recombinant plasmid; PCR amplification to obtain a gene fragment of the target protein or polypeptide, and ligating the target gene fragment to the first recombinant plasmid to obtain a second recombinant plasmid; The second recombinant plasmid is electroporated into a eukaryotic expression strain, and positive colonies are screened.

5. The method according to claim 4, wherein The target gene fragment is a β-lactamase β-LAC fragment.

6. The method according to claim 4, wherein The second recombinant plasmid was linearized by BglⅡ cutting and electroporated into Pichia pastoris GS115 competent cells. The electroporated cells were spread on YPD+100 μg Ble plates to screen for positive colonies.

7. The method according to claim 1, wherein Co-culturing the eukaryotic expression strain and antimicrobial peptide-sensitive bacteria on a solid plate comprises: After the bacterial solution of Escherichia coli is evenly spread on a YPD plate, the bacterial solution of the eukaryotic expression strain is then dropped onto the plate. After overnight culture, an inhibition zone appears.

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