CN116970503A - Pichia pastoris for producing lactoferrin for strengthening vesicle transport and method for promoting extracellular secretion - Google Patents
Pichia pastoris for producing lactoferrin for strengthening vesicle transport and method for promoting extracellular secretion Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
- C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/50—Isolated enzymes; Isolated proteins
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/79—Transferrins, e.g. lactoferrins, ovotransferrins
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Abstract
The invention discloses pichia pastoris for producing lactoferrin and a method for promoting extracellular secretion for strengthening vesicle transport, belonging to the technical field of biology. The present invention has found that accumulation of lactoferrin in cells is not discharged, and that the yield of extracellular lactoferrin is limited, and that accumulation of a large amount of lactoferrin in cells also affects the growth of yeast cells, and thus it is desired to find a method capable of effectively promoting secretory expression thereof. Through screening, the content of lactoferrin in extracellular supernatant is obviously increased after BMH2, sec12 or Sly1 is over-expressed, the content of lactoferrin in sediment and crushing supernatant is effectively reduced, vesicle transport of the lactoferrin in pichia pastoris can be enhanced, the lactoferrin is quickly secreted out of cells, high-efficiency expression of the lactoferrin is enhanced, and the problem that the lactoferrin is not secreted in cells at present is solved.
Description
Technical Field
The invention relates to pichia pastoris for producing lactoferrin for strengthening vesicle transport and a method for promoting extracellular secretion, belonging to the technical field of biology.
Background
Lactoferrin (LF), also known as Lactoferrin, is an iron binding protein with a molecular weight of about 80kDa and is also an important globulin in milk. The study shows that LF is not only in milk, but also widely distributed in various secretions such as tears, saliva, bile and the like. Wherein the content of the milk is highest, the content of the LF in the human milk is the second, and the concentration is as high as 1.0-3.0mg/mL, which is 10 times that of the cow milk. LF not only can transport iron ions, but also has biological activities of resisting bacteria, diminishing inflammation, regulating immune response of organisms and the like.
In addition to bacteriostatic activity, LF also has obvious bactericidal activity. On the one hand, alkaline high-density charges on the LF surface are easy to be subjected to specific binding with certain biomolecules of bacteria, and the functions of cell membranes are disturbed; on the other hand, LF can specifically bind to bacterial cell membranes, resulting in increased membrane permeability and even destruction of lipid bilayer, thus playing a bactericidal role. Further researches show that the bactericidal active region of the LF is mainly positioned on N leaves and is different from the region of the iron ion binding region, and the N end of the LF generates electrostatic attraction with lipopolysaccharide, teichoic acid and the like with negative charges on bacterial cell membranes depending on the positive charges of the LF, so that the whole LF is bound to the bacterial cell membranes, and finally the cell membranes are damaged. Therefore, LF is considered a novel antibacterial, anticancer drug. LF has been added to many commercial products including infant formulas, hyperthermia beverages, fermented milk, cosmetics, and toothpastes, among others. The variety of health promoting functions of LF and its widespread use in real life has stimulated increasing research interest.
Currently, lactoferrin is obtained mainly by separation and extraction from cow's milk, however, cow's milk contains only 0.03-0.49g/L lactoferrin. In the extraction process, the price is high, and the human body can bring a certain negative influence to eat the heterologous protein, so that antigen reaction is generated. In order to obtain a large amount of lactoferrin while avoiding side effects thereof, researchers have attempted to mass-produce human lactoferrin by genetic engineering techniques. Among them, host cells producing lactoferrin mainly include escherichia coli, yeast, mammalian cells, and plant cells. Coli expression systems lack glycosylation modification mechanisms, resulting in the inability to produce bioactive lactoferrin. Mammalian cells and plant cells can be appropriately glycosylation-modified, but large-scale production is difficult due to long cell growth cycle and complicated culture.
In the current research, elements suitable for lactoferrin expression are obtained by screening promoters, signal peptides and the like, and the expression of target proteins is enhanced by over-expressing molecular chaperones related to protein synthesis in saccharomycetes, and the expression of transcription factors is enhanced at the transcriptome level to realize the up-regulation of the whole protein level of the saccharomycetes so as to enhance the expression of the target proteins. When the inventors performed SDS-PAGE, WB and ELISA on the supernatant taken after crushing Pichia pastoris together with the extracellular supernatant to detect lactoferrin production, it was found that lactoferrin production was extracellular: intracellular is 1:2, it is shown that there are many more lactoferrin that are not secreted out in time effectively in cells, limiting the yield of lactoferrin. In order to further increase the yield of lactoferrin, the problem that lactoferrin is blocked in cells and cannot be discharged out in time must be solved, thereby realizing smooth secretion of exogenous proteins.
Disclosure of Invention
In order to solve the problems, the invention discovers that the content of the lactoferrin in extracellular supernatant is obviously increased and the content of the lactoferrin in sediment and crushing supernatant is effectively reduced by fusion expression of heterozygous signal peptide ost-a and lactoferrin and introduction of vesicle transport genes BMH2, sec12 or Sly1 into pichia pastoris original strains, and proves that intracellular lactoferrin transport to the outside is effectively promoted. The method increases the expression of related genes in cells, strengthens an intracellular membrane transport system, and improves the transport efficiency of lactoferrin in yeast bodies, thereby promoting the extracellular expression of lactoferrin.
It is a first object of the present invention to provide a lactoferrin producing pichia pastoris that enhances the vesicle transport pathway, which heterologously expresses the lactoferrin gene and expresses the vesicle transport gene BMH2, sec12 or sle 1.
Further, the lactoferrin gene comprises a human lactoferrin gene, and the amino acid sequence of the human lactoferrin is shown as SEQ ID NO. 4.
Further, the lactoferrin gene is expressed by a signal peptide with a nucleotide sequence shown as SEQ ID NO. 2.
Further, the lactoferrin gene is expressed by an inducible promoter.
Further, the inducible promoters include, but are not limited to, promoter P aox1 Etc.
Further, the inducible promoter P aox1 The nucleotide sequence of (2) is shown as SEQ ID NO. 3.
Further, the vesicle transport gene BMH2 has GenBank number FR839629.1; the GenBank number of the vesicle transport gene Sec12 is FN392322.1; the GenBank number of the vesicle transport gene Sly1 is FR839631.1.
Further, the vesicle transport gene BMH2 is expressed by a constitutive promoter.
Further, the constitutive promoters include, but are not limited to, promoter P GAP Etc.
Further, the promoter P GAP The nucleotide sequence of (2) is shown as SEQ ID NO. 1.
Further, pichia pastoris X-33 is used as an original strain.
The second object of the present invention is to provide a method for constructing pichia pastoris for producing lactoferrin, comprising the steps of:
s1, amplifying a vesicle transport gene BMH2, and connecting to an expression vector pGAP-HygR to obtain a recombinant plasmid pGAP-BMH2;
s2, amplifying a lactoferrin gene, fusing and expressing a signal peptide shown in SEQ ID NO.2, and connecting the fused gene fragment to an expression vector pPICZ alpha to obtain a recombinant plasmid pPICZ alpha-ost-a-LF;
s3, converting the recombinant plasmid pGAP-BMH2 and the recombinant plasmid pPICZ alpha-ost-a-LF into a pichia pastoris host strain, and constructing the pichia pastoris for producing the lactoferrin.
The third object of the invention is to provide the application of the pichia pastoris in preparing products (such as bacteriostatic agents) containing lactoferrin or derivatives thereof in the fields of biology, pharmacy, food or chemical industry.
A fourth object of the present invention is to provide a method for producing lactoferrin, comprising the steps of: fermenting and producing by using the pichia pastoris.
Further, the fermentation system contains an inducer, including but not limited to methanol, and the like.
Further, the fermentation conditions are 28-35 ℃,180-260rpm.
A fifth object of the present invention is to provide a method for enhancing secretory expression of lactoferrin in pichia pastoris, comprising the steps of: the vesicle transport genes BMH2, sec12 or Sly1 are introduced into the Pichia host.
Further, at P GAP The vesicle transport genes BMH2, sec12 or Sly1 are expressed for the promoters.
The invention has the beneficial effects that:
the invention takes a high copy Pichia pastoris-ist-a-hLF of a lactoferrin expression cassette as an initial strain, screens different vesicle transfer related genes for strengthening secretion of expressed proteins by the Pichia pastoris strain, determines vesicle transfer genes capable of strengthening the yield of the lactoferrin by verifying the influence of the vesicle transfer related genes on the expression quantity of the lactoferrin, and obtains the Pichia pastoris strain for efficiently synthesizing the lactoferrin. The invention not only realizes the aim of improving the expression quantity of the lactoferrin, but also provides a method for increasing the secretory expression of the protein. The extracellular yield of lactoferrin of the BMH2 strain reaches 721mg/L through detection of the lactoferrin ELISA kit.
Drawings
FIG. 1 shows SDS-PAGE results of recombinant strain fermentation broth. A, fermenting liquid supernatant; b: precipitating and crushing supernatant; m:10-180Protein Marker.
FIG. 2 shows the yield of lactoferrin from supernatant of recombinant strain fermentation broth.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and specific examples, which are not intended to be limiting, so that those skilled in the art will better understand the invention and practice it.
The materials involved in the following examples are as follows:
(1) Sequence information:
BMH2，GenBank:FR839629.1；Sar1，GenBank:FR839629.1；Sec1，GenBank:FR839631.1；Sec4，GenBank:FR839630.1；Sec12，GenBank:FN392322.1；Sly1，GenBank:FR839631.1；Sso1，GenBank:FR839628.1。
promoter P GAP SEQ ID NO.1; an ost-a signal peptide, SEQ ID NO.2; promoter P aox1 ，SEQ ID NO.3；hLF，SEQ ID NO.4。
(2) Culture medium
Liquid YPD medium: 1% yeast extract, 2% peptone, 2% glucose, steam sterilizing at 115deg.C for 20min, and storing at room temperature.
BMGY medium: yeast extract 5g, tryptone 10g, distilled water 350mL, steam sterilized at 121℃for 20min, 13.4% YNB 50mL,10% glycerol 50mL,1 mM potassium phosphate buffer 50mL,0.02% biotin 1mL were added.
BMMY medium: yeast extract 5g, tryptone 10g, distilled water 350mL, steam sterilized at 121℃for 20min, 13.4% YNB 50mL,10% glycerol 50mL,1 mM potassium phosphate buffer 50mL,0.02% biotin 1mL were added.
Example 1 amplification of vesicle Transporter Gene and construction of PGAP expression plasmid
The corresponding vesicle transport genes are respectively amplified by using pichia pastoris genome as a template and using the primer sequences listed in the table 1, and the PCR products are digested and then transformed into escherichia coli competent, and a plate containing hygromycin is coated to obtain corresponding transformants. Transformants were grown overnight, plasmids were extracted, and then the correct transformants were obtained by sequencing.
TABLE 1 primer sequences for amplification chaperones
Example 2 electric transformation of Pichia pastoris after linearization of recombinant plasmid
(1) The construction of the original strain ost-a-hLF is described in Chinese patent CN202210946281.4, and comprises the following specific steps: the ost-a signal peptide shown in SEQ ID No.2 and hLF coding gene are connected to a vector pPICZ alpha to obtain a recombinant plasmid pPICZ alpha-ost-a-hLF, and the recombinant plasmid is electrically transformed into Pichia pastoris X-33 to obtain the ost-a-hLF for expressing lactoferrin.
(2) The linearized fragment was obtained by amplification with the linearized primer using the plasmid line sequenced correctly in example 1 as a template, and purified by a kit. 80. Mu.L of competent cells ost-a-hLF was mixed with 10. Mu.L of linearized recombinant plasmid DNA and transferred to an ice-bathed 0.2cm electrotransformation cup at-20℃and the electrotransformation cup with the mixture was ice-bathed for 5min. And the parameters of the electric converter are adjusted, the electric converter is placed in Pichia pastoris, the voltage is 1.5 kilovolts, the capacitance is 25 microfarads, the resistance is 200 ohms, and the time is about 5 milliseconds. After electric shock, 1ml of a pre-chilled 1M sorbitol solution on ice was quickly added to the conversion cup, gently swirled and mixed well, and the mixed solution was sucked out and transferred to a centrifuge tube. Stationary culturing at 30deg.C for 1-2h, and centrifuging at 3000rpm for 5min. Mu.l of the supernatant was discarded, the remaining cells were homogenized, plated on YPDH (containing 500. Mu.g/mL hygromycin) plates, and cultured in an incubator at 30℃for 2-4 days until single colonies were grown, and the colonies were confirmed to be correct by colony PCR.
TABLE 2 linearization primer sequences
EXAMPLE 3 recombinant Pichia Induction of lactoferrin
The correct colonies were transferred to 3mL YPD medium and incubated at 30℃and 220rpm overnight. The next day 1mL of the bacterial suspension was transferred to a 250mL Erlenmeyer flask containing 25mL of BMGY medium and incubated at 30℃for 24h at 220 rpm. The cells were collected by centrifugation at 3000rpm for 5min, washed 3 times with sterile water to remove residual glycerol, resuspended in 25mL BMMY medium, transferred to a 250mL Erlenmeyer flask, incubated at 30℃for 5 days at 220rpm with 1% methanol added every 24 hours, and induction continued to occur.
The fermentation broth was centrifuged to obtain a fermentation supernatant, and the synthesis of lactoferrin was detected by SDS-PAGE, which showed that the lactoferrin content in the fermentation broth supernatant of the strain overexpressing the BMH2 gene was significantly increased (FIG. 1A), and the lactoferrin content in the cell disruption supernatant was also significantly decreased, i.e., the intracellular lactoferrin content was decreased (FIG. 1B). The results of the detection of lactoferrin yield using ELISA kit showed that lactoferrin yield in the supernatant of fermentation broth of the over-expressed BMH2 gene strain reached 721mg/L (FIG. 2).
It is apparent that the above examples are given by way of illustration only and are not limiting of the embodiments. Other variations and modifications of the present invention will be apparent to those of ordinary skill in the art in light of the foregoing description. It is not necessary here nor is it exhaustive of all embodiments. And obvious variations or modifications thereof are contemplated as falling within the scope of the present invention.
Claims (15)
1. A pichia pastoris for producing lactoferrin for enhancing vesicle transport pathway, characterized in that: the pichia pastoris heterologously expresses a lactoferrin gene and expresses vesicle transport genes BMH2, sec12 or Sly1.
2. The pichia pastoris of claim 1, wherein: the lactoferrin gene is expressed by regulating the expression of a signal peptide with a nucleotide sequence shown as SEQ ID NO. 2.
3. The pichia pastoris of claim 1, wherein: the lactoferrin gene is expressed by an inducible promoter.
4. The pichia pastoris of claim 3, wherein: the inducible promoter comprises a promoter P aox1 。
5. The pichia pastoris of claim 1, wherein: the lactoferrin gene comprises a human lactoferrin gene hLF.
6. The pichia pastoris of claim 1, wherein: vesicle transport gene BMH2 is formed by promoter P GAP Expression is initiated.
7. The pichia pastoris of claim 1, wherein: the GenBank number of the vesicle transport gene BMH2 is FR839629.1; the GenBank number of the vesicle transport gene Sec12 is FN392322.1; the GenBank number of the vesicle transport gene Sly1 is FR839631.1.
8. The pichia pastoris of claim 1, wherein: pichia pastoris X-33 is used as an original strain.
9. The method for constructing pichia pastoris of any one of claims 1 to 8, comprising the steps of:
s1, amplifying a vesicle transport gene BMH2, and connecting to an expression vector pGAP-HygR to obtain a recombinant plasmid pGAP-BMH2;
s2, amplifying a lactoferrin gene, fusing and expressing a signal peptide shown in SEQ ID NO.2, and connecting the fused gene fragment to an expression vector pPICZ alpha to obtain a recombinant plasmid pPICZ alpha-ost-a-LF;
s3, converting the recombinant plasmid pGAP-BMH2 and the recombinant plasmid pPICZ alpha-ost-a-LF into a pichia pastoris host strain, and constructing the pichia pastoris for producing the lactoferrin.
10. Use of pichia pastoris as claimed in any one of claims 1 to 8 for the preparation of a product comprising lactoferrin or a derivative thereof in the biological, pharmaceutical, food or chemical industries.
11. The use according to claim 10, characterized in that: the product comprises a bacteriostatic agent.
12. A method for producing lactoferrin, characterized by: comprising the step of fermentation production using the Pichia pastoris of any one of claims 1 to 8.
13. The method according to claim 12, wherein: the fermentation condition is 28-35 ℃,180-260rpm.
14. A method for enhancing secretory expression of lactoferrin in pichia pastoris, comprising the steps of: the vesicle transport genes BMH2, sec12 or Sly1 are introduced into the Pichia host.
15. The method according to claim 14, wherein: with P GAP The vesicle transport genes BMH2, sec12 or Sly1 are expressed for the promoters.
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| CN117903295A (en) * | 2024-03-19 | 2024-04-19 | 北京国科星联科技有限公司 | Kluyveromyces marxianus for secretory expression of lactoferrin and construction method and application thereof |
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