CN110028562B - Optimized Komagataella pastoris yeast alpha-factor signal peptide and application - Google Patents
Optimized Komagataella pastoris yeast alpha-factor signal peptide and application Download PDFInfo
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Abstract
The invention discloses an optimized Komagataella pastoris yeast alpha-factor signal peptide and application thereof. The invention introduces an N glycosylation site into the Komagataella pastoris yeast alpha-factor signal peptide with the amino acid sequence shown as SEQID NO. 1 to obtain the optimized Komagataella pastoris yeast alpha-factor signal peptide, and the amino acid sequence of the optimized Komagataella pastoris yeast alpha-factor signal peptide is shown as SEQID NO. 3. The optimized alpha-factor signal peptide is particularly suitable for guiding the secretory expression of the foreign protein in pichia pastoris, compared with the signal peptide before optimization, the expression level is improved by 25-35 times, the application of the optimized alpha-factor signal peptide is favorable for screening high-expression strains, and the optimized alpha-factor signal peptide has the advantages of reducing the production cost, facilitating subsequent purification and the like.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to an optimized Komagataella pastoris yeast alpha-factor signal peptide and application thereof.
Background
In recent years, with the improvement of living standard and the enhancement of health consciousness, the demand of various protein drugs (such as vaccine, immunoglobulin, antibody, protein factor and the like) for treatment or health care has been rising year by year. However, many pharmaceutical proteins cannot be obtained by separation and purification of natural materials, and must be expressed using a foreign protein system.
In the existing foreign protein expression system, although a prokaryotic system has simple genetic operation, low culture cost and high product expression, the foreign protein cannot be subjected to post-translational modification. Eukaryotic systems can perform post-translational processing modification on expressed foreign proteins, so that the obtained products generally have higher biological activity compared with prokaryotic systems. Because the pichia pastoris expression system serving as a unicellular eukaryote has the characteristics of both eukaryote and prokaryote, the pichia pastoris expression system is favored by researchers when researching secretory proteins needing to be processed and modified. Most secreted proteins have important physiological regulatory functions, such as peptide hormones, neuropeptides, insulin and its receptors, growth and differentiation factors, cell surface receptors, extracellular metalloproteinases, blood coagulation factors, adhesion molecules, plasma proteins, etc., of which a considerable proportion are candidates for pharmaceutical proteins or have been successful protein drugs.
When pichia pastoris is used for expressing foreign secretory proteins, a signal peptide is needed to guide the foreign proteins to enter a secretory channel, and finally the proteins can be secreted outside cells. The efficiency of the signal peptide determines the secretory expression of the foreign protein. Therefore, the development of a signal peptide capable of efficiently secreting the protein to the extracellular space in pichia pastoris is important for the secretory expression of the foreign protein.
Disclosure of Invention
The invention aims to provide an optimized Komagataella pastoris alpha-factor signal peptide and application thereof, wherein the optimized signal peptide can efficiently guide secretion of foreign proteins in pichia pastoris
In order to achieve the purpose, the invention adopts the following technical scheme:
firstly, the invention analyzes the genome sequence of Komagataella pastoris, finds an alpha-factor gene, and obtains an alpha-factor gene signal peptide from the alpha-factor gene, wherein the amino acid sequence of the alpha-factor gene signal peptide is shown as SEQIDNO 1, and the nucleotide sequence for coding the alpha-factor gene signal peptide is shown as SEQID NO 2.
The alpha-factor signal peptide can guide the secretory expression of a target protein.
Further, an N glycosylation site is introduced into the amino acid sequence of the alpha-factor signal peptide to obtain the optimized alpha-factor signal peptide, the amino acid sequence of the optimized alpha-factor signal peptide is shown as SEQ ID NO. 3, and the nucleotide sequence for coding the alpha-factor signal peptide is shown as SEQ ID NO. 4.
The N glycosylation sites can be introduced into any position of the Komagataella pastoris yeast alpha-factor signal peptide, and the number of the introduced N glycosylation sites can be 1 or more. In the present invention, the N-glycosylation site is located at amino acid 113.
The optimized alpha-factor signal peptide can efficiently guide the secretion expression of foreign proteins.
The foreign protein can be any protein, and is green fluorescent protein in the invention.
The optimized alpha-factor signal peptide can be added into a Komagataella pastoris intracellular expression vector for guiding the secretory expression of foreign proteins in the Komagataella pastoris.
The intracellular expression vector may be any one of: pGAPA, B & C, pAO815, pPIC3.5K, pPIC3.5, pFLD1 and pPICZA, B & C. In the examples of the present invention, the intracellular expression vector used is pPICZA.
The invention compares the signal peptide of the Komagataella pastoris yeast alpha-factor before and after sequence optimization with the expression of exogenous protein. The expression amount measurement result shows that the optimized alpha-factor signal peptide is particularly suitable for Komagataella pastoris, compared with the signal peptide before optimization, the expression level is improved by 25-35 times, the application of the signal peptide is favorable for screening high-expression strains, and the signal peptide has the advantages of reducing the production cost, facilitating subsequent purification and the like.
Drawings
FIG. 1 is a diagram of the expression of a foreign protein guided by the Komagataella pastoris yeast alpha-factor signal peptide;
FIG. 2 is a comparison graph of the expression level of foreign proteins guided by the Komagataella pastoris yeast α -factor signal peptide before and after optimization.
Detailed Description
The following examples illustrate the invention in detail, but do not limit the scope of the invention.
Before optimization
The amino acid sequence of the Komagataella pastoris yeast alpha-factor signal peptide is shown as SEQ ID NO. 1.
The nucleotide sequence of the nucleotide for coding the optimized Komagataella pastoris yeast alpha-factor signal peptide is shown as SEQ ID NO. 2.
After optimization
The amino acid sequence of the optimized Komagataella pastoris yeast alpha-factor signal peptide is shown as SEQ ID NO. 3.
The nucleotide sequence of the nucleotide for coding the optimized Komagataella pastoris yeast alpha-factor signal peptide is shown as SEQ ID NO. 4.
EXAMPLE 1 Synthesis of the coding sequence for the Yeast alpha-factor Signal peptide of Komagataella pastoris
The coding sequence encoding the Komagataella pastoris yeast α -factor signal peptide was synthesized by bio-corporation and cloned into a T-vector, designated pT- α -factor in this example.
Example 2 construction of a Yeast expression vector containing Komagataella pastoris Yeast alpha-factor Signal peptide Komagataella pastoris
In this example, pPICZA is used as a framework to describe the construction process of a yeast expression vector containing a Komagataella pastoris alpha-factor signal peptide.
The vector pPICZA was digested with Sfu I and EcoR I, and the gel was split and purified for use.
Designing a pair of primers:
an upstream primer: tt is a Chinese characterttcgaaatgaaaccactttataagc (Sfu I site sequence underlined)
A downstream primer: cg (cg)gaattcagcttctgccttctcttgcc (underlined section EcoR I site sequence)
The coding sequence for the Komagataella pastoris yeast α -factor signal peptide was amplified from the vector pT- α -factor and Sfu I and EcoR I cleavage sites were introduced at both ends, respectively. And purifying the PCR amplification product, performing double enzyme digestion by using Sfu I and EcoR I enzymes, and tapping and purifying the gel for later use.
The vector pPICZA after double enzyme digestion of Sfu I and EcoR I is connected with the coding sequence of the Komagataella pastoris alpha-factor signal peptide. The ligation product is transformed into escherichia coli, a single colony growing from a Zeocin plate is selected for sequencing confirmation, and a vector with correct sequence is named as pPICZA K.pa -α-factor。
Example 3 construction of optimized Komagataella pastoris Yeast alpha-factor Signal peptide Komagataella pastoris Yeast expression vector
Through the amino acid sequence analysis of the Komagataella pastoris alpha-factor signal peptide, the serine (S) at the 113 th site of the Komagataella pastoris alpha-factor signal peptide can be changed into an N-glycosyl modified site through point mutation. Thus, a pair of point mutation primers was designed:
an upstream primer: GAGAGGTTCAGTTCAATTGGACACACACATTAGG
A downstream primer: CCTAATGTGTGTCCAATTGAACTGAACTCTCTC
With pPICZA K.pa The alpha-factor vector is used as a framework, serine (S) at the 113 th site of the Komagataella pastoris alpha-factor signal peptide is mutated into asparagine (N) by using a point mutation kit and operating according to the instruction, and the newly constructed recombinant plasmid is named as pPICZA K.pa - S113N - α -factor. The point mutation kit used in this example is Fast Mutagenesis System, a product of Beijing Quanji Biotechnology (TransGen Biotech) Ltd.
Example 4 expression of foreign proteins in Komagataella pastoris using expression vectors containing Komagataella pastoris yeast alpha-factor signal peptide before and after optimization
In the embodiment, enhanced Green Fluorescent Protein (EGFP) is used as a reporter protein to detect the capability of guiding the secretion and expression of foreign proteins by Komagataella pastoris yeast alpha-factor signal peptide before and after optimization.
EGFP fragments were amplified from commercial vector pEGFP-N1 using a pair of specific primers containing EcoR I cleavage sites at the 5' end.
An upstream primer: cg (cg)gaattcatggtgagcaagggcgagag (underlined part is EcoR I site sequence)
A downstream primer: cg (cg)gaattcttactgtacagcctgtccat (EcoR I site sequence in underlined part)
Respectively cloning the EGFP fragments amplified to an expression vector pPICZA by using a DNA recombination technology K.pa -alpha-factor and pPICZA K.pa - S113N At the-alpha-factor EcoR I siteThe vectors are named pPICZA respectively K.pa -alpha-factor-EGFP and pPICZA K.pa - S113N -α-factor-EGFP。
pPICZA K.pa -alpha-factor-EGFP and pPICZA K.pa - S113N Before the-alpha-factor-EGFP vector is used for electrically transforming the Komagataella pastoris yeast, the vector is linearized by Sac I enzyme and purified after linearization.
Adding 100 μ l of freshly prepared competent Komagataella pastoris yeast into 1-10 μ g of linearized vector; transferring into an electric rotating cup (specification of 0.2 mm); a Bio-Rad GenePulser electric conversion instrument is used under the conditions of 1500V,25 muF and 200 omega, 1ml of 1M sorbitol (precooling) is added to the mixture immediately after electric shock, and the mixture is subjected to static culture for 1-2h in an incubator at the temperature of 28-30 ℃. After the culture, 200-400. Mu.l of the culture solution was spread on YPD plates (10 cm) containing Zeocin, and the plates were placed in a constant temperature incubator at 28-30 ℃ for inverted culture.
Selecting a single colony on the YPD plate to be cultured in a constant temperature shaking table of 5ml BMGY,28-30 ℃ and 250r/min for 24h; and (5) observing the growth condition and whether the bacteria are infected or not in the BMGY by microscopic examination. And centrifugally collecting thalli at room temperature of 1500-3000g, re-suspending the thalli by using an induction culture medium BMMY, and adjusting the dosage of the BMMY culture medium to enable the final OD600 value to be about 1.0. Culturing the culture solution in a constant temperature shaking table at 28-30 ℃. Every 24h, methanol (1% of the induction culture volume is added) is added; after methanol induction for 72h, the fermentation supernatant was subjected to Western blot detection using EGFP antibody, and the results are shown in FIG. 2.
FIG. 1 is a diagram showing that a Komagataella pastoris yeast α -factor signal peptide guides the expression of a foreign protein before optimization, and it can be seen that the Komagataella pastoris yeast α -factor signal peptide can guide the secretion of the foreign protein but has a low secretion level.
FIG. 2 is a comparison graph of the expression level of foreign proteins guided by the Komagataella pastoris yeast α -factor signal peptide before and after optimization. The determination result of the expression quantity in figure 2 shows that the optimized alpha-factor signal peptide is particularly suitable for guiding the secretory expression of the foreign protein in the Komagataella pastoris yeast, compared with the signal peptide before optimization, the expression level is improved by more than 25 times, and the application of the optimized alpha-factor signal peptide is favorable for screening high-expression strains and has the advantages of reducing the production cost, facilitating subsequent purification and the like.
Sequence listing
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gatgatgtcg gtgttcgcat gtccgctatc aaagaagaaa atcctgacag acattttgtt 180
ggcgacgaaa ttcctgatga ggccgtaatc cagtcattca agtcaaaaag gagagaaatc 240
ttattccttc ttgatgatat cggatcagct gtcgagttta taggtagtaa cctagcccag 300
attgaggcta atcatttgtc ggagagagtt cagttcagtt ggacacacat taggaaaaac 360
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caaccatttg gcaagagaga ggcagaagct 390
Claims (5)
1. An optimized Pichia yeastKomagataella pastoris) An α -factor signal peptide characterized by: it is prepared from the Pichia yeast (SEQ ID NO: 1)Komagataella pastoris) Introducing an N into an amino acid sequence of an alpha-factor signal peptideGlycosylation sites are obtained, and the amino acid sequence is shown as SEQID NO. 3.
2. A recombinant yeast strain encoding the optimized Pichia pastoris (Pichia pastoris) (of claim 1)Komagataella pastoris) A nucleic acid for an α -factor signal peptide, wherein: the nucleic acid sequence is shown as SEQ ID NO. 4.
3. The optimized pichia pastoris (pichia pastoris) of claim 1Komagataella pastoris) Use of an alpha-factor signal peptide, characterized in that; the signal peptide is used for guiding the secretion expression of the foreign protein in pichia pastoris.
4. Use according to claim 3, characterized in that; the foreign protein is any protein.
5. A Pichia expression vector, which is characterized in that: the vector contains optimized pichia pastoris (Pichia pastoris) (A) capable of expressing the gene of claim 1Komagataella pastoris) Nucleic acid sequence of alpha-factor signal peptide.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5602034A (en) * | 1987-12-30 | 1997-02-11 | Chiron Corporation | Expression and secretion of heterologous proteins in yeast employing truncated alpha-factor leader sequences |
| CN101538318A (en) * | 2009-03-06 | 2009-09-23 | 中国人民解放军军事医学科学院生物工程研究所 | Signal peptide, coding genes thereof and application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5602034A (en) * | 1987-12-30 | 1997-02-11 | Chiron Corporation | Expression and secretion of heterologous proteins in yeast employing truncated alpha-factor leader sequences |
| CN101538318A (en) * | 2009-03-06 | 2009-09-23 | 中国人民解放军军事医学科学院生物工程研究所 | Signal peptide, coding genes thereof and application |
Non-Patent Citations (3)
| Title |
|---|
| 吴静雯.α-factor信号肽及其N-糖基化修饰对毕赤酵母外源蛋白分泌的影响.《中国优秀硕士学位论文全文数据库 基础科学辑》.2020,A006-77. * |
| 熊爱生等.信号肽序列对毕赤酵母表达外源蛋白质的影响.《生物化学与生物物理学报》.2003,(第02期), * |
| 石艺平等.不同信号肽对毕赤酵母表达漆酶的影响.《微生物学报》.2014,(第12期), * |
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