CN112940956A - Pichia pastoris for enhancing lactoferrin expression by adopting double promoters and application thereof - Google Patents
Pichia pastoris for enhancing lactoferrin expression by adopting double promoters and application thereof Download PDFInfo
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Abstract
The invention relates to a pichia pastoris for enhancing lactoferrin expression by adopting double promoters and application thereof, wherein a Paox1 and Pgap tandem promoter is constructed in a fusion PCR mode, the interval length and the sequence of the promoters of the two promoters are further optimized, and fermentation experiments show that the interval sequences with different lengths cause different transcription levels. When the spacer sequence reaches 50bp, compared with a single promoter Paox, the transcription level of lactoferrin is improved by nearly 6.5 times, and the expression quantity of the lactoferrin is found to be increased by about 3.7 times through western blot and reaches 150 mg/L. The method can effectively improve the expression quantity of the lactoferrin, and reduce the consumption of the methanol by more than 50%.
Description
Technical Field
The invention belongs to the technical field of metabolic engineering, and particularly relates to pichia pastoris for enhancing lactoferrin expression by adopting a double promoter and application thereof.
Background
Lactoferrin (Lactoferrin, LF) is a non-heme iron binding protein, a member of the transferrin family, and functions to transport iron in serum. Lactoferrin has antioxidant, anticancer, anti-inflammatory and microbial infection-protective properties by chelating iron or interacting directly with infectious agents. Thus, LF is considered a new antibacterial, anticancer drug, and has been added to many commercial products, including infant formula, heat-treated beverages, fermented milks, cosmetics, and toothpaste. The diverse health-promoting functions of LF and its widespread use in real life have stimulated increasing research interest. Lactoferrin has been an important ingredient in commercial milk powders in many countries.
Currently, the way of obtaining lactoferrin is mainly to separate and extract from cow milk, however, cow milk contains only 0.03-0.49g/L of lactoferrin. In the extraction process, the price is high, and the human body eating the heterologous protein can bring certain negative effects to generate antigen reaction. In order to widely apply LF and avoid side effects caused by LF, the mass production of human LF by using a genetic engineering technology becomes a hotspot and development trend of LF research.
During the past decade, researchers have conducted heterologous expression of human LF using bacterial, fungal, plant and animal cells as hosts. Coli as a host is able to synthesize heterologous proteins with high efficiency, but prokaryotes lack the corresponding post-translational modifications (e.g., glycosylation) resulting in the inefficient function of lactoferrin. Although mammalian cells as hosts can be glycosylated and modified with lactoferrin, and can better simulate the state in human body, mammalian cells have long growth cycle and complex culture, and are difficult to realize large-scale production. The yeast expression system has the characteristics of fast prokaryotic organism growth and simple operation, has post-translational processing and modifying functions of various mammalian cells, and is an ideal host for exogenous expression of eukaryotic genes.
The expression vector is one of the main components in a pichia pastoris expression system, mainly comprises a promoter sequence, a transcription termination sequence and 3' region messenger RNA essential in a polyadenylation process, wherein the promoter region is a key factor for ensuring the expression quantity of the foreign protein. The methanol oxidase promoter Paox1 is one of the most commonly used promoters in pichia pastoris. The promoter Paox1 requires methanol as the sole carbon source for induction, which is potentially hazardous for use in the food industry. Therefore, the primary problem of increasing the expression level of lactoferrin is to find a way to express lactoferrin efficiently and reduce the amount of methanol.
Disclosure of Invention
In order to solve the technical problems, the invention adds a constitutive promoter Pgap on the basis of the Paox1 promoter to form a model for tandem expression of double promoters. The double promoters expressed in series can not only reduce the using amount of methanol, but also increase the expression amount of lactoferrin.
The first purpose of the invention is to provide pichia pastoris for enhancing lactoferrin expression by adopting double promoters, wherein the pichia pastoris uses a promoter Paox1 and a promoter Pgap to serially start expression of a lactoferrin gene.
Further, the promoter Paox1 is upstream of the promoter Pgap.
Furthermore, a 25-75 bp spacing sequence is arranged between the promoter Paox1 and the promoter Pgap.
Furthermore, the length of the spacer sequence is 50bp, and the nucleotide sequence is shown as SEQ ID NO. 3.
Further, the host of pichia is pichia pastoris GS 115.
Further, the nucleotide sequence of the lactoferrin gene is shown in SEQ ID NO. 1.
The second purpose of the invention is to provide the construction method of the pichia pastoris, which comprises the following steps:
s1, integrating the Pgap promoter on a plasmid for expressing lactoferrin by adopting a promoter Paox1 to obtain a recombinant plasmid;
s2, transforming the recombinant plasmid into a pichia pastoris host to obtain the recombinant pichia pastoris.
Further, in step S1, the Pgap promoter is integrated downstream of the promoter Paox 1.
Further, in the step S1, a random spacer sequence of 25-75 bp is inserted between the promoter Paox1 and the promoter Pgap.
The third purpose of the invention is to provide the application of the pichia pastoris in the fermentation production of lactoferrin.
By the scheme, the invention at least has the following advantages:
according to the invention, Paox1 and Pgap tandem promoters are constructed in a fusion PCR mode, the interval length of the two promoters and the sequence of the promoters are further optimized, and fermentation experiments show that the interval sequences with different lengths cause different transcription levels. When the spacer sequence reaches 50bp, compared with a single promoter Paox, the transcription level of lactoferrin is improved by nearly 6.5 times, and the expression quantity of the lactoferrin is found to be increased by about 3.7 times through western blot and reaches 150 mg/L. The method can effectively improve the expression quantity of the lactoferrin, and reduce the consumption of the methanol by more than 50%.
The foregoing is a summary of the present invention, and in order to provide a clear understanding of the technical means of the present invention and to be implemented in accordance with the present specification, the following is a preferred embodiment of the present invention and is described in detail below.
Drawings
FIG. 1 tandem promoter map;
FIG. 2 shows the effect of different spacer sequences on tandem promoter activity.
FIG. 3 Effect of different sequences of promoters on tandem promoter Activity
FIG. 4 measurement of lactoferrin production.
Detailed Description
The related detection method comprises the following steps: the lactoferrin production was measured using an ELISA kit purchased from Biotechnology under the limited stock (cat # D711315) according to the instructions. The lactoferrin gene transcription level was detected using the SYBR Green method.
Example 1: construction of tandem promoters by fusion PCR
The promoter Pgap was amplified by primers 1 and 2 using the yeast genome as a template, as shown in Table 1. And sequencing and identifying the PCR product. Then, a Pgap promoter and a lactoferrin gene are integrated on a pic9k plasmid by means of Gibson assembly to obtain a lactoferrin expression plasmid, and the map is shown in fig. 1.
TABLE 1 primers for amplification of lactoferrin
In order to change the interval size between the two promoters, random sequences with different lengths (including 25, 50, 75, 100, 150 and 200bp, and nucleotide sequences shown as SEQ ID NO. 2-7) are inserted between the two promoters in a Gibson assembly mode. The recombinant plasmid was then transformed into pichia pastoris by means of electrotransformation, and then the change in fluorescence of green fluorescent protein was detected by fermentation (fig. 2). The results show that the fluorescence level decreases with increasing spacer sequence when the spacer sequence exceeds 50 bp. When the spacer sequence was 50bp, the fluorescence level increased nearly 5.4 fold compared to the promoter Paox 1.
Example 2: recombinant plasmid electrotransformation pichia pastoris
80 μ L of competent cells and 10 μ L of linearized recombinant plasmid DNA were mixed well and transferred to a 0.2cm electric transformation cuvette which was ice-bathed at-20 ℃ and the electric transformation cuvette with the mixed solution was ice-bathed for 5 min. The parameters of the electric converter are adjusted and placed in the Pichia pastoris range, 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 1M sorbitol solution precooled on ice is rapidly added into the conversion cup, the mixture is gently sucked and beaten and uniformly mixed, and the mixture is sucked out and transferred into a centrifuge tube. Standing at 30 deg.C for 1-2h, and centrifuging at 3000rpm for 5 min. Discarding 800 μ l of supernatant, uniformly blowing the residual thallus, coating on MD plate, and culturing in 30 deg.C incubator for 2-4 days until single colony grows out.
Example 3: determination of tandem promoter transcription level in recombinant pichia pastoris
Culturing the obtained transformants in BMGY and BMMY respectively, adding methanol with different concentrations in the culturing process, collecting thalli, rapidly cooling and freezing by liquid nitrogen, crushing the thalli by using a mortar, extracting RNA by using Trizol, obtaining cDNA according to the specification of a reverse transcription kit, and further determining the transcription level of lactoferrin in different thalli by using a fluorescence quantitative kit. The primer sequences for detecting the transcription level of the lactoferrin are shown in the table 2.
TABLE 2 Lactoferrin qPCR primer sequences
| |
TTGGCTGTTGCTGTTGTTAGAAGAT |
| Primer 4 | CAGAACCAGGAGCACAAGATTGAG |
The effect of different spacer sequences on the transcription activity of the tandem promoter was examined, and the results are shown in FIG. 3, which shows that the tandem promoter constructed can effectively enhance the transcription of a heterologous gene when Paox1 is upstream. Wherein, when the spacer sequence is 50bp and methanol is added to 1%, the transcription level of lactoferrin is increased by about 6.5 times. The transcriptional level of lactoferrin was consistent with that of Pgap alone when no methanol was added to the broth, and increased 5.6-fold when the final concentration of methanol was 0.5%.
Example 4: detection of translation level of lactoferrin in recombinant pichia pastoris
The strain (Paox 1-Pgap-lactoferrin) with the highest lactoferrin transcription level is cultured for 16h by using BMGY culture medium, then BMMY is used for induction fermentation, methanol with different volumes is added every day, the final content of methanol in the fermentation liquid is kept at 0.5%, and the fermentation is continued for 5 days. Then, the supernatant was collected by centrifugation, concentrated by centrifugation, and quantitatively analyzed for lactoferrin using SDS-PAGE and western blot. As shown in FIG. 4, the expression level of lactoferrin increased gradually with the increase of fermentation time, and the final yield reached 150 mg/L.
Comparative example
The human lactoferrin gene is expressed only by using the promoter Paox1, the final concentration of the added methanol is 1%, and then the methanol is added every day, so that the final concentration is kept unchanged. Then, the supernatant was collected by centrifugation, and the amount of protein expression was determined by SDS-PAGE after concentration.
The above is only a preferred embodiment of the present invention, and is not intended to limit the present invention, it should be noted that, for those skilled in the art, many modifications and variations can be made without departing from the technical principle of the present invention, and these modifications and variations should also be regarded as the protection scope of the present invention.
Sequence listing
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gttgcttttg ttaaggatgt tactgttttg caaaatactg atggtaataa caatgaagct 1740
tgggctaaag atttgaaatt ggctgatttt gctttgttgt gtttggatgg taaaagaaaa 1800
cctgttactg aggctagatc ttgtcatttg gcaatggctc caaatcatgc tgtcgtttct 1860
agaatggata aagttgaaag attgaaacaa gttttgttgc atcaacaagc taaatttggt 1920
agaaatggat ctgattgtcc tgataaattc tgtttgttcc aatctgaaac taaaaatttg 1980
ttgtttaatg ataatactga atgtttggct agattgcatg gtaaaactac ttatgaaaaa 2040
tatttgggtc cacaatatgt tgctggtatt actaatttga aaaaatgttc tacttctcca 2100
ttgttggaag cttgtgaatt tttgagaaaa 2130
<210> 2
<211> 25
<212> DNA
<213> (Artificial sequence)
<400> 2
tgcgttatcc cctgattctg tggat 25
<210> 3
<211> 50
<212> DNA
<213> (Artificial sequence)
<400> 3
accgtattac cgcctttgag tgagctgata ccgctcgccg cagccgaacg 50
<210> 4
<211> 75
<212> DNA
<213> (Artificial sequence)
<400> 4
ctctccccgc gcgttggccg attcattaat gcagctggca cgacaggttt cccgactgga 60
<210> 5
<211> 100
<212> DNA
<213> (Artificial sequence)
<400> 5
agtacaatct gctctgatgc cgcatagtta agccagcccc gacacccgcc aacacccgct 60
gacgcgccct gacgggcttg tctgctcccg gcatccgctt 100
<210> 6
<211> 150
<212> DNA
<213> (Artificial sequence)
<400> 6
aagctgtggt atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc 60
cccgacaccc gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg 120
cttacagaca agctgtgacc gtctccggga 150
<210> 7
<211> 200
<212> DNA
<213> (Artificial sequence)
<400> 7
ttgaaaagct gtggtatggt gcactctcag tacaatctgc tctgatgccg catagttaag 60
ccagccccga cacccgccaa cacccgctga cgcgccctga cgggcttgtc tgctcccggc 120
atccgcttac agacaagctg tgaccgtctc cgggagctgc atgtgtcaga ggttttcacc 180
Claims (10)
1. A pichia pastoris for enhancing lactoferrin expression by adopting a double promoter is characterized in that the pichia pastoris uses a promoter Paox1 and a promoter Pgap to serially start the expression of a lactoferrin gene.
2. The Pichia pastoris of claim 1, wherein the promoter Paox1 is upstream of the promoter Pgap.
3. The pichia pastoris of claim 1, wherein a 25-75 bp spacer sequence is arranged between the promoter Paox1 and the promoter Pgap.
4. The pichia pastoris of claim 1, wherein the length of the spacer sequence is 50bp, and the nucleotide sequence is shown as SEQ ID No. 3.
5. The pichia pastoris of claim 1, wherein the host of pichia is pichia pastoris GS 115.
6. The pichia pastoris of claim 1, wherein the nucleotide sequence of the gene of lactoferrin is shown as SEQ ID No. 1.
7. A construction method of Pichia pastoris according to any one of claims 1 to 6, characterized by comprising the following steps:
s1, integrating the Pgap promoter on a plasmid for expressing lactoferrin by adopting a promoter Paox1 to obtain a recombinant plasmid;
s2, transforming the recombinant plasmid into a pichia pastoris host to obtain the recombinant pichia pastoris.
8. The method of claim 7, wherein in step S1, the Pgap promoter is integrated downstream of the Paox1 promoter.
9. The method of claim 7, further comprising inserting a random spacer sequence of 25-75 bp between the promoter Paox1 and the promoter Pgap in the step of S1.
10. Use of pichia pastoris according to any one of claims 1 to 6 for the fermentative production of lactoferrin.
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| CN116970503A (en) * | 2023-07-25 | 2023-10-31 | 江南大学 | Pichia pastoris for producing lactoferrin for strengthening vesicle transport and method for promoting extracellular secretion |
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| CN116970503A (en) * | 2023-07-25 | 2023-10-31 | 江南大学 | Pichia pastoris for producing lactoferrin for strengthening vesicle transport and method for promoting extracellular secretion |
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Application publication date: 20210611 |