Signal peptides suitable for yeast and uses thereof

Abstract

The invention discloses a signal peptide SEQ ID NO: 1-3 suitable for yeast, the coding gene of the signal peptide is expressed in the yeast after being fused with a foreign protein gene, the expression level of the foreign protein can be improved, and the signal peptide has popularization and application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a signal peptide generated by using a natural language model and its use in improving the expression of foreign proteins by yeast. Background Art

[0002] Bovine lactoferrin (BLF) has broad application prospects in the pharmaceutical, food, cosmetics and other industries. Osteopontin (OPN) is a glycosylated protein that is widely present in the extracellular matrix. It was originally believed that OPN is an important bone matrix protein that is closely related to the formation and development of bones. The activity of osteopontin can directly reach the intestine, enhance the intestinal protective barrier function, achieve systemic immune protection, and reduce 50% of fever and discomfort and other biological functions.

[0003] Casein is an important biochemical reagent and a safe and harmless thickener and emulsifier in cow's milk. Because casein contains various amino acids needed by the human body and has high nutritional value, it can also be consumed as a nutritional enhancer and is widely used in all food industries.

[0004] The production of high value-added exogenous proteins such as bovine lactoferrin and osteopontin by microbial engineering is a promising method. Compared with Escherichia coli, yeast has high biosafety and its fermentation products are more easily accepted by consumers. Therefore, the development of yeast engineering bacteria that secrete and express exogenous proteins has great industrial application prospects.

[0005] Patent document with publication number CN118878701A discloses a genetic engineering technology for producing bovine lactoferricin antimicrobial peptide by fermentation of Pichia pastoris. However, efficient secretory expression of foreign proteins in microbial expression systems has always been a key issue limiting their large-scale production and application.

[0006] As a key element for guiding protein secretion, signal peptide plays an important role in the secretion efficiency of proteins. For example, patent documents with publication numbers CN117004501B and CN116875476B both disclose that a signal peptide is integrated into a recombinant Pichia pastoris that secretes and expresses bovine lactoferrin antibacterial peptide, thereby promoting the secretion of bovine lactoferrin antibacterial peptide LFcinB.

[0007] Traditional signal peptide optimization methods are often based on limited experimental attempts and known biological rules, and have certain blindness and limitations. For example, the pre-pro signal peptide sequence of mating factor α of Saccharomyces cerevisiae has certain limitations in the secretory expression of bovine lactoferrin and cannot meet the requirements of industrial production for efficient expression and secretion. Therefore, the development of new and more effective signal peptide sequences is of great significance for improving the yield and quality of bovine lactoferrin. Summary of the invention

[0008] Based on bioinformatics big data, we used natural language models to generate a large number of signal peptide variants suitable for yeast, fused them in front of the foreign protein gene, and cloned them into the Pichia pastoris genome. Through Pichia pastoris engineering bacteria fermentation, it was confirmed that the expression of the foreign protein was significantly increased. Based on this, the present invention includes the following technical solutions.

[0009] The first aspect of the present invention provides a signal peptide having a pre-pro structure, wherein the amino acid sequence of the signal peptide is shown in SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

[0010] Wherein, SEQ ID NO:1 is:

[0011] MRF S SIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 1), designated herein as SP2;

[0012] SEQ ID NO:2 is:

[0013] MRFPSI L TAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 2), designated herein as SP8;

[0014] SEQ ID NO:3 is:

[0015] MRF A SIFTAVLFAASSALAAPVAPAEEAANHLHKR (SEQ ID NO: 3), designated herein as SP12.

[0016] The second aspect of the present invention provides a gene encoding the signal peptide SP2, SP8 or SP12 as described above.

[0017] In one embodiment, the gene encoding the signal peptide SEQ ID NO: 1 is a polynucleotide shown in the nucleotide sequence SEQ ID NO: 4, or a polynucleotide having 90% or more, preferably 92% or more, preferably 95% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more homology with SEQ ID NO: 4:

[0018] atgagattttcttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaa acggcacaaattccggctgaagctgtcatcggttatcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcaca aataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaaga (SEQ ID NO: 4);

[0019] The gene encoding the signal peptide SEQ ID NO: 2 is a polynucleotide represented by the nucleotide sequence SEQ ID NO: 5, or a polynucleotide having 90% or more, preferably 92% or more, preferably 95% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more homology to SEQ ID NO: 5:

[0020] atgagatttccttcaattttgactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatga aacggcacaaattccggctgaagctgtcatcggttatcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcac aaataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaaga (SEQ ID NO: 5);

[0021] The gene encoding the signal peptide SEQ ID NO:3 is a polynucleotide represented by the nucleotide sequence SEQ ID NO:6, or a polynucleotide having 90% or more, preferably 92% or more, preferably 95% or more, more preferably 97% or more, more preferably 98% or more, more preferably 99% or more homology to SEQ ID NO:6:

[0022] atgagatttgcttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagttgctccagccgaagaggcagc aaaccacttgcacaagcgt (SEQ ID NO: 6).

[0023] The third aspect of the present invention provides a DNA molecule comprising the coding gene as described above, for example, an expression cassette / expression frame of the signal peptide SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3.

[0024] The fourth aspect of the present invention provides the use of the signal peptides SP2, SP8 and SP12 and their encoding genes as described above in improving the expression of foreign proteins in yeast.

[0025] Preferably, the yeast is Pichia pastoris (Pichia pastoris), such as Pichia pastoris GS115 or Saccharomyces cerevisiae (baker's yeast).

[0026] As a specific embodiment of the above use, the coding gene of the above signal peptide SP2, SP8 or SP12 is fused with the foreign protein gene and expressed in yeast.

[0027] In one embodiment, the exogenous protein is selected from the group consisting of bovine lactoferrin (BLF), osteopontin (OPN), casein, and thaumatin (Thaumatin).

[0028] For example, the above-mentioned exogenous protein is bovine lactoferrin (BLF), and the nucleotide sequence of the gene encoding bovine lactoferrin is SEQ ID NO: 7;

[0029] The exogenous protein is osteopontin, and the nucleotide sequence of the gene encoding osteopontin is SEQ ID NO: 8; or

[0030] The above-mentioned exogenous protein is casein, and the nucleotide sequence of the gene encoding the casein is SEQ ID NO:9.

[0031] The fifth aspect of the present invention provides an integration fragment for expressing foreign proteins in yeast, comprising the coding gene of the above-mentioned signal peptide SP2, SP8 or SP12 and a foreign protein gene located downstream.

[0032] A sixth aspect of the present invention provides a yeast engineering bacterium, preferably a Pichia pastoris engineering bacterium or a Saccharomyces cerevisiae engineering bacterium, in which the above-mentioned integration fragment is integrated into the genome.

[0033] A seventh aspect of the present invention provides the use of the above-mentioned Pichia pastoris engineered bacteria or Saccharomyces cerevisiae engineered bacteria in the production of bovine lactoferrin, osteopontin, casein or thaumatin.

[0034] The signal peptide variants SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 generated by the natural language model can effectively promote the secretion expression of foreign protein genes in yeast, especially Pichia pastoris. When the fusion gene of the signal peptide and foreign proteins such as bovine lactoferrin is expressed in Pichia pastoris, the fermentation yield of foreign protein can be increased by more than 100% compared with the signal peptide sequence of the prior art, which greatly improves the production efficiency and reduces the production cost of foreign protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 This is an agarose gel electrophoresis photograph of the integration of the fusion gene of signal peptide variants SP2, SP8 or SP12 and bovine lactoferrin into the Pichia genome using PCR. This image is a validation diagram of the integration of the lactoferrin expression cassette sequence, primers Paox1(lox66)-F / Taox1-R, and the positive band is about 3.6kb. In the figure, lanes 1-16 are based on Pichia transformants as templates, wherein lanes 1-5 are SP2 fused bovine lactoferrin expression cassette transformants, lanes 6-10 are SP8 fused bovine lactoferrin expression cassette transformants, and lanes 11-16 are SP12 fused bovine lactoferrin expression cassette transformants.

[0036] Figure 2This is an agarose gel electrophoresis photograph of the integration of the fusion gene of signal peptide variants SP2, SP8 or SP12 and osteopontin into the Pichia genome using PCR. This image is a verification diagram of the integration of the osteopontin expression cassette sequence, primer Paox1(lox66)-F / Taox1-R, and the positive band is about 2.4kb. In the figure, lanes 1-16 are based on Pichia transformants as templates, among which lanes 1-5 are SP2 fusion osteopontin expression cassette transformants, lanes 6-10 are SP8 fusion osteopontin expression cassette transformants, and lanes 11-16 are SP12 fusion osteopontin expression cassette transformants.

[0037] Figure 3 This is an agarose gel electrophoresis photograph of the integration of the fusion gene of signal peptide variants SP2, SP8 or SP12 and casein into the Pichia genome using PCR. This image is a casein expression cassette sequence integration verification diagram, primer Paox1 (lox66) -F / Taox1-R, the positive band is about 2.2kb. In the figure, lanes Lane 1-16 are based on Pichia transformants as templates, wherein lanes 1-5 are SP2 casein expression cassette transformants, lanes 6-10 are SP8 casein expression cassette transformants, and lanes 11-16 are SP12 casein expression cassette transformants.

[0038] Figure 4 The figure is a color development photograph and a standard curve of the standard sample of the Histag ELISA kit used for detecting Histag in Example 2. DETAILED DESCRIPTION

[0039] To overcome the limitation of the pre-pro signal peptide sequence (numbered SP0) of the mating factor α from Saccharomyces cerevisiae in promoting the secretion expression of bovine lactoferrin, mutation modification of the signal peptide sequence to improve its promotion efficiency is an optional approach. To this end, we used a natural language model to generate many mutants for the pre sequence, which were combined with one of the two pro sequences α-pro and EpxI-pro to form a complete pre-pro signal peptide variant.

[0040] For the sake of brevity, the "pre-pro signal peptide" may be referred to as the "signal peptide", the "pre-pro signal peptide variant" may be referred to as the "signal peptide variant", and the pre-pro signal peptide of mating factor α of Saccharomyces cerevisiae (numbered SP0) may be referred to as the "wild-type signal peptide".

[0041] Through experiments, these signal peptide variants were compared with the wild-type signal peptide (numbered SP0), and the signal peptide genes were fused with the genes of three foreign proteins, bovine lactoferrin, osteopontin or casein, and expressed in Pichia pastoris. After comparing the expression levels, it was found that three signal peptide variants could simultaneously increase the expression of the three foreign proteins, namely SP2, SP8 and SP12.

[0042] As used herein, the terms "(exogenous protein expression level) increase", "enhance" or "increase" as used above mean an increase of at least 20% or more compared to a reference level, for example, an increase of at least 30% or more, at least 50% or more, at least 80% or more, at least about 1-fold, at least about 2-fold, at least about 3-fold, at least about 4-fold or at least about 5-fold compared to a reference level.

[0043] An effective method to achieve co-expression of signal peptide variants and foreign proteins in yeast is to use fusion gene expression, that is, to form a fusion gene by combining the signal peptide variant encoding gene and the foreign protein gene located downstream.

[0044] The construction of fusion gene firstly carries out gene cloning and modification, amplifies the foreign protein by PCR technology, and amplifies the signal peptide variant sequence obtained by screening. During the amplification process, the appropriate restriction endonuclease recognition site is introduced in the primer design for the subsequent gene connection operation. The amplified gene fragment is purified and quantitatively analyzed. Then the fusion gene is constructed, and the signal peptide variant sequence is fused with the foreign protein gene by gene editing technology such as Gibson assembly method, Overlap-PCR technology or traditional restriction endonuclease enzyme cutting-connection method to ensure the accuracy and stability of the fusion site. In the design of fusion gene, the connecting peptide sequence between the signal peptide and the foreign protein is considered, and the appropriate amino acid residues are selected to form the connecting peptide to ensure that the signal peptide can effectively guide the secretion of the target protein without affecting the functional structure of the target protein. Finally, the fusion gene fragment is constructed and further fused with a strong promoter (such as saccharifying enzyme promoter, gpd promoter, etc.), an efficient terminator, a suitable screening marker (such as antibiotic resistance gene) and elements that are conducive to gene expression regulation (such as enhancers, silencers, etc.) to form an expression cassette. The correctness of the expression vector construction was verified by enzyme digestion identification and sequencing to ensure that the fusion gene was correctly inserted into the appropriate position of the vector and that the other elements of the vector were intact.

[0045] The signal peptide variants SP2 and SP8 of the present invention have only 85 amino acids, SP12 has only 35 amino acids, and the amino acid sequences of the exogenous proteins bovine lactoferrin, osteopontin, casein and thaumatin are clear, so it is easy for those skilled in the art to obtain fusion genes, expression cassettes (DNA molecules) and plasmids containing these fusion genes, and transformants containing the plasmids. These fusion genes, expression cassettes, plasmids, and transformants can be obtained by genetic engineering construction methods well known to those skilled in the art.

[0046] The signal peptide variants of the present invention have the following beneficial effects:

[0047] 1. Improve the secretion expression efficiency of exogenous proteins: Signal peptide variants SP2, SP8 and SP12 can significantly improve the secretion expression efficiency of bovine lactoferrin, osteopontin and casein in Pichia pastoris. Compared with the wild-type signal peptide, the protein yield can be increased by more than 100%, greatly improving the production efficiency and reducing the production cost.

[0048] 2. Great potential for industrial application: The co-expression method of the fusion gene of the present invention in a new yeast strain with food safety provides reliable technical support for the large-scale industrial production of high value-added proteins such as bovine lactoferrin, has broad application prospects, and can meet the large demand for bovine lactoferrin in the food, medicine and other industries.

[0049] 3. Expanded the application field of signal peptides: The research results of the present invention provide new ideas and methods for the efficient expression of other high-value proteins in Pichia pastoris, which will help promote the development of genetic engineering.

[0050] The present invention is further described in detail below in conjunction with specific examples. It should be understood that the following examples are only used to illustrate the present invention and are not used to limit the scope of the present invention.

[0051] Example

[0052] The examples involve the addition amounts, contents and concentrations of various substances, wherein the percentages described therein, unless otherwise specified, are all by mass percentages.

[0053] In the examples herein, if no specific description is given for laboratory temperature or operating temperature, the temperature generally refers to room temperature (15-30° C.).

[0054] Materials and methods

[0055] The whole gene synthesis, primer synthesis and sequencing in the examples were all completed by Suzhou Genewise Biotechnology Co., Ltd.

[0056] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0057] PCR amplification experiments were performed according to the reaction conditions provided by the plasmid or DNA template supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0058] The strains used included Pichia pastoris GS115, which was purchased from Shanghai Institute of Microbiology.

[0059] The experimental steps include:

[0060] 1. Signal peptide sequence design

[0061] 1. A series of different pre sequences were generated through a natural language model and fused in front of the pro sequence of mating factor α of Saccharomyces cerevisiae or Epxl of Pichia pastoris to construct new signal peptide sequence variants.

[0062] 2. After experimental screening and optimization, several signal peptide sequences were identified, and their secretory expression effects on exogenous proteins bovine lactoferrin, osteopontin and casein in Pichia pastoris were significantly better than the traditional wild-type pre-pro signal peptide sequence of mating factor α of Saccharomyces cerevisiae.

[0063] 2. Gene fusion and expression vector construction

[0064] 3. Gene cloning: The exogenous proteins bovine lactoferrin, osteopontin or casein genes and the screened signal peptide variant sequences including SP1-SP16 were obtained by PCR amplification technology, and homologous regions were introduced into the primer design to facilitate the subsequent gene connection operation.

[0065] 4. Construction of integration fragment: Through conventional molecular cloning technology, the synthesized signal peptide pre sequence and pro sequence, promoter, foreign protein gene, histag, and antibiotic gene are connected through overlap-PCR to form an expression vector.

[0066] 3. Host cell transformation and screening

[0067] 5. Transformation and screening of host cells: The recombinant expression vector is introduced into Pichia pastoris host cells by electroporation, positive transformants are screened by culture medium containing antibiotics, and the integration of the target gene in the transformants is verified by PCR.

[0068] 6. Cultivate and optimize the positive transformants to improve their secretory expression ability of exogenous proteins such as bovine lactoferrin, osteopontin or casein.

[0069] IV. Fermentation and protein expression

[0070] 7. The positive transformants obtained by screening were inoculated into the fermentation medium for fermentation culture, and samples were taken regularly to detect the expression level and secretion of bovine lactoferrin using SDS-PAGE, Western blot and other methods.

[0071] 8. During the fermentation process, the expression level and secretion of exogenous proteins such as bovine lactoferrin, osteopontin or casein were monitored, and the yield and quality of the expressed proteins were further improved by adjusting the culture conditions (such as temperature, pH, dissolved oxygen, etc.).

[0072] Example 1: Integrating bovine lactoferrin expression cassettes with different signal peptides into GS115

[0073] The PCR amplification primers for some gene fragments in this example are listed in Table 1.

[0074] Table 1. Primer sequences

[0075] Primer Sequence(5’-3’) lox71-ResP-F taccgttcgtatagcatacattatacgaagttatgatcccccacacaccatagcttc lox66-ResT-R taccgttcgtataatgtatgctatacgaagttatgcaaattaaagccttcgagcg Paox1(lox66)-F tttgcataacttcgtatagcatacattatacgaacggtagttggtattgtgaaatagac Paox1-R catcgtttcgaataattagttgtt pAOX1-a-factor-F gagaagatcaaaaaacaactaattattcgaaacgatgagatttccttcaatttttactg afa(wEAM)-R gttgagaaatagtacaccatctaacgttctttcttggagctcttttctcgagagatacc BLF(wM)-F gctccaagaaagaacgttagatgg BLF-R acatcctcttgattaatgatgatgatgatggtgatgatgtctagtcaagaaagcacaag Taox1(BLF)-F tcttgactagacatcatcaccatcatcatcatcattaatcaagaggatgtcagaatgcc Taox1-R tctcacttaatcttctgtactc

[0076] In Table 1, "-F" in the name stands for forward direction; "-R" stands for reverse direction.

[0077] Some gene sequences in this example are listed in Table 2.

[0078] Table 2. Gene sequences

[0079]

[0080]

[0081]

[0082] Using HZP-sgRNA plasmid (gift from Lian Jiachang's research group at Zhejiang University, for a description of the plasmid structure, see the literature DOI: 10.1021 / acssynbio.1c00307) as a template and lox71-ResP-F / lox66-ResT-R as primers, PCR amplification was performed to obtain the zeocin fragment.

[0083] Using the Pichia genome as a template and Paox1(lox66)-F / Paox1-R as primers, PCR amplification was performed to obtain the Paox1 fragment of the Pichia AOX1 gene promoter. Using the pPIC9K plasmid as a template and pAOX1-a-factor-F / afa(wEAM)-R as primers, PCR amplification of the α-mating factor signal peptide was performed to obtain the a-factor fragment. The PCR fragment was recovered using a gel recovery kit. 1 μl of the recovered fragments Paox1 and a-factor from the above PCR products were mixed as a template, and Paox1(lox66)-F / afa(wEAM)-R were used as primers to obtain the Paox1-a-factor fragment by OverlapPCR amplification.

[0084] The BLF gene (SEQ ID NO: 7) of bovine lactoferrin optimized by gene synthesis codon was used as template and BLF (wM) -F / BLF-R was used as primers to obtain the BLF fragment by PCR amplification. The Pichia pastoris genome was used as template and Taox1 (BLF) -F / Taox1-R was used as primers to obtain the Pichia pastoris AOX1 gene terminator Taox1 fragment by PCR amplification. 1 μl of the fragments BLF and Taox1 recovered from the above PCR products were mixed as templates, and BLF (wM) -F / Taox1-R was used as primers to obtain the BLF-Taox1 fragment by OverlapPCR amplification.

[0085] 1 μl of each of the PCR product recovered fragments zeocin, Paox1-a-factor, and BLF-Taox1 was mixed as a template, and lox71-ResP-F / Taox1-R was used as primers to obtain the final fragment used for bovine lactoferrin integration by Overlap PCR amplification, which is an integration fragment of the wild-type signal peptide SP0 and the fusion gene of bovine lactoferrin located downstream thereof, that is, the bovine lactoferrin gene recombinant expression vector: zeocin-Paox1-a-factor-BLF-Taox1.

[0086] The competent preparation process of Pichia yeast is as follows:

[0087] (1) Take out the P. pastoris GS115 glycerol culture stored in the -80°C freezer, pick a small amount from the frozen tube and streak it onto YPD antibiotic-free solid medium, culture it at 30°C for 3 days until large colonies grow, then take it out;

[0088] (2) Pick a larger colony from the plate and inoculate it into 10 mL of YPD medium and culture it at 30°C and 200 rpm for 1 day;

[0089] (3) Secondary activation: add 1 mL of bacterial solution from step (2) to 100 mL of YPD medium and culture for 4-5 h. The time should not be too long as the bacteria must remain active.

[0090] (4) Place the cultured bacterial solution on ice for a certain period of time, then divide the bacterial solution into sterilized 50 mL centrifuge tubes on a sterile workbench, centrifuge at 4°C, 4,000 × g for 5 min, and discard the supernatant; add 10 mL of sterilized water to each tube, use a pipette to resuspend the cells, then centrifuge at 4°C, 4,000 × g for 5 min, and discard the supernatant; repeat the water washing three times;

[0091] (5) After washing three times, add 1 mL of pre-cooled 1 M sorbitol to each tube, resuspend the cells, transfer to a sterile 1.5 mL centrifuge tube, centrifuge at 4°C, 4,000 × g for 5 min, and discard the supernatant;

[0092] (6) Add 80 μL of pre-cooled 1 M sorbitol to each tube and resuspend the cells by pipetting. The P. pastoris GS115 competent cell preparation is complete and ready for use.

[0093] Add the recombinant expression vector (5-10ng) to be transformed into the prepared Pichia yeast competent cells, mix gently, and place in an ice bath for 5 minutes; after the ice bath, transfer the competent cells with the recombinant expression vector into the treated 0.2cm electroporation cup. 1,500V, 5ms, electric shock. After the electric shock, quickly add 1mL of 1M sorbitol placed on ice, blow several times with a pipette, and then transfer the liquid in the electroporation cup to a centrifuge tube and incubate in a 30℃ metal bath for 1-2h. Then centrifuge at 6,000×g for 5min, discard the supernatant, apply to the MD plate, and culture at 30℃ for 3d until larger colonies grow. The 30℃ incubator will breed mold. In order to prevent contamination, seal the plate with sealing film and then wrap it with several layers of plastic wrap.

[0094] The antibiotic transformants screened from the MD plate were subjected to colony PCR. Using the transformant colonies on the plate as templates and lox71-ResP-F / Taox1-R as primers, a 4.8Kb fragment was amplified by PCR, which was considered positive.

[0095] According to the method similar to the above, Pichia pastoris engineering bacteria expressing one of the signal peptide variants SP1-SP16 and bovine lactoferrin were constructed respectively.

[0096] Figure 1 The agarose gel electrophoresis photographs showing the integration of the fusion gene of the signal peptide variant SP2 and bovine lactoferrin into the Pichia pastoris genome using PCR detection.

[0097] The signal peptide variants SP1-SP16 with pre-pro sequence structures generated using the natural language model are listed in Table 3.

[0098] Table 3. Partial signal peptide sequences

[0099]

[0100]

[0101] Example 2: Fermentation detection of bovine lactoferrin expression level of engineered bacteria

[0102] The positive transformants were selected and inoculated into BMGY medium at 30°C and 250 rpm for 16-20 h until OD 600 The cells were centrifuged at 4000 × g for 10 min to collect the cells and resuspended in BMMY medium to an OD of 4-6. 600 The expression was carried out at 28°C, 250 rpm, pH 6.0, and the inducer methanol was added every 24 hours to a final concentration of 0.5% v / v. The total induction time was 96 hours.

[0103] Fermentation medium:

[0104] BMGY medium: yeast extract 10 g / L, peptone 20 g / L, potassium phosphate buffer (pH 6.0) 100 mM, YNB 13.4 g / L, biotin 0.4 mg / L, glycerol 10 mL / L.

[0105] BMMY medium: yeast extract 10 g / L, peptone 20 g / L, potassium phosphate buffer (pH 6.0) 100 mM, YNB 13.4 g / L, biotin 0.4 mg / L, methanol 5 mL / L.

[0106] Shake flask fermentation process:

[0107] The positive transformants were picked and cultured in YPD tubes overnight for 24 hours.

[0108] 3% v / v was inoculated into 30 ml of BMGY medium and cultured at 30°C, 250 rpm for 16-20 h.

[0109] Centrifuge at 4000 × g for 10 min, collect the cells, and resuspend the cells in 50 ml BMMY medium until the OD 600 is 1.

[0110] Expression was carried out at 30°C, 250 rpm, pH 6.0.

[0111] The inducer methanol was added every 24 hours to a final concentration of 0.5% v / v.

[0112] The total induction time was 96 h, and the end point OD was about 8-10.

[0113] The BLF production in the fermentation supernatant was detected using a Histag ELISA kit.

[0114] The fermentation yield was detected using His Tag ELISA Detection Kit (GenScript Cat. No.: L00436). The specific process is as follows:

[0115] 1. Add 50 μl of His-tag standard or sample containing His-tagged protein to each well of the His-tag plate.

[0116] 2. Add 50 μl of anti-His monoclonal antibody to all wells.

[0117] 3. Cover the plate with a sealing film and incubate at room temperature (20-25°C) for 30 minutes.

[0118] 4. Wash the plate four times with 260 μl of 1× washing solution.

[0119] 5. Pat the plate with a paper towel to remove any remaining liquid in the wells.

[0120] 6. Add 100 μl of Antibody Tracker to all wells.

[0121] 7. Cover the plate with a sealing film and incubate at room temperature for 30 minutes.

[0122] 8. Wash the plate four times with 260 μl of 1× washing solution.

[0123] 9. Pat the plate with a paper towel to remove any remaining liquid in the wells.

[0124] 10. Add 100 μl of TMB substrate to all wells and incubate at room temperature for 10-15 minutes.

[0125] 11. Add 50 μl of stop solution to all wells to stop the enzyme reaction. Read the absorbance of the plate at 450 nm on a microplate reader.

[0126] 12. Draw a standard curve with absorbance as the ordinate and the concentration of the His-tag standard as the abscissa.

[0127] 14. The amount of His-tagged protein in the sample was determined by extrapolating its optical density value to the standard curve.

[0128] The bovine lactoferrin expression levels of Pichia pastoris engineered bacteria containing different signal peptide variants fused to the bovine lactoferrin gene are listed in Table 4.

[0129] Table 4. Detection results of bovine lactoferrin expression levels of strains

[0130]

[0131]

[0132] The results in Table 4 show that the three signal peptide variants SP2, SP8 and SP12 have a higher promoting effect on the expression of bovine lactoferrin than the wild-type signal peptide SP0.

[0133] The three signal peptide variants were further examined to see if there was any consistency in their expression-promoting effects on other exogenous proteins, osteopontin and casein, in Pichia pastoris.

[0134] Example 3: Using the three selected signal peptides to express osteopontin and casein

[0135] The PCR amplification primers for some gene fragments in this example are listed in Table 5.

[0136] Table 5. Primer sequences for amplification of osteopontin and casein genes

[0137] Primer Sequence(5’-3’) 8his-F catcatcaccatcatcatcatc a-factor(KR)-R tcttttctcgagagataccccttc OPN-F ttgctgctaaagaagaaggggtatctctcgagaaaagacttcctgtcaagccaacttcc OPN-R attcgcggccgcttaatgatgatgatgatggtgatgatggttgacttcactactggcag bCN-F cattgctgctaaagaagaaggggtatctctcgagaaaagacgtgaattagaagagctga bCN-R ttcgcggccgcttaatgatgatgatgatggtgatgatgaacgataattgggaagggacc

[0138] In Table 5, "-F" in the name stands for forward; "-R" stands for reverse.

[0139] The osteopontin and casein gene sequences of this example are listed in Table 6.

[0140] Table 6. Osteopontin and casein gene sequences

[0141]

[0142]

[0143] Osteopontin integration fragments obtained:

[0144] Using the zeocin-Paox1-a-factor-BLF-Taox1 fragment in Example 1 as a template and lox71-ResP-F / a-factor (KR)-R as primers, PCR amplification was performed to obtain the zeocin-Paox1-a-factor fragment. Using the gene-synthesized osteopontin gene (SEQ ID NO: 24) as a template and OPN-F / OPN-R as primers, PCR amplification was performed to obtain the osteopontin gene OPN fragment. Using the zeocin-Paox1-a-factor-BLF-Taox1 fragment in Example 1 as a template and 8his-F / Taox1-R as primers, PCR amplification was performed to obtain the Taox1 fragment. The PCR fragment was recovered using a gel recovery kit. Take 1 μl of the recovered fragments zeocin-Paox1-a-factor, OPN, and Taox1 of the above PCR products as a mixture and use lox71-ResP-F / Taox1-R as primers to amplify the fragment for osteopontin integration by Overlap PCR. The fragment is an integration fragment of the fusion gene of the wild-type signal peptide SP0 and the osteopontin located downstream thereof, i.e., the osteopontin gene recombinant expression vector: zeocin-Paox1-a-factor-OPN-Taox1.

[0145] Casein integrated fragments obtained:

[0146] Same as above: Using the zeocin-Paox1-a-factor-BLF-Taox1 fragment in Example 1 as a template and lox71-ResP-F / a-factor(KR)-R as primers, PCR amplification was performed to obtain the zeocin-Paox1-a-factor fragment. Using the gene-synthesized casein gene (SEQ ID NO: 25) as a template and bCN-F / bCN-R as primers, PCR amplification was performed to obtain the casein gene bCN fragment. Using the zeocin-Paox1-a-factor-BLF-Taox1 fragment in Example 1 as a template and 8his-F / Taox1-R as primers, PCR amplification was performed to obtain the Taox1 fragment. The PCR fragment was recovered using a gel recovery kit. Take 1 μl of the recovered fragments zeocin-Paox1-a-factor, bCN, and Taox1 of the above PCR products as a mixture and use lox71-ResP-F / Taox1-R as primers to amplify the fragment for casein integration by Overlap PCR. The fragment is an integration fragment of the fusion gene of the signal peptide SP0 and the casein located downstream thereof, that is, the casein gene recombinant expression vector: zeocin-Paox1-a-factor-bCN-Taox1.

[0147] According to the method similar to the above, Pichia pastoris engineering bacteria expressing signal peptide variants SP2, SP8 or SP12 and osteopontin / casein were constructed respectively.

[0148] Figure 2 Agarose gel electrophoresis photographs showing the integration of the fusion gene of signal peptide variant SP2 and osteopontin into the Pichia pastoris genome using PCR detection.

[0149] Figure 3 The agarose gel electrophoresis photographs showing the integration of the fusion gene of the signal peptide variant SP2 and casein into the Pichia genome detected by PCR.

[0150] Example 4: Expression of osteopontin and casein by yeast engineering bacteria fermentation

[0151] The yeast engineering bacteria were fermented in a similar manner to Example 2 to investigate the promoting effects of the three signal peptide variants SP2, SP8 or SP12 on the expression of osteopontin and casein. The results are shown in Table 7.

[0152] Table 7. Expression levels of casein and osteopontin

[0153] Signal peptide number Casein protein expression level μg / L Osteopontin expression level μg / L SP0 512 109 SP2 612 221 SP8 834 312 SP12 852 287

[0154] The results in Table 7 show that the three signal peptide variants SP2, SP8 and SP12 still have a higher promoting effect on the expression of osteopontin and casein than the wild-type signal peptide SP0, and the effect is consistent.

[0155] Through the analysis of the results of the examples, it can be seen that the novel signal peptide sequences SP2, SP8 and SP12 designed by the present invention can effectively improve the secretory expression efficiency of bovine lactoferrin, osteopontin and casein in Pichia pastoris. This may be because the three novel signal peptide sequences are more conducive to the transmembrane transport and secretion of proteins in structure and function. The specific mechanism needs further in-depth study.

Claims

1. A signal peptide, characterized in that It has a pre-pro structure, and the amino acid sequence of the signal peptide is shown in SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO:

3. Wherein, SEQ ID NO:1 is: MRFSSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR(SEQ ID NO:1); SEQ ID NO:2 is: MRFPSILTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 2); SEQ ID NO:3 is: MRFASIFTAVLFAASSALAAPVAPAEEAANHLHKR (SEQ ID NO: 3).

2. A gene encoding the signal peptide according to claim 1.

3. The gene according to claim 2, characterized in that The gene encoding the signal peptide SEQ ID NO:1 is a polynucleotide having a nucleotide sequence of SEQ ID NO:4, or a polynucleotide having 90% or more homology with SEQ ID NO:4; The gene encoding the signal peptide SEQ ID NO:2 is a polynucleotide having a nucleotide sequence of SEQ ID NO:5, or a polynucleotide having 90% or more homology with SEQ ID NO:5; The gene encoding the signal peptide SEQ ID NO:3 is a polynucleotide represented by the nucleotide sequence SEQ ID NO:6, or a polynucleotide having 90% or more homology with SEQ ID NO:

6.

4. A DNA molecule, characterized in that Comprising the gene as claimed in claim 2.

5. Use of the signal peptide according to claim 1 or the gene according to claim 3 in improving the expression of foreign proteins in yeast.

6. The use according to claim 5, characterized in that The signal peptide encoding gene as claimed in claim 2 is fused with a foreign protein gene and expressed in yeast. In one embodiment, the exogenous protein is selected from the group consisting of bovine lactoferrin, osteopontin, and casein.

7. The use according to claim 7, characterized in that The exogenous protein is bovine lactoferrin, and the nucleotide sequence of the gene encoding bovine lactoferrin is SEQ ID NO: 7; The exogenous protein is osteopontin, and the nucleotide sequence of the gene encoding osteopontin is SEQ ID NO: 8; or The exogenous protein is casein, and the nucleotide sequence of the gene encoding the casein is SEQ ID NO:

9.

8. An integrated fragment, characterized in that Used for expressing foreign protein in yeast, comprising the signal peptide encoding gene as claimed in claim 2 and a foreign protein gene located downstream.

9. A yeast engineering bacterium, characterized in that: The integration fragment as claimed in claim 8 is integrated into its genome.

10. Use of the engineered yeast according to claim 9 in producing bovine lactoferrin, osteopontin or casein.

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