Pectinase artificial sequence and expression method and application thereof
阅读说明：本技术 一种果胶酶人工序列及其表达方法和应用 (Pectinase artificial sequence and expression method and application thereof ) 是由 李洪波 董海丽 邓伟思 严盼 张赛名 于 2019-11-29 设计创作，主要内容包括：本发明涉及一种果胶酶人工序列及其表达方法和应用,该基因至少含有下列核苷酸序列之一的DNA片：1)序列表中SEQ ID NO.1的核苷酸序列；2)与SEQ ID NO.1所示核苷酸序列具有90％以上同源性、且编码相同生物学功能蛋白质的核苷酸序列；或3)与SEQ ID NO.1所示核苷酸序列杂交且编码相同生物学功能蛋白质的核苷酸序列。按照本发明所述的基因序列进一步构建重组载体并转化酵母,可实现该重组果胶酶在甲醇诱导下的分泌表达,通过DEAE阴离子交换纯化,可得到纯度高于95％活性重组果胶蛋白,该活性酶蛋白具有很强的澄清果汁的活性。(The invention relates to a pectinase artificial sequence, an expression method and application thereof, wherein the gene at least contains a DNA sheet of one of the following nucleotide sequences: 1) the nucleotide sequence of SEQ ID NO.1 in the sequence table; 2) a nucleotide sequence which has more than 90 percent of homology with the nucleotide sequence shown in SEQ ID NO.1 and codes the protein with the same biological function; or 3) a nucleotide sequence which is hybridized with the nucleotide sequence shown in SEQ ID NO.1 and encodes the protein with the same biological function. According to the gene sequence of the invention, a recombinant vector is further constructed and yeast is transformed, so that the secretory expression of the recombinant pectinase under the induction of methanol can be realized, and the active recombinant pectic protein with the purity higher than 95 percent can be obtained through DEAE anion exchange purification, and the active pectic protein has strong activity of clarifying juice.)
1. An artificial pectinase sequence, which is characterized in that the similarity between the sequence and a nucleotide sequence shown in SEQ ID NO.1 is more than or equal to 90%.
2. The pectinase artificial sequence of claim 1 wherein the sequence comprises the nucleotide sequence shown as SEQ ID No. 1.
3. The pectinase artificial sequence of claim 1 wherein the sequence is the nucleotide sequence shown in SEQ ID No. 1.
4. Use of the artificial pectinase sequence according to any one of claims 1-3 for the construction of recombinant vectors, expression cassettes or recombinant bacteria.
5. Use of the artificial pectinase sequence according to any one of claims 1-3 wherein the protein encoded by the nucleotide sequence shown in SEQ id No.1 is used for juice clarification.
6. A method for expressing an artificial sequence of pectinase, which comprises the following steps:
step one, recombining a nucleotide sequence shown in SEQ ID NO.1 into a pPICZ α vector, transforming the vector into a Pichia pastoris host bacterial cell, and screening by using a YPD plate with 1000 mug/mL Zeocin to obtain a transformant with high Zeocin resistance;
step two, culturing the obtained transformant to OD by using BMGY medium60010-12, centrifugally collecting cell precipitates, re-suspending the cells by using a BMMY culture medium, adding methanol to enable the mass fraction of the cells to be 1-2%, inducing for 1-3 days, and screening a transformant for high-level secretory expression of pectinase;
and step three, culturing a transformant for high-level secretory expression of pectinase by using BMGY for mass growth, then suspending cells by using BMMY culture medium and continuing induction culture at 28 ℃, and supplementing methanol to keep the mass fraction of the cells at 1-2%.
7. The method for expressing an artificial sequence of pectinase according to claim 6, which further comprises the step four of protein purification: purifying the supernatant of the BMMY culture medium in the third step by using a DEAE ion exchange column, firstly balancing the chromatographic column by using a balance buffer solution, then passing the supernatant which is subjected to ion removal and pH adjustment through dialysis through the column, rinsing the column by using a pH 8.0 buffer solution containing 30mM NaCl, and then eluting the target protein by using a pH6.0 buffer solution containing 100mM NaCl.
Technical Field
The invention belongs to the technical field of biomolecule cloning, and relates to a preparation method and application of an artificial sequence of a high-activity pectinase gene and a recombinant protein thereof.
Background
China is a large world with fruit production, and the total fruit yield in China is kept above forty-five million tons for nearly ten years. The fruit resources are rich, and a good foundation is provided for the development of the fruit juice industry in China. At present, the market competition of the fruit juice is intense, the brands are numerous, a plurality of processing enterprises are in a predicament, and how to develop the fruit juice industry becomes a problem worthy of attention. A large bottle strength in fruit juice production is that the produced fruit juice is relatively turbid, so that the quality and the taste of the fruit juice are influenced. The use of pectinase for clarifying fruit and vegetable juice has been used as early as the 20 th century in the 30 s. Generally, the fruit and vegetable juice products have the phenomena of turbidity and precipitation, which not only affect the appearance of the products, but also directly affect the quality and stability of the fruit and vegetable juice. Therefore, clarification is required during the processing. After the pectin in the fruit and vegetable juice is partially hydrolyzed under the action of pectinase, flocculate can be removed through centrifugation and filtration, thereby achieving the purpose of clarification. Therefore, the development of a highly active pectinase for clarifying fruit juice is urgently needed.
The inventor discovers a novel high-activity pectinase which is high in activity and is derived from poria cocos in previous researches, but the expression level of the pectinase in the poria cocos is very low, so that the high-efficiency expression of the novel recombinase protein by using a foreign gene expression system is a necessary way for developing the pectinase.
The production of recombinant pectinase by using an exogenous gene expression system is one of the main technical means for obtaining pectinase in large quantities. Currently, many expression systems have been developed such as: baculovirus expression systems, prokaryotic expression systems, yeast expression systems, filamentous fungal expression systems, insect cell expression systems, plant expression systems, mammalian expression systems, and the like. The genetic background of the escherichia coli is clear, and the escherichia coli becomes a preferred expression system of the exogenous gene due to the characteristics of short period, high efficiency, easy operation, safe use and the like. Coli is expressed in BL21(DE3) after transformation, but all obtained inclusion bodies are inactive, and soluble protein which can only be obtained by dozens to hundreds of micrograms per liter of culture medium can be obtained by dissolving, denaturing, renaturing and purifying in vitro under proper conditions. In Escherichia coli, toxic and side effects may be caused due to the presence of LPS, so that the toxicity of the expressed and purified product is often required to be analyzed and determined. Finally, to obtain a high purity protein, it is usually required to go through multiple purification steps, and the more purification steps, the lower the yield of the protein, and the more likely it is to cause inactivation of the target product, so the expression system is not suitable for mass production of recombinant pectinase. Exogenous gene expression systems such as insect cell expression systems, plant expression systems, mammalian expression systems and the like have high requirements on technology, equipment and technical level, so that the produced products are often very expensive and are not suitable for mass and low-cost production of pectinase. The methanol yeast expression system is the most widely applied yeast expression system, and the exogenous gene expression system taking Pichia pastoris as a host develops most rapidly in recent years and is most widely applied. The pichia pastoris system is widely used because the system has the most remarkable advantages besides the characteristics of common yeasts: by screening transformants for high-level secretory expression and optimizing the expression conditions, recombinant proteins can be produced and prepared inexpensively and on a large scale.
The invention obtains an artificially synthesized poria-derived pectinase gene through repeatedly optimizing genes and changing expression vectors and strains, the optimized genes can realize inducible high-level expression in pichia pastoris, the recombinase protein can be effectively purified by utilizing ion exchange, and the novel recombinant pectinase protein can clarify fruit juice and has important application and development values in the production and processing of the fruit juice.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a novel artificially synthesized poria pectinase gene and a preparation method and application of recombinant protein thereof.
In order to achieve the purpose, the invention provides the following technical scheme:
an artificial pectase sequence, the similarity between the sequence and the nucleotide sequence shown in SEQ ID NO.1 is more than or equal to 90 percent
In a further improvement, the sequence comprises a nucleotide sequence shown in SEQ ID NO. 1.
The further improvement is that the sequence is a nucleotide sequence shown as SEQ ID NO. 1.
Further improvement, the application is to construct a recombinant vector, an expression cassette or a recombinant bacterium.
In a further improvement, the protein coded by the nucleotide shown in SEQ ID NO.1 is used for clarifying juice.
A method for expressing artificial sequences of pectinase comprises the following steps:
step one, recombining a nucleotide sequence shown in SEQ ID NO.1 into a pPICZ α vector, transforming the vector into a Pichia pastoris host bacterial cell, and screening by using a YPD plate with 1000 mug/mL Zeocin to obtain a transformant with high Zeocin resistance;
step two, culturing the obtained transformant to OD by using BMGY medium60010-12, centrifugally collecting cell precipitates, re-suspending the cells by using a BMMY culture medium, adding methanol to enable the mass fraction of the cells to be 1-2%, inducing for 1-3 days, and screening a transformant for high-level secretory expression of pectinase;
and step three, culturing a transformant for high-level secretory expression of pectinase by using BMGY for mass growth, then suspending cells by using BMMY culture medium and continuing induction culture at 28 ℃, and supplementing methanol to keep the mass fraction of the cells at 1-2%.
Further improvement, the method also comprises a fourth step of protein purification:
purifying the supernatant of the BMMY culture medium in the third step by using a DEAE ion exchange column, firstly balancing the chromatographic column by using a balance buffer solution, then passing the supernatant which is subjected to ion removal and pH adjustment through dialysis through the column, rinsing the column by using a pH 8.0 buffer solution containing 30mM NaCl, and then eluting the target protein by using a pH6.0 buffer solution containing 100mM NaCl.
The recombinant vector, expression cassette or recombinant bacterium containing the gene also belongs to the protection scope of the invention.
The recombinant vector is particularly preferably used for inserting the gene between Xho I and Xba I enzyme cutting sites of the expression vector pPICZ α A to obtain the recombinant vector for expressing the protein.
Primer pairs for amplifying the full length of the gene or any fragment thereof also belong to the protection scope of the invention.
The nucleotide sequence of one primer in the primer pair is SEQ ID No.3 in the sequence table, the nucleotide sequence of the other primer in the primer pair is SEQ ID No.4 in the sequence table, and related enzyme cutting and connecting operations can also be carried out on a target gene amplified by using the primers of SEQ ID No.3 and SEQ ID No. 4.
The invention also provides a method for preparing the pectinase protein, which comprises the following steps:
s1, carrying out double enzyme digestion on the gene and the expression vector pPICZ α A of
s2, performing single enzyme digestion linearization on the recombinant vector pPICZ α A-pectinase by Sac I, converting the linearized vector into a pichia pastoris host strain by a lithium chloride conversion method, and screening by Zeocin to obtain a positive clone;
s3: the positive clones were transferred to YPD plates containing 1000. mu.g/mL Zeocin, transformants with high Zeocin resistance were selected, the transformants with high Zeocin resistance were cultured in 50 mL centrifuge tubes containing 10mL BMGY medium, and the cultured cells were incubated at 28 ℃ and 250rpm to OD600The bacterial cells were collected by centrifugation and added with 1.5ml BMMY medium, cultured at 28 ℃ and 250rpm for 2 days, 10. mu.l of methanol was added to the centrifuge tube every 12 hours, after induction, the supernatant was collected by centrifugation, 40. mu.l of the supernatant was added to 10. mu.l of 5-fold loading buffer, after denaturation, electrophoretic analysis was performed by 12% SDS-PAGE, and after staining and decoloration, transformants expressing recombinant pectinase at the highest level were determined according to the expression of the target protein.
S4: and (3) performing mass growth culture on the transformant which secretes and expresses pectinase at a high level by using BMGY, suspending the cells by using BMMY culture medium, continuously performing induction culture at 28 ℃ for secreting pectinase recombinant protein at a large amount, and supplementing methanol to keep the mass fraction of the pectinase recombinant protein at 1-2%.
Further, the transformant with high pectinase expression is subjected to amplification culture by using 1L of BMGY culture medium until OD600 is 10-15, the thallus obtained by centrifugation is resuspended by using 100ml of BMMY culture medium, and then induction culture is performed for secreting a large amount of pectinase recombinant protein, wherein the induction culture conditions are as follows: 1ml of methanol was added to the centrifuge tube at 28 ℃ and 250rpm every 24 hours for 3 to 4 days.
After BMGY scale-up growth culture, BMMY was typically induced in
Preferably, after step S4, the following steps of purifying the protein are also included:
s5: centrifuging the culture solution after the S4 fermentation, taking the supernatant, adjusting the pH to 8 by using Tris alkali, centrifuging the supernatant for 30 minutes at the rotating speed of more than or equal to 15000g, adding the obtained supernatant into 10mM of pH 8Tris-HCl buffer solution for dialysis overnight, centrifuging the supernatant for 30 minutes at the rotating speed of 15000g after dialysis, loading the supernatant into a DEAE ion exchange column which is balanced by 10mM of pH 8Tris-HCl buffer solution, and rinsing the chromatographic column by 10 times of the volume of the chromatographic column of a solution which contains 10mM of Tris-HCl and 30mM of NaCl and has the pH 8;
s6: the column was eluted with 100mM NaCl in pH6.0 buffer, and the resulting eluate was dialyzed against pH6.0 PBS buffer using a dialysis bag having a molecular weight of 10kDa, followed by concentration by ultrafiltration.
Preferably, after step S5, the following steps of preserving the protein are also included:
s7: the product obtained by ultrafiltration concentration is quickly frozen at-80 ℃ and then freeze-dried.
The protein prepared by any method for preparing the protein also belongs to the protection scope of the invention.
The yeast transformant which is obtained by screening in the step S3 and stably secretly expresses pectinase at high level also belongs to the protection scope of the invention.
The protein, the gene or the recombinant vector, the expression cassette and the transgenic cell line are also within the protection scope of the invention.
The protein obtained by the preparation method can be applied to the fields of food and health care, such as the clarification of juice.
The technical scheme provided by the invention has the advantages that firstly, an artificial gene sequence capable of efficiently expressing high-activity pectinase is provided, secondly, a yeast carrier pPICZ α A-secretion signal α -factor signal peptide on the pectinase is utilized to guide gene secretion expression of target protein, the target protein can be secreted into a culture solution in a large amount and can form an accurate spatial structure, so that the natural activity of the pectinase is kept, thirdly, a stable yeast capable of carrying out high-level secretion expression is obtained through screening, fourthly, a method for expressing the pectinase by using a eukaryotic host pichia pastoris and a method for rapidly and efficiently purifying the pectinase are found, the cost can be reduced, mass production can be realized, the recombinant pectinase with biological activity obtained through secretion expression and purification by the expression method can effectively prevent host bacteria from degrading expression products, the metabolic load of host cells and the toxic effect of the expression products on hosts are reduced, and sixthly, the expressed recombinant pectinase can be rapidly purified by using DEAE anion chromatography, and the purified protein has very strong juice activity.
The invention prepares a novel high-activity recombinant pectinase from tuckahoe by optimizing genes and utilizing a pichia exogenous gene expression system for the first time. The successful preparation of the recombinant pectinase has important application and development prospects for the next industrial development and the research fields of fruit juice clarification and the like.
Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention.
Drawings
FIG. 1 is a schematic diagram of the construction of expression vector pPICZ α A-pectinase in the example of the invention.
FIG. 2 is a SDS-PAGE graph of supernatants from yeast transformant cultures with high Zeocin-resistant pectinase activity according to this invention.
FIG. 3 is a SDS-PAGE result of the expression of the target protein at different time points under the condition of expanded culture in the example of the present invention.
FIG. 4 is an elution profile of DEAE ion exchange purification in accordance with an example of the present invention.
FIG. 5 is a SDS-PAGE result of purified pectinase protein according to the present invention.
FIG. 6 is a photograph showing the results of SDS-PAGE detection of the target protein in the supernatant of the transformant of the comparative yeast.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
In the following examples,% is by mass unless otherwise specified. In the quantitative tests in the following examples, three replicates were set, and the data are the mean or the mean ± standard deviation of the three replicates.
The Pichia pastoris strain and the integrative expression plasmid pPICZ α A selected by the invention are purchased from Invitrogen corporation of America.
The specific sequences of the primers in the invention are shown as SEQ ID No.3 and SEQ ID No.4 in the sequence table, and the target genes amplified by the primers of SEQ ID No.3 and SEQ ID No.4 can also be used for carrying out related enzyme cutting and connection operations.
The formula of the culture medium is as follows:
1) yeast growth medium (BMGY)
10g of yeast extract and 20g of peptone were completely dissolved and the volume was made 800 mL. Autoclaving at 121 deg.C for 15-20min, cooling to room temperature, adding 100mL of 1M potassium phosphate solution, 100mL of YNB, 2mL of 500 × biotin, and 20mL of 50% sterilized glycerol;
2) yeast induction medium (BMMY)
10g of yeast extract and 20g of peptone were completely dissolved and the volume was made 800 mL. Steam autoclaving at 121 deg.C for 15-20min, cooling to room temperature, adding 100mL of 1M potassium phosphate solution, 100mL of YNB, 2mL of 500 × biotin, and 10mL of methanol;
3) YPD medium
Completely dissolving 10g of yeast extract and 20g of peptone, diluting to 900mL, autoclaving with steam at 121 deg.C for 15-20min, cooling to about 70 deg.C, and adding 100mL of 20% sterilized glucose solution. YPD solid medium can be prepared by adding 1.6-1.8% agar thereto.