CN116102625B

CN116102625B - Signal peptide and application thereof

Info

Publication number: CN116102625B
Application number: CN202310276032.3A
Authority: CN
Inventors: 陈昌盛; 潘红阳; 张思雯
Original assignee: Hangzhou Startech Biotechnology Co ltd
Current assignee: Hangzhou Startech Biotechnology Co ltd
Priority date: 2023-03-20
Filing date: 2023-03-20
Publication date: 2023-09-22
Anticipated expiration: 2043-03-20
Also published as: CN116102625A

Abstract

The application discloses a signal peptide and application thereof, and belongs to the technical field of biology. The signal peptide is artificially synthesized for efficient secretion expression of mammal cell recombinant protein, and the amino acid sequence of the signal peptide is shown as SEQ ID NO. 1. Compared with the existing signal peptide, the signal peptide can greatly improve the secretion expression quantity of the exogenous target protein, and has wide industrial application prospect.

Description

Signal peptide and application thereof

Technical Field

The application belongs to the technical field of biology, and particularly relates to a signal peptide and application thereof, and more particularly relates to the signal peptide and application thereof in guiding secretion expression of exogenous target proteins in mammalian cells.

Background

Signal peptides (sp) are short peptide chains that direct the transfer of newly synthesized proteins to the secretory pathway, mostly located at the n-terminus of the secreted protein, and are typically 16-30 amino acid residues in length. As shown in fig. 1, the signal peptide comprises three regions: n region, h region and c region. Wherein the n region consists of 1 to 5 positively charged amino acid residues, which interact with negatively charged phospholipids; the middle part is an h region and is composed of 7-15 hydrophobic amino acid residues; the length of the c region is usually 3 to 7 amino acids, and mainly includes hydrophilic amino acids that can be recognized and cleaved by signal peptidase. The nature of the signal peptide plays a critical role in the secretion of heterologous proteins during their secretion.

Many important proteins of medical value (e.g., antibodies) are not biologically active or are very poorly active in E.coli expression systems, since many eukaryotic proteins cannot accomplish post-translational modifications in such systems, such as glycosylation, amidation, proper linkage of disulfide bonds, proper assembly of subunits, and the like. In mammalian cell expression systems, these post-translational modifications can be performed normally, yielding molecules identical to the native protein. Thus, mammalian cell expression systems are evolving into industry.

In actual production, the problems of no secretion or low secretion of recombinant proteins of the constructed vector are frequently encountered, and the problems of poor stability, low activity and the like of expressed proteins are also caused. One solution is to incorporate a suitable signal peptide into the 5' end of the gene sequence of the target protein, to effect proper folding and assembly into the endoplasmic reticulum under the influence of the signal peptide, and then to secrete the protein in an active form extracellularly. In general, the gene sequence of the protein carries a signal peptide sequence for secretion expression of the protein, but most signal peptides belong to low-efficiency secretion signal peptides, and the original signal peptides need to be re-screened or optimized to improve the secretion efficiency of the recombinant protein. The secretion enhancement capability through signal peptide screening is a common strategy for enhancing the extracellular expression quantity of heterologous proteins, and has great significance for industrial production.

Disclosure of Invention

1. Problems to be solved

Aiming at the problem of lower time division efficiency when the mammalian cells express the exogenous protein, the application provides a signal peptide and application thereof for guiding the secretory expression of the exogenous target protein in the mammalian cells, which can obviously improve the expression and the secretory quantity of the exogenous target protein, reduce the production cost of the recombinant protein and have wide industrial application prospect.

2. Technical proposal

In order to solve the problems, the technical scheme adopted by the application is as follows:

the application provides a signal peptide, which comprises an amino acid sequence shown as SEQ ID NO.1, and can be expressed in mammalian cells together with an exogenous target protein, so that the secretion of the exogenous target protein in the mammalian cells can be guided, and the expression or secretion amount of the target protein in the mammalian cells can be improved.

The application also provides a polynucleotide sequence which codes for the signal peptide, and the sequence of the polynucleotide sequence is shown as SEQ ID NO. 38.

The application also provides an expression vector which comprises the polynucleotide sequence and is used for guiding the secretory expression of the target protein.

Further, the above expression vector is a mammalian cell expression vector.

Further, the polynucleotide sequence encoding the signal peptide in the expression vector is linked at the 3' -rear end to the polynucleotide sequence encoding the foreign target protein.

Further, a Kozak sequence was added to the 5' -end of the polynucleotide encoding the signal peptide in the above expression vector.

Further, the polynucleotide sequence for encoding the exogenous target protein in the expression vector is connected with a polynucleotide sequence for encoding a His tag at the 3' -rear end, the His tag (His tag) is used for separating and purifying the target protein, and the amino acid sequence of the His tag is shown as SEQ ID NO. 2.

Further, the promoter of the mammalian cell expression vector may be one or more of EF-1 alpha (human elongation factor-1 alpha ) promoter, hCMV (human cytomegalovirus, human cytomegalovirus) promoter, SV40 (Simian vacuolating virus, monkey vacuolate virus) late promoter, SV40 early promoter, and a promoter obtained by mixing EF-1 alpha with hCMV promoter.

Further, the mammalian cell expression vector includes pcDAN3.4.

The application also provides application of the signal peptide, the polynucleotide sequence and the mammalian cell expression vector in guiding secretion and expression of target proteins in mammalian cells.

Further, the above application includes: transfecting a mammalian cell after ligating the polynucleotide sequence encoding the signal peptide with a polynucleotide encoding an exogenous target protein; or the target protein is expressed after the mammalian cell expression vector is transfected into mammalian cells.

Further, the mammalian cells are selected from one or more of CHO (Chinese hamster ovary, chinese hamster ovary cells), BHK (baby hamster kidney, baby hamster kidney cells), SP2/0 (mouse myeloma cells), C127 (mouse mammary tumor cells), HEK293 (human embryonic kidney293, human embryonic kidney293 cells). Further, the mammalian cells are selected from HEK293 cells.

Further, the target protein is selected from one or more of Human interleukin-8 (Human IL-8), human soluble fms-like tyrosine kinase 1 (Human sFlt-1), human pepsinogen I (Human PGI), human insulin-like growth factor binding protein 3 (Human IGFBP 3), human epididymal protein (Human HE 4), human CD19 (Human CD 19), human prostate-specific antigen (Human PSA), and the like.

Further, the amino acid sequences of the target proteins, namely Human IL-8, human sFlt-1, human PGI, human IGFBP3, human HE4, human CD19 and Human PSA are shown as SEQ ID NO.3-SEQ ID NO.9 respectively.

Further, the amino acid sequences of the polypeptide sequences comprising the signal peptide, the exogenous target protein and the His tag are respectively shown as SEQ ID NO.17-SEQ ID NO. 23.

Further, the polynucleotide sequences encoding the polypeptide sequences comprising the signal peptide, the exogenous target protein and the His tag are shown in SEQ ID NO.31-SEQ ID NO.37, respectively.

The application also provides a cell for expressing target protein, which comprises the polynucleotide sequence or a mammalian cell expression vector, and is obtained by transfection of the polynucleotide sequence or transfection of the expression vector.

Further, the mammalian cells are selected from one or more of CHO (Chinese hamster ovary, chinese hamster ovary cells), BHK (baby hamster kidney, baby hamster kidney cells), SP2/0 (mouse myeloma cells), C127 (mouse mammary tumor cells), HEK293 (human embryonic kidney293, human embryonic kidney293 cells).

The application also provides a production method of the target protein, which comprises the following steps:

s1: ligating the above polynucleotide sequence encoding a signal peptide to the 5' front end of the polynucleotide sequence encoding a target protein;

s2: transfecting the polynucleotide sequence ligated in S1 into a mammalian cell; or cloning the polynucleotide sequence connected in the S1 to an expression vector, and then transfecting the cloned expression vector into a mammalian cell;

s3: culturing the transfected mammalian cells in S2, expressing and purifying the target protein.

3. Advantageous effects

Compared with the prior art, the application has the beneficial effects that:

(1) The signal peptide provided by the application can be used for guiding the secretion and expression of exogenous target proteins in mammalian cells, and as shown in an embodiment, 7 target proteins are selected for the functions of the signal peptide. Experimental results show that the polypeptide consisting of the amino acid sequence shown in SEQ ID NO.1 is used as a signal peptide, the expression level of the selected verification protein is improved by 5 times or more, wherein the expression levels of the Human sFlt-1 and the Human IGFBP3 are improved by 15 times, and the Human PGI protein almost without expression of the signal peptide can be purified to obtain the target protein under the condition of using the signal peptide, so that the signal peptide can be widely used for high-efficiency secretory expression of the target protein in mammalian cells, and can also effectively solve the production problem of the protein with difficult expression.

(2) Compared with the existing signal peptide, the signal peptide provided by the application has the advantages that the expression level of the exogenous target protein in the mammal cells is greatly improved, the expression level of 7 verification proteins is improved by 5 times or more, the expression level of 4 verification proteins is improved by 10 times, and the expression levels of Human sFlt-1 and Human IGFBP3 are improved by 15 times.

(3) Compared with the natural protein signal peptide, the signal peptide provided by the application greatly improves the protein expression quantity, and the target protein is protein with in-vitro diagnosis value, such as Human IL-8, human sFlt-1, human PGI, human IGFBP3, human HE4, human CD19 and Human PSA, and has obviously improved yield and wide industrial application prospect.

Drawings

FIG. 1 is a schematic diagram of the structure of a signal peptide.

FIG. 2 is a graph showing the secretory pathway analysis of the signal peptide +human IL-8 of the present application.

FIG. 3 is a pcDAN3.4 plasmid map.

FIG. 4 is a schematic diagram of plasmid construction.

FIG. 5 shows the result of comparison of the signal peptide of the present application with the signal peptide expression effect of the target protein itself.

Detailed Description

The application is further described below in connection with specific embodiments.

The terms such as "upper", "lower", "left", "right", "middle" and the like are also used in the present specification for convenience of description, and are not intended to limit the scope of the present application, but rather to change or adjust the relative relationship thereof, and are also considered to be within the scope of the present application without substantial change of technical content.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the term "and/or" as used herein includes any and all combinations of one or more of the associated listed items.

The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

As used herein, the term "about" is used to provide the flexibility and inaccuracy associated with a given term, metric or value. The degree of flexibility of a particular variable can be readily determined by one skilled in the art.

As used herein, the term "is intended to be synonymous with" one or more of ". For example, "at least one of A, B and C" expressly includes a only, B only, C only, and respective combinations thereof.

Concentrations, amounts, and other numerical data may be presented herein in a range format. It is to be understood that such range format is used merely for convenience and brevity and should be interpreted flexibly to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a numerical range of about 1 to about 4.5 should be interpreted to include not only the explicitly recited limits of 1 to about 4.5, but also include individual numbers (such as 2, 3, 4) and subranges (such as 1 to 3, 2 to 4, etc.). The same principle applies to ranges reciting only one numerical value, such as "less than about 4.5," which should be construed to include all such values and ranges. Moreover, such an interpretation should apply regardless of the breadth of the range or the characteristics being described.

Example 1

This example provides construction of recombinant expression plasmids.

The signal peptide of the present application was verified by SignalP-6.0 as Sec/SPI secretory pathway, as shown in FIG. 2, i.e., transported by Sec transposition and cleaved by signal peptidase I (Lep).

The application selects 7 test proteins (target proteins) to verify the function of the signal peptide. Under the same condition, the expression level of the target protein in HEK293 is verified by comparing the signal peptide of the application with the signal peptide of the test protein.

The expression vector listed in this example is plasmid pcDNA3.4 (purchased from Hongxun, suzhou, as shown in FIG. 3), and the construction method is shown in FIG. 4.

The amino acid and nucleotide sequences of the application are shown in table 1, the amino acid sequence of the signal peptide is SEQ ID NO.1, and the polynucleotide sequence for encoding the signal peptide is SEQ ID NO.38; the amino acid sequence of the His tag is shown as SEQ ID NO. 2; the amino acid sequences of self signal peptides of verification proteins (target proteins) Human IL-8, human sFlt-1, human PGI, human IGFBP3, human HE4, human CD19 and Human PSA are shown in SEQ ID NO.3-9 respectively; the amino acid sequences of the self signal peptide, the target protein and the His tag are shown in SEQ ID NO.10-16 respectively, and the polynucleotide sequences of the amino acid sequences of the coding self signal peptide, the target protein and the His tag are shown in SEQ ID NO. 24-30; the amino acid sequences of the signal peptide, the target protein and the His tag are respectively shown in SEQ ID NO.17-23, and the polynucleotide sequences of the amino acid sequences of the self signal peptide, the target protein and the His tag are respectively shown in SEQ ID NO. 31-37.

The polynucleotide sequence of this example was synthesized by Suzhou body. During synthesis, the 5 'end of the polynucleotide sequence SEQ ID No.24-37 is preceded by an enzyme digestion site XbaI and a Kozak sequence, and the 3' end is followed by a polynucleotide sequence encoding a His tag (SEQ ID No. 2) and a stop codon and an enzyme digestion site Eco32I. The synthetic sequence and pcDNA3.4 plasmid were digested with restriction endonucleases XbaI and Eco32I, and then identified by agarose gel electrophoresis, and the fragment of interest was recovered by excision. The recovered target fragment and pcDNA3.4 were ligated. Recombinant plasmids SDT1-IL-8, SDT1-sFlt-1, SDT1-PGI, SDT1-IGFBP3, SDT1-HE4, SDT1-CD19, SDT1-PSA and recombinant plasmids SDT2-IL-8, SDT2-sFlt-1, SDT2-PGI, SDT2-IGFBP3, SDT2-HE4, SDT2-CD19, SDT2-PSA with the signal peptide of the verification protein itself are obtained. TOP competence is respectively transformed by the plasmids, after plating and culturing for 16 hours, plasmid miniprep is carried out on the selected monoclone, double digestion is carried out on the extracted plasmid by using restriction endonucleases XbaI and Eco32I, agarose gel electrophoresis identification is carried out, and positive clone is sent to the golden Siro biotechnology Co. The clone with correct sequence is used for large extraction of plasmid, and the extracted plasmid is stored at-20 ℃ for standby.

TABLE 1 amino acid and polynucleotide sequences

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Example 2

This example provides the transient transfection of HEK293 cells with recombinant plasmids to express the protein of interest.

The recombinant plasmids SDT1-IL-8, SDT1-sFlt-1, SDT1-PGI, SDT1-IGFBP3, SDT1-HE4, SDT1-CD19, SDT1-PSA and recombinant plasmids SDT2-IL-8, SDT2-sFlt-1, SDT2-PGI, SDT2-IGFBP3, SDT2-HE4, SDT2-CD19, SDT2-PSA with the signal peptide of the verification protein itself were transfected into HEK293 cells by the cationic liposome method (FectoPRO), respectively, and the specific transfection procedure was as follows:

HEK293 cell density was adjusted to 2X 10 day prior to transfection with pre-warmed OPM-293CD05 Medium (Shanghai ao Pu Mai) ⁶ Placing the cells in a condition that the relative humidity is more than or equal to 80% and CO is 8% ₂ Shake culturing at 95rpm in shake incubator at 37deg.C. The next day the cell density was adjusted to 3X 10 with pre-warmed OPM-293CD05 Medium ⁶ And (5) standby. The transfection mixture was formulated as follows: 75 μg recombinant plasmid (self-contained), 50 μl FectoPRO (Polyplus)SA), 5mL of OPM-CDTrans293 (Shanghai ao Pu Mai), and 50mL of 3X 10 density was added after leaving the mixture at room temperature for 5 minutes ⁶ In the cells of (2), the relative humidity is more than or equal to 80 percent, and the CO content is 8 percent ₂ Shake culturing at 95rpm in shake incubator at 37deg.C. 3mL OPM-293Profeed (Shanghai Ao Pu Mai), cultivation was continued for 96h, and then supernatant purification was collected by centrifugation at 12000rpm for 10 min.

Example 3

This example provides purification of the target protein.

The target protein is separated and purified by His tag affinity chromatography, and the used filler is Ni Smart (Hemsy and Tiandi). The supernatants of example 2 were each filtered through a 0.45 μm filter (Merck Millipore) and bound to 5mL Ni Smart chromatography columns equilibrated with PBS, followed by washing off the contaminating proteins with 5 column volumes of PBS containing 10mM imidazole, and finally eluting the target proteins with 3 column volumes of PBS containing 500mM imidazole, and the collections were analyzed by SDS-PAGE.

Example 4

This example provides an analysis of the secretion expression level of a target protein guided by the signal peptide of the present application.

Under the same condition, the signal peptide of the application is compared with the signal peptide of the target protein, and the measurement result of the expression quantity of the exogenous target protein is shown in figure 5. Compared with the existing signal peptide, the signal peptide has the advantages that the expression quantity of the exogenous target protein in the mammalian cells is greatly improved, the expression level of 7 verification proteins is improved by 5 times or more, the expression level of 4 verification proteins is improved by 10 times, and the expression levels of Human sFlt-1 and Human IGFBP3 are improved by 15 times.

In summary, the application provides a novel signal peptide, which is compared with a natural protein signal peptide, the signal peptide greatly improves the expression level of target protein, and the test sample is protein with in vitro diagnostic value such as Human IL-8, human sFlt-1, human PGI, human IGFBP3, human HE4, human CD19 and Human PSA, the yield is obviously improved, and the application has wide industrial application prospect.

Claims

1. The signal peptide is characterized in that the amino acid sequence of the signal peptide is shown as SEQ ID NO. 1.

2. A polynucleotide encoding the signal peptide of claim 1, wherein the sequence is set forth in SEQ ID No. 38.

3. An expression vector comprising the polynucleotide sequence of claim 2.

4. The expression vector of claim 3, further comprising a polynucleotide sequence encoding an exogenous protein of interest.

5. The expression vector of claim 4, wherein the polynucleotide sequence encoding the exogenous protein of interest is linked to the 3' rear end of the polynucleotide sequence of claim 2; and/or a Kozak sequence is added to the 5' -front end of the polynucleotide sequence; and/or the 3' -rear end of the polynucleotide sequence for encoding the target protein is connected with a polynucleotide sequence for encoding a His tag.

6. Use of the signal peptide of claim 1, the polynucleotide sequence of claim 2, the expression vector of any one of claims 3-5 for directing secretory expression of a protein of interest in a mammalian cell.

7. The use of claim 6, comprising transfecting a mammalian cell after ligating the polynucleotide sequence with a polynucleotide sequence encoding a protein of interest; or transfecting the expression vector into a mammalian cell, and culturing the transfected mammalian cell to secrete and express the target protein.

8. The use according to claim 6 or 7, wherein the mammalian cells are selected from one or more of chinese hamster ovary cells CHO, baby hamster kidney cells BHK, mouse myeloma cells SP2/0, mouse mammary tumor cells C127, human embryonic kidney293 cells HEK 293; and/or the target protein is selected from one or more of Human IL-8, human sFlt-1, human PGI, human IGFBP3, human HE4, human CD19, and Human PSA.

9. A cell expressing a protein of interest, said cell comprising the polynucleotide sequence of claim 2 and a polynucleotide sequence encoding the protein of interest, said cell being transfected after ligation of said polynucleotide sequence with said polynucleotide sequence encoding the protein of interest, or said cell being transfected with an expression vector according to any one of claims 3 to 5.

10. A method for producing a target protein, comprising the steps of:

s1: ligating the polynucleotide sequence of claim 2 to the 5' front end of the polynucleotide sequence encoding the protein of interest;

s3: culturing the transfected mammalian cells in S2 to express the protein of interest.

11. The method of claim 10, wherein the target protein is selected from one or more of Human IL-8, human sFlt-1, human PGI, human IGFBP3, human HE4, human CD19, human PSA; and/or the mammalian cells are selected from one or more of Chinese hamster ovary cells CHO, baby hamster kidney cells BHK, mouse myeloma cells SP2/0, mouse mammary tumor cells C127, human embryonic kidney293 cells HEK 293.