Signal peptides suitable for yeast and uses thereof

Abstract

The application discloses a signal peptide suitable for yeast, and has the SEQ ID NO:1-3, wherein the coding gene of the signal peptide is fused with an exogenous protein gene and is expressed in yeast, so that the expression level of the exogenous protein is improved, and the signal peptide has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of genetic engineering, and particularly relates to a signal peptide generated by a natural language model and use thereof in improving expression of exogenous proteins by yeast. BACKGROUND

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

[0003] Casein is an important biochemical reagent and a safe and harmless thickening agent and emulsifier in cow's milk. In addition, casein contains various amino acids required by the human body, has high nutritional value, and can be used as a nutritional fortifier for food, and is widely used in all food industries.

[0004] Fermentation by microbial engineering bacteria to produce high-value exogenous proteins such as bovine lactoferrin and osteopontin is a promising method. Compared with Escherichia coli, yeast has high biological safety, and its fermentation products are more easily accepted by consumers, so the development of yeast engineering bacteria secreting and expressing exogenous proteins has industrial application prospects.

[0005] The patent document with publication number CN118878701A discloses a genetic engineering technology for producing bovine lactoferricin by fermentation of Pichia pastoris engineering bacteria. However, efficient secretion and expression of exogenous proteins in microbial expression systems has been a key problem limiting their large-scale production and application.

[0006] Signal peptides, as key elements guiding protein secretion, play an important role in the secretion efficiency of proteins. For example, the patent documents with publication numbers CN117004501B and CN116875476B both disclose that the integration of signal peptides in recombinant Pichia pastoris bacteria secreting and expressing bovine lactoferricin peptide promotes the secretion of bovine lactoferricin 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 the mating factor alpha of Saccharomyces cerevisiae has certain limitations in the secretion expression of bovine lactoferritin, and cannot meet the needs of industrial production for efficient expression and secretion. Therefore, developing new and more effective signal peptide sequences is of great significance to improve the yield and quality of bovine lactoferritin. SUMMARY

[0008] Based on bioinformatics big data, we generated a large number of signal peptide variants suitable for yeast using natural language models, and fused them in front of the exogenous protein gene and cloned into the Pichia pastoris (Pichia pastoris) genome, and through Pichia pastoris engineering bacteria fermentation, it is confirmed that the expression amount of exogenous protein is significantly improved. Accordingly, the present application includes the following technical solutions.

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

[0010] SEQ ID NO: 1 is:

[0011] MRF S SIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 1), which is named SP2 herein;

[0012] SEQ ID NO: 2 is:

[0013] MRFPSI L TAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 2), which is named SP8 herein;

[0014] SEQ ID NO: 3 is:

[0015] MRF A SIFTAVLFAASSALAAPVAPAEEAANHLHKR (SEQ ID NO: 3), which is named SP12 herein.

[0016] The second aspect of the application 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 represented by the nucleotide sequence SEQ ID NO: 4, or a polynucleotide having more than 90%, preferably more than 92%, preferably more than 95%, more preferably more than 97%, more preferably more than 98%, more preferably more than 99% homology to SEQ ID NO: 4:

[0018] atgagattttcttcaatttttactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaa acggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcaca 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 more than 90%, preferably more than 92%, preferably more than 95%, more preferably more than 97%, more preferably more than 98%, more preferably more than 99% homology to SEQ ID NO: 5:

[0020] atgagatttccttcaattttgactgcagttttattcgcagcatcctccgcattagctgctccagtcaacactacaacagaagatgaa acggcacaaattccggctgaagctgtcatcggttactcagatttagaaggggatttcgatgttgctgttttgccattttccaacagcaca aataacgggttattgtttataaatactactattgccagcattgctgctaaagaagaaggggtatctctcgagaaaaga (SEQ ID NO: 5);

[0021] The gene encoding the signal peptide SEQ ID NO: 3 is a polynucleotide represented by the nucleotide sequence of 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 with SEQ ID NO: 6:

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

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

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

[0025] Preferably, the yeast is Pichia pastoris (P. pastoris) such as Pichia pastoris GS115 or Saccharomyces cerevisiae (S. cerevisiae).

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

[0027] In one embodiment, the exogenous protein is selected from the group consisting of Bovine Lactoferrin (BLF), osteopontin (OPN), casein, Thaumatin (African arrowroot sweetener, Shamatin.

[0028] For example, the exogenous protein is Bovine Lactoferrin (BLF), and the nucleotide sequence of the gene encoding the Bovine Lactoferrin is SEQ ID NO: 7;

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

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

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

[0032] The sixth aspect of the present application provides a yeast engineering bacterium, preferably a Pichia pastoris engineering bacterium or a Saccharomyces cerevisiae engineering bacterium, whose genome is integrated with the above-mentioned integration fragment.

[0033] The seventh aspect of the present application provides the above-mentioned Pichia pastoris engineering bacterium or Saccharomyces cerevisiae engineering bacterium for use in the production of lactoferrin, osteopontin, casein or thaumatin.

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

[0035] Figure 1 is an agarose gel electrophoresis photo of PCR detection of integration of the fusion gene of the signal peptide variant SP2, SP8 or SP12 and lactoferrin into the Pichia pastoris genome. The image is a sequence integration verification image of the lactoferrin expression cassette, primer Paox1 (lox66)-F / Taox1-R, positive band about 3.6 kb. In the figure, lanes Lane 1-16 are Pichia pastoris transformants as templates, wherein lanes 1-5 are SP2 fusion lactoferrin expression cassette transformants, lanes 6-10 are SP8 fusion lactoferrin expression cassette transformants, and lanes 11-16 are SP12 fusion lactoferrin expression cassette transformants.

[0036] Figure 2Figure 1 is an agarose gel electrophoresis photograph for detecting the integration of the fusion gene of signal peptide variant SP2, SP8 or SP12 and osteopontin into the genome of Pichia pastoris by PCR. This image is the sequence integration verification diagram of the osteopontin expression cassette, primer Paox1 (lox66)-F / Taox1-R, positive band about 2.4 kb. In the figure, lanes Lane 1-16 are templates of Pichia pastoris transformants, wherein 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 Figure 2 is an agarose gel electrophoresis photograph for detecting the integration of the fusion gene of signal peptide variant SP2, SP8 or SP12 and casein into the genome of Pichia pastoris by PCR. This image is the sequence integration verification diagram of the casein expression cassette, primer Paox1 (lox66)-F / Taox1-R, positive band about 2.2 kb. In the figure, lanes Lane 1-16 are templates of Pichia pastoris transformants, 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 Figure 3 is a photograph of the standard curve and standard curve of the Histag ELISA kit used for detecting in Example 2. DETAILED DESCRIPTION

[0039] To overcome the limitations of the pre-pro signal peptide sequence of the mating factor a of Saccharomyces cerevisiae (numbered SP0) in promoting the secretion expression of bovine lactoferrin, it is an optional approach to mutate and modify the signal peptide sequence to improve its promotion efficiency. For this purpose, we used a natural language model to generate a number of mutants of the pre sequence, which were combined with one of the two pro sequences, a-pro and EpxI-pro, to form a complete pre-pro signal peptide variant.

[0040] For the sake of brevity of description, “pre-pro signal peptide” can be referred to as “signal peptide” and “pre-pro signal peptide variant” can be referred to as “signal peptide variant” in this paper, and the pre-pro signal peptide of the mating factor a of Saccharomyces cerevisiae (numbered SP0) can be referred to as “wild-type signal peptide”.

[0041] The signal peptide variants were compared with the wild-type signal peptide (numbered SP0) by experiment. The signal peptide genes were fused with the genes of three kinds of foreign proteins, bovine lactoferrin, bone bridge protein or casein, respectively, and then expressed in Pichia pastoris. Through comparison of expression levels, it was found that three signal peptide variants could simultaneously improve the expression levels of the three kinds of foreign proteins, which were SP2, SP8 and SP12.

[0042] In this paper, the above-mentioned terms "(foreign protein expression level) improvement", "promotion" or "increase" means at least 20% higher than the reference level, for example, at least 30% higher than the reference level, at least 50% higher than the reference level, at least 80% higher than the reference level, at least about 1 times, at least about 2 times, at least about 3 times, at least about 4 times or at least about 5 times higher than the reference level.

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

[0044] The construction of the fusion gene first carries out gene cloning and modification. The foreign protein is amplified by PCR technology, and the signal peptide variant sequence screened is also amplified. In the amplification process, appropriate restriction enzyme recognition sites are introduced in the primer design for subsequent gene ligation operation. The amplified gene fragments are 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 enzyme digestion-ligation method, etc. to ensure the accuracy and stability of the fusion site. In the design of the 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 compose the connecting peptide to ensure that the signal peptide can effectively guide the secretion of the target protein, while not affecting the functional structure of the target protein. Finally, the fusion gene fragment is constructed and further fused with a strong promoter (such as a saccharification enzyme promoter, a gpd promoter, etc.), a high-efficiency terminator, a suitable selection marker (such as an antibiotic resistance gene) and elements beneficial to gene expression regulation (such as enhancers, silencers, etc.) to form an expression cassette. The correctness of the expression vector construction is verified by restriction enzyme identification and sequencing, ensuring that the fusion gene is correctly inserted into the appropriate position of the vector, and the other elements of the vector are complete and intact.

[0045] The signal peptide variants SP2 and SP8 of the present application only have 85 amino acids, SP12 only has 35 amino acids, and the amino acid sequences of the foreign proteins bovine lactoferrin, bone bridge protein, casein and thaumatin are clear, so the fusion genes, expression cassettes (DNA molecules) containing these fusion genes and plasmids, and transformants containing the plasmids can be easily obtained by those skilled in the art. These fusion genes, expression cassettes, plasmids, transformants can be obtained by gene engineering construction methods known to those skilled in the art.

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

[0047] 1. Improve the secretion expression efficiency of foreign proteins: the signal peptide variants SP2, SP8 and SP12 can significantly improve the secretion expression efficiency of bovine lactoferrin, bone bridge protein, casein in Pichia pastoris, compared with 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 fusion genes of the present application provide reliable technical support for large-scale industrial production of high-value proteins such as bovine lactoferrin by co-expression method with new yeast species with food safety, which has broad application prospects and can meet the large demand of food, medicine and other industries for bovine lactoferrin.

[0049] 3. Expand the application field of signal peptide: the research results of the present application provide new ideas and methods for the efficient expression of other high-value proteins in Pichia pastoris, which is helpful to promote the development of genetic engineering field.

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

[0051] Examples

[0052] The addition amount, content and concentration of various substances involved in the examples are described, wherein the percentage content refers to mass percentage content unless otherwise specified.

[0053] In the examples herein, if no specific description is made for laboratory temperature or operating temperature, the temperature usually refers to room temperature (15-30℃).

[0054] Materials and methods

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

[0056] The molecular biology experiments in the examples include plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, medium preparation, etc. and are mainly performed according to the Molecular Cloning: A Laboratory Manual (3rd Edition), J. Sambrook, D. W. Russell (USA), Huang Peitang et al. (transl.), Science Press, Beijing, 2002. The specific experimental conditions can be determined by simple tests if necessary.

[0057] The PCR amplification experiments are performed according to the reaction conditions or kit instructions provided by the plasmid or DNA template supplier. The specific experimental conditions can be adjusted by simple tests if necessary.

[0058] The strains used include P. pastoris GS115, purchased from Shanghai Institute of Microbiology.

[0059] The experimental steps include:

[0060] I. Signal peptide sequence design

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

[0062] 2. After experimental screening and optimization, several signal peptide sequences were determined, which were significantly better than the wild-type pre-pro signal peptide sequence of the mating factor alpha of Saccharomyces cerevisiae in terms of the secretion and expression of exogenous proteins bovine lactoferrin, osteopontin and casein in P. pastoris.

[0063] II. Gene fusion and expression vector construction

[0064] 3. Gene cloning: The exogenous protein bovine lactoferrin, osteopontin or casein gene and the signal peptide variant sequences including SP1-SP16, etc. were obtained by PCR amplification technology. Homologous regions were introduced in the primer design for subsequent gene ligation operations.

[0065] 4. Construction of integration fragments: The synthesized signal peptide pre-sequence and pro-sequence, promoter, exogenous protein gene, histag, and antibiotic gene were connected by overlap-PCR to form an expression vector through conventional molecular cloning technology.

[0066] III. Host cell transformation and screening

[0067] 5. Transformation and screening of host cells: The recombinant expression vector is introduced into the Pichia pastoris host cells by electroporation, and the 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. Culture and optimization of positive transformants to improve their secretion and expression of exogenous proteins, bovine lactoferrin, osteopontin or casein.

[0069] Four, fermentation culture and protein expression

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

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

[0072] Example 1: Integration of bovine lactoferrin expression cassette with different signal peptides into GS115

[0073] The PCR amplification primers of 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 represents forward; “-R” represents reverse.

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

[0078] Table 2, gene sequences

[0079]

[0080]

[0081]

[0082] The zeocin fragment is obtained by PCR amplification using HZP-sgRNA plasmid (donated by Professor Lian Jiachang of Zhejiang University, see literature DOI: 10.1021 / acssynbio.1c00307 for plasmid structure description) as template and lox71-ResP-F / lox66-ResT-R as primers.

[0083] The Paox1 fragment of the AOX1 gene promoter of Pichia pastoris was obtained by PCR amplification using the Paox1 (lox66)-F / Paox1-R as primers and the genome of Pichia pastoris as a template. The a-factor fragment of the alpha-mating factor signal peptide was obtained by PCR amplification using the pAOX1-a-factor-F / afa(wEAM)-R as primers and the pPIC9K plasmid as a template. The PCR fragments were recovered by using a gel recovery kit. 1 microliter of each of the recovered fragments Paox1 and a-factor was mixed as a template, and the Paox1-a-factor fragment was obtained by Overlap PCR amplification using the Paox1 (lox66)-F / afa(wEAM)-R as primers.

[0084] The BLF fragment was obtained by PCR amplification using the BLF(wM)-F / BLF-R as primers and the codon-optimized BLF gene (SEQ ID NO: 7) of bovine lactoferrin synthesized by gene synthesis as a template. The Taox1 fragment of the AOX1 gene terminator of Pichia pastoris was obtained by PCR amplification using the Taox1(BLF)-F / Taox1-R as primers and the genome of Pichia pastoris as a template. 1 microliter of each of the recovered fragments BLF and Taox1 was mixed as a template, and the BLF-Taox1 fragment was obtained by Overlap PCR amplification using the BLF(wM)-F / Taox1-R as primers.

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

[0086] The preparation process of the competent cells of Pichia pastoris is as follows:

[0087] (1) The stored P.pastoris GS115 glycerol bacteria were taken out from the-80℃ refrigerator, and a small amount of streak was taken from the frozen tube to the YPD solid medium without antibiotics, and cultured at 30℃ for 3d until larger colonies were formed.

[0088] (2) The larger colonies were picked from the plate and inoculated into 10mL YPD medium, and cultured at 30℃, 200rpm for 1d.

[0089] (3) Secondary activation, 1 mL of bacteria solution in step (2) is added to 100 mL of YPD medium, and cultured for 4-5 h. The time should not be too long, and the bacteria should be kept active;

[0090] (4) The cultured bacteria solution is placed on ice for a certain period of time, and then is divided into sterilized 50 mL centrifuge tubes in a sterile workbench. Centrifugation is performed at 4°C and 4,000 x g for 5 min, and the supernatant is discarded. 10 mL of sterilized water is added to each tube, and the cells are resuspended by blowing and sucking with a gun. Then, centrifugation is performed at 4°C and 4,000 x g for 5 min, and the supernatant is discarded. The water washing is repeated for a total of 3 times;

[0091] (5) After the water washing for 3 times, 1 mL of pre-cooled 1M sorbitol is added to each tube. After resuspending the cells, they are transferred to sterile 1.5 mL centrifuge tubes, and centrifugation is performed at 4°C and 4,000 x g for 5 min. The supernatant is discarded.

[0092] (6) 80 μL of pre-cooled 1M sorbitol is added to each tube, and the cells are resuspended by blowing and sucking with a gun. The preparation of P. pastoris GS115 competence is completed and is ready for use.

[0093] The recombinant expression vector (5-10 ng) to be transformed is added to the prepared P. pastoris competence, and mixed gently. After ice bath for 5 min, the competence cells with the recombinant expression vector are transferred to a treated 0.2 cm electroporation cup. Electroporation is performed at 1,500 V and 5 ms. After the end of electroporation, 1 mL of 1M sorbitol placed on ice is quickly added, and the liquid in the electroporation cup is transferred to a centrifuge tube by blowing several times with a pipette. Then, incubation is performed at 30°C in a metal bath for 1-2 h. Then, centrifugation is performed at 6,000 x g for 5 min, and the supernatant is discarded. The MD plate is inoculated, and incubation is performed at 30°C for 3 d until larger colonies are grown. In order to prevent contamination, the plate is sealed with a sealing film, and then is wrapped with several layers of fresh-keeping film.

[0094] Colony PCR is performed on the antibiotic transformants screened from the MD plate. The transformed colony on the plate is used as a template, and lox71-ResP-F / Taox1-R is used as a primer. A 4.8 Kb fragment is obtained by PCR amplification, which is positive.

[0095] According to the similar method described above, P. pastoris engineering bacteria expressing one of the signal peptide variants SP1-SP16 and bovine lactoferritin are constructed.

[0096] Figure 1 An agarose gel electrophoresis photo showing that the fusion gene of signal peptide variant SP2 and bovine lactoferritin is integrated into the genome of P. pastoris is shown.

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

[0098] Table 3, partial signal peptide sequences

[0099]

[0100]

[0101] Example 2: Fermentation to detect the expression level of bovine lactoferritin in engineered bacteria

[0102] The positive transformants were inoculated into BMGY medium and cultured at 30°C, 250 rpm for 16-20 h until OD 600 was 4-6. The cells were collected by centrifugation at 4000 x g for 10 min and resuspended in BMMY medium to OD 600 was 1. The expression was carried out at 28°C, 250 rpm, pH 6.0, and the inducer methanol was added every 24 h to a final concentration of 0.5% v / v, and the total induction time was 96 h.

[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] Shaking flask fermentation process:

[0107] The positive transformants were inoculated into YPD medium and cultured at 30°C for 24 h.

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

[0109] The cells were collected by centrifugation at 4000 x g for 10 min and resuspended in 50 ml BMMY medium to OD 600 was 1.

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

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

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

[0113] BLF production in the supernatant of the fermentation broth was detected by histag ELISA kit.

[0114] The fermentation production was detected by His Tag ELISA Detection Kit (GenScript Cat. No.: L00436), and the specific process was as follows:

[0115] 1. Add 50 μl of His tag standard or His tag protein-containing sample 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 with 1x washing solution 260 μl for four times.

[0119] 5. Tap the plate with a paper towel to remove residual liquid in the wells.

[0120] 6. Add 100 μl of antibody tracer 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 with 1x washing solution 260 μl for four times.

[0123] 9. Tap the plate with a paper towel to remove residual 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 terminate the enzyme reaction. Read the absorbance of the plate at 450 nm on a microplate reader.

[0126] 12. Plot the standard curve with absorbance as the vertical coordinate and His tag standard concentration as the horizontal coordinate.

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

[0128] The bovine lactoferritin expression levels of Pichia pastoris engineering bacteria containing different signal peptide variant fusion bovine lactoferritin genes are listed in Table 4.

[0129] Table 4, results of detection of bovine lactoferritin expression levels of strains

[0130]

[0131]

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

[0133] Further investigation was made on the promoting effect of the three signal peptide variants on the expression of other foreign proteins, osteopontin and casein in Pichia pastoris, to see if there is any consistency.

[0134] Example 3: Use of the selected three signal peptides for expression of osteopontin and casein

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

[0136] Table 5, Osteopontin and casein gene amplification primer sequences

[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, the "-F" in the name represents forward; "-R" represents reverse.

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

[0140] Table 6, Osteopontin and casein gene sequences

[0141]

[0142]

[0143] Osteopontin integration fragment obtained:

[0144] With the zeocin-Paox1-a-factor-BLF-Taox1 fragment in Example 1 as a template, and lox71-ResP-F / a-factor(KR)-R as primers, a zeocin-Paox1-a-factor fragment was obtained by PCR amplification. With the OPN gene (SEQ ID NO: 24) synthesized by gene as a template, and OPN-F / OPN-R as primers, an OPN fragment of the OPN gene was obtained by PCR amplification. With the zeocin-Paox1-a-factor-BLF-Taox1 fragment in Example 1 as a template, and 8his-F / Taox1-R as primers, a Taox1 fragment was obtained by PCR amplification. The PCR fragments were recovered by using a gel recovery kit. 1 microliter of each of the recovered PCR product fragments of zeocin-Paox1-a-factor, OPN, and Taox1 was mixed as a template, and lox71-ResP-F / Taox1-R as primers, to obtain a fragment for OPN integration by Overlap PCR amplification, which was an integration fragment of the fusion gene of the wild-type signal peptide SP0 and OPN located downstream of the wild-type signal peptide SP0, i.e., an OPN gene recombinant expression vector: zeocin-Paox1-a-factor-OPN-Taox1.

[0145] Casein integration fragment:

[0146] With the zeocin-Paox1-a-factor-BLF-Taox1 fragment in Example 1 as a template, and lox71-ResP-F / a-factor(KR)-R as primers, a zeocin-Paox1-a-factor fragment was obtained by PCR amplification. With the casein gene (SEQ ID NO: 25) synthesized by gene as a template, and bCN-F / bCN-R as primers, a bCN fragment of the casein gene was obtained by PCR amplification. With the zeocin-Paox1-a-factor-BLF-Taox1 fragment in Example 1 as a template, and 8his-F / Taox1-R as primers, a Taox1 fragment was obtained by PCR amplification. The PCR fragments were recovered by using a gel recovery kit. 1 microliter of each of the recovered PCR product fragments of zeocin-Paox1-a-factor, bCN, and Taox1 was mixed as a template, and lox71-ResP-F / Taox1-R as primers, to obtain a fragment for casein integration by Overlap PCR amplification, which was an integration fragment of the fusion gene of the signal peptide SP0 and casein located downstream of the signal peptide SP0, i.e., a casein gene recombinant expression vector: zeocin-Paox1-a-factor-bCN-Taox1.

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

[0148] Figure 2 The agarose gel electrophoresis photo for detecting the integration of the fusion gene of signal peptide variant SP2 and osteopontin into the Pichia pastoris genome by PCR is shown.

[0149] Figure 3 The agarose gel electrophoresis photo for detecting the integration of the fusion gene of signal peptide variant SP2 and casein into the Pichia pastoris genome by PCR is shown.

[0150] Example 4: Fermentation of the yeast engineering bacteria to express osteopontin and casein

[0151] The yeast engineering bacteria were fermented according to the similar method of Example 2 to investigate the promoting effect 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 pg / L Osteopontin expression level pg / L SP0 512 109 SP2 612 221 SP8 834 312 SP12 852 287

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

[0155] Through the analysis of the results of the examples, it can be known that the novel signal peptide sequences SP2, SP8 and SP12 designed in the present application can effectively improve the secretion expression efficiency of bovine lactoferrin, osteopontin and casein in Pichia pastoris, which may be due to the fact that the three novel signal peptide sequences are more conducive to the transmembrane transport and secretion of proteins in structure and function, and the specific mechanism needs to be further studied.

Claims

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

3. Among them, SEQ ID NO: 1 is: MRFSSIFTAVLFAASSALAAPVNTTTEDETAQIPAEAVIGYSDLEGDFDVAVLPFSNSTNNGLLFINTTIASIAAKEEGVSLEKR (SEQ ID NO: 1); SEQ ID NO: 3 is: MRFASIFTAVLFAASSALAAPVAPAEEAANHLHKR (SEQ ID NO: 3).

2. A gene encoding the signal peptide as described in claim 1.

3. The gene as described in claim 2, characterized in that, The gene encoding the signal peptide SEQ ID NO: 1 is a polynucleotide as shown in SEQ ID NO: 4; The gene encoding the signal peptide SEQ ID NO: 3 is a polynucleotide as shown in the nucleotide sequence SEQ ID NO:

6.

4. A DNA molecule, characterized in that, It contains the gene as described in claim 2.

5. Use of the signal peptide as described in claim 1 or the gene as described in claim 3 in improving the expression of exogenous proteins in yeast.

6. The use as described in claim 5, characterized in that, The signal peptide encoding gene as described in claim 2 is fused with an exogenous protein gene and expressed in yeast.

7. The use as described in claim 6, characterized in that, The exogenous protein is selected from the following group: bovine lactoferrin, osteopontin, and casein.

8. The use as described in 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.

9. An integrated segment, characterized in that, For expressing exogenous proteins in yeast, comprising the signal peptide encoding gene as described in claim 2 and a downstream exogenous protein gene, wherein... 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.

10. A type of engineered yeast, characterized in that, Its genome integrates the integrated fragment as described in claim 9.

11. The use of the engineered yeast strain as described in claim 10 in the production of bovine lactoferrin, osteopontin, or casein.

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