CN115851468A - A kind of recombinant Pichia pastoris producing human casein macropeptide and its application - Google Patents

Abstract

The invention relates to a recombinant Pichia pastoris for producing human casein macropeptide and its application. The human casein macropeptide gene is expressed in a high copy in the Pichia yeast strain to realize the secretion and expression of the human casein macropeptide. In the product produced by the constructed recombinant Pichia pastoris in the present invention, the miscellaneous protein content is low, which is convenient for subsequent purification. The secreted expression accounts for 93% of the total secreted protein of Pichia pastoris, which lays the foundation for the efficient production of human milk protein by metabolic engineering of Pichia pastoris.

Description

A kind of recombinant Pichia pastoris producing human casein macropeptide and its application

technical field

The invention relates to the field of biotechnology, in particular to a recombinant Pichia pastoris producing human casein macropeptide and its application.

Background technique

Milk is the secretory product of the mammary glands of mammals and is the best nutrient for newborn mammals. The most important protein in milk is casein, and κ-casein is a kind of casein. It is the only casein containing sugar, and its content in bovine skim milk accounts for about 30% of the total casein. Casein macropeptide (CMP) is derived from the C-terminal part of κ-casein. It is a kind of polypeptide produced by chymosin degradation of κ-casein. It consists of 64 amino acid residues from the beginning to the end of the C-terminal (Val) at position 169. It has the following biologically active functions and unique nutritional properties: promotes the growth of beneficial bacteria including bifidobacteria, inhibits platelet aggregation, and inhibits the adhesion of oral actinomycetes to cell membranes; regulates the body's appetite. Currently, commercially produced bovine CMP (bCMP) from milk has been used in the food industry, mainly as an ingredient in infant formula. Human CMP (hCMP) is preferred over bCMP because it is derived from humans; however, it is not possible to collect large quantities of human breast milk for hCMP production. Therefore, it is necessary to find a method capable of producing hCMP in large quantities.

At present, the production methods of CMP include natural extraction method and microbial fermentation method. The natural extraction method involves ultrafiltration, chromatographic separation (including ion exchange technology, gel filtration, affinity adsorption, etc.), precipitation technology, etc., such as Zhang Ningning et al. Industry, 2016) used spray drying technology to produce casein macropeptide. Specifically, the CMP spray drying process was optimized by means of response surface analysis, and the effects of inlet air temperature, fan frequency, and feed speed on CMP powder output were studied. Effect; Liu Jianhong et al. (Separation and purification of casein glycomacropeptide in casein pepsin hydrolyzate by salting out and Q Sepharose FF, Food Science, 2005) using (NH4) ₂ SO ₄ fractional precipitation, dialysis desalination and Q-Sepharose FF Chromatographic separation and purification of CMP in the pepsin hydrolyzate of casein; Zhang Xiuyuan et al. Under the ultrafiltration conditions of 0.02MPa and concentration ratio of 8, relatively pure CMP can be separated. However, the preparation route of the above method is complex, the operation is cumbersome, and it is not easy to prepare a large amount of CMP. At present, using genetic engineering methods to efficiently express CMP in engineered bacteria or engineered animals and plants, so as to achieve the purpose of commercial production is the common goal pursued by scholars from all over the world. For example, Kim YJ et al. (Purification and characterization of human caseinomacropeptide produced by arecombinant Saccharomyces cerevisiae, Protein Expression & Purification, 2005) reported the application of Saccharomyces cerevisiae (Saccharomyces cerevisiae) 2805 to secrete and express human casein macropeptide, and through continuous molecular cut-off ultrafiltration and anion exchange Purified by chromatography, its HPLC purity can reach 94%. Liu Fangjie (Expression of unglycosylated casein macropeptide in recombinant Escherichia coli, Beijing University of Chemical Technology, 2008) cloned the human casein macropeptide gene and expressed it prokaryotically in Escherichia coli, specifically, in the gene Bam HI and Sac I sites were introduced at the 5' and 3' ends of the PCR product, and the PCR product was connected to the expression vector pET-28a(+) to construct a recombinant expression vector and transform it into E.coli BL21(DE3) , the highest expression of the fusion protein can account for 57.51% of the total bacterial protein, and after optimization of the fermentation conditions, the expression of the fusion protein only accounts for 62.8% of the total bacterial protein. However, since the peptide chain of casein macropeptide is too short, it is easily degraded by host bacteria when expressed alone, so it usually needs to be constructed as a fusion protein for expression, but even so, its yield can only reach 25mg/L. Therefore, there is still a need for a method for improving the production of casein macropeptide.

Contents of the invention

In order to solve the above-mentioned technical problems, the present invention provides a strain for producing casein macropeptide, which takes Pichia pastoris as a host, and expresses a high copy of human casein macropeptide gene, so as to realize high-efficiency production of casein macropeptide .

The first object of the present invention is to provide a recombinant Pichia yeast strain producing human casein macropeptide, which is obtained by integrating the human casein macropeptide gene from Pichia pastoris and then screening for high copy number.

Further, the high-copy gene screening is resistance screening using antibiotics at a concentration of 500 μg/mL or above. Preference is given to 500 μg/mL of bleomycin.

Further, among the recombinant Pichia pastoris strains obtained through high-copy subselection, the copy number of the recombinant plasmid is 6-8.

Further, the amino acid sequence of the human casein macropeptide gene is shown in SEQ ID NO.1. Specifically:

AIPPKKIQDKIIIPTINTIATVEPTPAPATEPTVDSVVTPEAFSESIITSTPETTTVAVTPPTA.

Further, the nucleotide sequence of the human casein macropeptide gene is shown in SEQ ID NO.2. Specifically:

GCCATCCCCCCAAAAGAAAATTCAGGATAAAATAATCATCCCTACCATCAATACCATTGCTACTGTTGAACCTACACCAGCTCCTGCCACTGAACCAACGGTGGACAGTGTAGTCACTCCAGAAGCTTTTTCAGAGTCCATCATCACGAGCACCCCTGAGACAACCACAGTTGCAGTTACTCCACCTACGGCA.

Further, the recombinant Pichia strain contains a recombinant plasmid expressing the human casein macropeptide gene using pPICZαA or pPIC9K as a vector.

Further, the sequence of pPICZαA is shown in SEQ ID NO.3, and the sequence of pPIC9K is shown in SEQ ID NO.4.

Further, the Pichia pastoris is Pichia pastoris X33 or Pichia pastoris GS115.

Further, the above-mentioned recombinant Pichia strain was constructed by the following steps:

The human casein macropeptide gene was inserted into the multiple cloning site of the plasmid, linearized by digestion with Sac I/Sal I, electroporated and integrated into Pichia pastoris strains, and then 500 μg/mL or more of zeocin (bleomycin ) resistance plate for screening to obtain high-copy strains.

The second object of the present invention is to provide the application of the above-mentioned recombinant Pichia strain in the production of human casein macropeptide.

Further, the recombinant Pichia strain is fermented and cultured, and induced to express human casein macropeptide.

Further, methanol was used as an inducer.

Further, the fermentation culture is as follows: pick a single colony and culture it in the seed culture medium, transfer it to the expansion medium for culture after the culture is over, collect the bacteria, and transfer it to the induction expression medium for culture.

Preferably, pick a single colony into the seed medium, and cultivate it at 28-35°C, 200-250rpm for 18-22 hours; take the seed liquid and transfer it to the expansion medium, and ferment at 28-35°C, 200-250rpm22 -26 hours; collect all the cells by centrifugation, transfer to induction expression medium, and ferment at 26-30° C., 230-270 rpm for 70-75 hours. Wherein, the seed medium is preferably YPD medium, the expansion medium is preferably BMGY medium, and the induction expression medium is preferably BMMY medium.

Further, in terms of mass percentage, the composition of the YPD medium is: 1-5% yeast extract, 2-5% peptone and 2-5% glucose.

Further, the composition of BMGY medium is: yeast extract 5-15g/L, peptone 15-25g/L, K ₂ HPO ₄ 1-5g/L, KH ₂ PO ₄ 10-15g/L, glycerol, sterilized After adding YNB and biotin.

Further, the composition of BMMY medium is: yeast extract 5-15g/L, peptone 15-25g/L, K ₂ HPO ₄ 1-5g/L, KH ₂ PO ₄ 10-15g/L, after sterilization, add YNB, biotin and methanol.

Further, after induction of expression, the supernatant was collected and passed through a nickel chelate column to obtain the target protein.

Pichia pastoris (Pichia pastoris) expression system is currently one of the standard tools for expressing recombinant proteins in the field of molecular biology. Genetic manipulation; the promoter of the alcohol oxidase gene of Pichia pastoris has strong inducibility and strong promoter, which is suitable for high-level inducible expression of foreign genes; Pichia pastoris has a strong aerobic growth preference, and can carry out cell High-density culture is conducive to large-scale industrial production; Pichia pastoris can secrete and express foreign proteins at a high level; Pichia pastoris has the function of post-translational modification of eukaryotic cells. The present invention adopts Pichia pastoris with few foreign proteins in the supernatant of the fermentation broth as the host cell to prepare the casein macropeptide, in order to reduce the cost for subsequent large-scale purification of breast milk protein.

The Pichia pastoris strain producing casein macropeptide provided by the present invention has great application potential in the fields of biology, pharmacy, food or chemical industry, such as for preparing food and medicine containing casein macropeptide.

By means of the above solution, the present invention has at least the following advantages:

The invention discloses a strain of recombinant Pichia pastoris expressing human casein macropeptide, using Pichia yeast X33 or Pichia GS115 as a host, and using pPICZαA or pPIC9K as a carrier to integrate human α-lactalbumin gene ( hCMP), high-copy copies were obtained by screening with bleomycin resistance plates at a concentration of 500 μg/mL or above. After methanol-induced expression, human casein macropeptide was obtained, and the shake flask yield was 234mg/L. This process does not require fusion expression, no need to cut and remove the fusion tag during post-processing, and the fermentation supernatant has less impurity protein, which lays the foundation for the efficient production of human milk protein by Pichia pastoris metabolic engineering.

The above description is only an overview of the technical solutions of the present invention. In order to understand the technical means of the present invention more clearly and implement them according to the contents of the description, the preferred embodiments of the present invention are described below with detailed drawings.

Description of drawings

In order to make the content of the present invention more clearly understood, the present invention will be further described in detail below according to the specific embodiments of the present invention and in conjunction with the accompanying drawings.

Fig. 1 is the secretion expression result of human casein macropeptide in different bacterial strains;

Figure 2 is the SDS-PAGE result of the secreted expression of human casein macropeptide with high copy number;

Figure 3 shows the results of human casein macropeptide yields of single-copy and multi-copy copies at different induction times.

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, so that those skilled in the art can better understand the present invention and implement it, but the examples given are not intended to limit the present invention.

The materials and methods involved in the following examples are as follows:

Screening solid media:

MD plate/selection medium: (Minimal Dextrose Medium+Histidine): Add 2g of agarose (20g/L) to 80mL of ultrapure water, sterilize at 121°C for 20 minutes, wait until the temperature reaches 60°C, and place it on the ultra-clean bench Add 10*YNB 10mL, 10*D (20% glucose) 10ml, 500*B (0.02% biotin) 0.2mL, mix well, pour plate.

YPD-zeocin medium: 1% yeast extract, 2% peptone, 2% agarose, 2% glucose.

Recombinant Pichia pastoris seed culture and fermentation:

The formula of seed liquid medium is YPD medium: 1% yeast extract, 2% peptone, 2% glucose;

The formula of expansion medium (BMGY) is: yeast extract 10g/L, peptone 20g/L, K ₂ HPO ₄ 3g/L, KH ₂ PO ₄ 11.8g/L, glycerin 10mL, ultrapure water 890mL, sterilized at 121°C After 20 minutes, when the temperature drops to 60°C, add 100mL of 10*YNB and 1mL of 500*B (0.02% biotin) into the ultra-clean bench.

Induced expression medium (BMMY) formulation: yeast extract 10g/L, peptone 20g/L, K ₂ HPO ₄ 3g/L, KH ₂ PO ₄ 11.8g/L, ultrapure water 895mL, sterilized at 121°C for 20 minutes After the temperature dropped to 60°C, add 100ml of 10*YNB, 1mL of 500*B (0.02% biotin) and 5mL of methanol into the ultra-clean bench.

Sample detection method: First, the fermentation broth is centrifuged and the supernatant is taken, purified using a His-Binding-resin purification column, and the purified samples are collected for SDS-PAGE electrophoresis.

The secretion and expression of human casein macropeptide in different bacterial strains of embodiment 1

Three commonly used Pichia pastoris strains were selected for the expression of human casein macropeptide, which were wild-type strain X33, histidine-deficient strain GS115 and protease-deficient strain SMD1163. The Pep4 and prb1 genes in the SMD1163 genome are mutated, resulting in the loss of proteolytic enzyme A and proteolytic enzyme B activities, which can protect the expression product from degradation and promote the increase of expression.

First order the synthetic hCMP gene (see SEQ ID NO.2 for the nucleotide sequence), amplify the target gene fragment; amplify two commercial plasmid vectors (pPICZαA and pPIC9K) at the MCS site as the backbone, use Gibson assembly and then transform E. coli Competent cells to construct two expression vectors (pPICZαA-hCMP and pPIC9K-hCMP). A single colony with correct sequencing was inoculated into LB medium, cultured overnight for 16 hours, and the recombinant plasmid was extracted for enzyme digestion. The digestion reaction system is: DNA ≤ 5 μg, 3 μL of fast cutting enzyme SacⅠ or SalⅠ, 5 μL of 10×Buffer, and add ddH ₂ O to a total volume of 50 μL. Then the linearized recombinant plasmid was purified and recovered, the linearized recombinant plasmid pPICZαA-hCMP was electrotransferred into host X33, and the linearized recombinant plasmid pPIC9K-hCMP was electrotransferred into hosts GS115 and SMD1163. Positive transformants were screened using YPD plates and MD plates containing 100 μg/mL, respectively. Fermentation positive strains (X33-hCMP, GS115-hCMP and SMD1163-hCMP). The 72h fermentation supernatant was purified using a His-Binding-resin purification column, and the protein content was determined using the Bradford method.

The fermentation process is as follows: first, a single colony is picked and placed in 5 mL of seed liquid culture medium, and cultured at 30° C. and 220 rpm for 20 hours. Then, 5 mL of seed solution was transferred to 50 mL of BMGY medium for expanded production. After 24 hours, all the bacteria were collected by centrifugation, and the induction expression medium BMMY was replaced for 72 hours at 28°C and 250 rpm, adding 1% methanol every 24 hours.

The results are shown in Figure 1. Compared with the other two strains, X33-hCMP has more advantages in growth rate and yield.

Secreted expression of human casein macropeptide of embodiment 2 high copy number

All the positive transformants X33-hCMP in Example 1 were collected and reapplied on YPD-500 μg/mL zeocin plate to screen for high copy. Transformants were selected for shake-flask fermentation, and the fermentation process was the same as above. The 72h fermentation supernatant was purified by His-Binding-resin purification column, and human casein macropeptide was detected after SDS-PAGE. The results are shown in Figure 2. The protein content was determined by the Bradford method, and compared with the single-copy results in Example 1, the results are shown in FIG. 3 .

As can be seen from Fig. 3, compared with the wild-type Pichia pastoris that does not produce human casein macropeptide, the output of human casein macropeptide reaches 72 hours after the shake flask fermentation of the high-copy recombinant Pichia pastoris strain constructed by the present invention. 234mg/L, while the shake flask yield of the single-copy strain was 77mg/L. At the same time, the 72h unpurified X33-pPICZαA-hCMP fermentation supernatant was taken to directly measure the total protein concentration, and the result was 252mg/L, and the secreted expression of the target protein accounted for the proportion of the total secreted protein of Pichia pastoris, which was 93%. After qPCR analysis, the copy number of the multi-copies was 6.

Apparently, the above-mentioned embodiments are only examples for clear description, and are not intended to limit the implementation. For those of ordinary skill in the art, on the basis of the above description, other changes or changes in various forms can also be made. It is not necessary and impossible to exhaustively list all the implementation manners here. However, the obvious changes or changes derived therefrom are still within the scope of protection of the present invention.

Claims (10)

1. A recombinant Pichia strain producing human casein macropeptide, characterized in that: the strain uses Pichia pastoris as a host, and expresses a high copy of the human casein macropeptide gene. 2. The recombinant Pichia strain according to claim 1, characterized in that: the recombinant Pichia is obtained by integrating human casein macropeptide gene from Pichia pastoris, and then obtained through high-copy sub-screening; Duplicant screening is resistance screening using antibiotics at a concentration of 500 μg/mL or above. 3. The recombinant Pichia strain according to claim 1, characterized in that: the amino acid sequence of the human casein macropeptide gene is shown in SEQ ID NO.1. 4. The recombinant Pichia strain according to claim 1, characterized in that: the nucleotide sequence of the human casein macropeptide gene is shown in SEQ ID NO.2. 5. The recombinant Pichia strain according to claim 1, characterized in that: the recombinant Pichia strain uses pPICZαA or pPIC9K as the expression vector. 6. The recombinant Pichia strain according to claim 1, characterized in that: the strain uses Pichia X33 or Pichia GS115 as a host. 7. The application of the recombinant Pichia pastoris described in any one of claims 1-6 in the production of human casein macropeptide. 8. The application according to claim 7, characterized in that: the recombinant Pichia strain is fermented and cultured, and the expression of human casein macropeptide is induced. 9. The application according to claim 8, characterized in that: the fermentation culture is to pick a single colony to cultivate in the seed culture medium, transfer it to the expansion medium for cultivation after the culture is over, collect the thalli, transfer cultured in inducible expression medium. 10. The application according to claim 8, characterized in that: methanol is used as an inducer.

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