CN112481286B

CN112481286B - Amino acid sequence for improving heterologous expression efficiency of recombinant milk protein

Info

Publication number: CN112481286B
Application number: CN202011297107.9A
Authority: CN
Inventors: 林敏�; 周正富; 燕永亮; 庞雨; 王劲; 陆伟
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2020-11-17
Filing date: 2020-11-17
Publication date: 2022-06-28
Anticipated expiration: 2040-11-17
Also published as: CN112481286A

Abstract

The invention provides a polypeptide for promoting high-efficiency expression of protein. Experimental results show that the gene of the polypeptide and the target protein gene are expressed together in a fusion expression mode, so that the protein gene which cannot be expressed in vitro originally can be promoted to obtain the function of in vitro recombinant expression, and the target protein is efficiently synthesized.

Description

Amino acid sequence for improving heterologous expression efficiency of recombinant milk protein

Technical Field

The invention relates to the technical field of genetic engineering, in particular to an amino acid sequence for improving the expression efficiency of recombinant protein and application thereof.

Background

Soluble recombinant proteins with biological activity are the basis for protein structure and function studies. However, the low expression level of soluble proteins has been a significant obstacle to the restriction of the structure and function of the target proteins.

With the development of synthetic biology and genetic engineering techniques, more and more researchers have produced useful proteins using DNA recombination methods, which require highly active, high-yield protein expression systems. However, in conventional expression, heterologous recombinant proteins are difficult to express efficiently, and even can not be folded correctly into conformation with biological activity, which has become a difficult problem in research and production of basic scientific research, immune medicine, industrial and agricultural enzyme preparations and protein products.

Therefore, improving the efficiency of recombinant protein expression in vitro is a technical problem to be solved.

Disclosure of Invention

The invention aims to improve the in vitro expression efficiency of the recombinant protein.

The invention discovers a section of amino acid sequence PEP (Peptide for Expression-Promoting), which has the function of promoting the high-efficiency expression of the recombinant protein in vitro.

Therefore, the invention provides a method for improving the high-efficiency expression of eukaryotic proteins, which is characterized in that a gene encoding polypeptide PEP and a target protein gene are expressed together in a fusion expression mode to improve the expression quantity of the target protein, particularly the translation protein of a eukaryotic gene source, and realize the high-efficiency synthesis of the target protein.

The invention discovers for the first time that the polypeptide PEP with the amino acid sequence shown as SEQ ID NO.2 can be used for constructing a high-efficiency protein heterologous expression system and applied to the fields of basic scientific research, immune medicine, industry, agricultural enzyme preparations, protein products and the like.

The invention selects three protein genes which are difficult to be recombined and expressed in vitro: the specific invention contents of the research objects of the bovine alpha S2-casein gene csn1S2, the bovine kappa-casein gene csn3 and the bovine alpha-lactalbumin gene LALBA are as follows:

1. PEP polypeptide for improving high-efficiency expression of alpha S2-casein

Alpha S2-casein gene csn1S2 is used as a target gene for in vitro recombination expression. CSN1S2 is connected with pET28a vector and transformed into Escherichia coli BL21, and a positive expression strain BL21-CSN1S2 is obtained. The induction expression is carried out at the conditions of 16 ℃, 22 ℃ and 30 ℃, and the expression condition of the target protein is detected by SDS-PAGE.

The research result shows that: α S2-casein was not expressed and expression in vitro failed. Meanwhile, the polypeptide PEP and the alpha S2-casein gene CSN1S2 are subjected to fusion expression, the same expression system is utilized, induction expression is respectively carried out under different conditions, and SDS-PAGE detection shows that target protein in the recombinant strain BL21-PEP-CSN1S2 is efficiently expressed. The Western hybridization result proves that the high-expression protein band is the target protein recombinant alpha S2-casein, and most of the high-expression protein band is soluble protein (see example 2 for details).

2. PEP polypeptide for improving high-efficiency expression of kappa-casein

The kappa-casein gene csn3 is used as a target gene for in vitro recombination expression. An expression system is constructed by using the vector pET28a and an expression strain BL21, and a positive expression strain BL21-CSN3 is obtained. Induction of expression was attempted under different conditions of 16 ℃ and 22 ℃ and 30 ℃ respectively.

The research result shows that: kappa-casein is likewise not expressed. Meanwhile, the polypeptide PEP and the kappa-casein gene CSN3 are subjected to fusion expression, expression strains are constructed by using the same expression system, induction expression is respectively carried out under different conditions, and SDS-PAGE detection shows that target proteins in the recombinant strain BL21-PEP-CSN3 are efficiently expressed. The Western blot results confirmed that the highly expressed protein band is the target protein recombinant kappa-casein and is all soluble protein (see example 3 for details).

3. PEP polypeptide for improving efficient expression of alpha-lactalbumin

The same in vitro gene LALBA which can not express the alpha-lactalbumin by recombination is modified. The polypeptide PEP and the alpha-lactalbumin gene LALBA are subjected to fusion expression, and an expression system is constructed by utilizing a vector pET28a and an expression strain BL21 to obtain a positive expression strain BL 21-PEP-LALBA.

The research result shows that: SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) detection shows that the alpha-lactalbumin cannot be recombined and expressed in vitro, and the target protein in the recombinant strain is efficiently expressed. Western blot results also confirmed that the highly expressed protein band is the target protein, recombinant alpha-lactalbumin, and almost all soluble proteins (see example 4 for details).

The results of the above comparative studies using the three protein genes confirm that,

The polypeptide PEP with the amino acid sequence shown as SEQ ID NO.2 can promote the eukaryotic protein gene to express in vitro, and endows the protein gene which cannot be expressed in vitro with the function of in vitro recombination expression.

Can obviously enhance the expression amount of target protein, particularly eukaryotic protein, and realize the high-efficiency synthesis of the target protein.

The polypeptide PEP can be used for constructing a high-efficiency protein heterologous expression system and is applied to the fields of basic scientific research, immune medicine, industry, agricultural enzyme preparations, protein products and the like.

Description of the drawings:

FIG. 1 shows a comparison of SDS-PAGE electrophoresis results for heterologous expression of eukaryotic genes csn1s2, csn3 and LALBA.

In the figure, M, marker; 1, control strain was not induced; 2, control strain IPTG induction; 3, BL21-CSN1S2 strain was not induced; 4, BL21-CSN1S2 strain IPTG induction; 5, BL21-CSN3 strain was not induced; 6, BL21-CSN3 strain IPTG induction; 7, BL21-LALBA strain was not induced; 8, BL21-LALBA Strain IPTG induction.

FIG. 2 analysis of transcription level expression of genes csn1s2, csn3 and LALBA after induction.

FIG. 3 is a comparison of SDS-PAGE electrophoresis results of the expression of target proteins of BL21-PEP-CSN1S2 strain.

In the figure, M, marker; 1, 30 ℃ without induction; 2, inducing by a strain IPTG at the temperature of 30 ℃; 3, 22 ℃ without induction; 4, inducing by a strain IPTG at the temperature of 22 ℃; 5, 16 ℃ without induction; 6, 16 ℃ strain IPTG induction.

FIG. 4Western blot detection of target protein PEP-CSN1S 2.

FIG. 5 is a comparison of SDS-PAGE electrophoresis results of the expression of the target protein of BL21-PEP-CSN3 strain.

In the figure, M, marker; 1, 30 ℃ without induction; 2, inducing a strain IPTG at the temperature of 30 ℃; 3, 22 ℃ without induction; 4, inducing by a strain IPTG at the temperature of 22 ℃; 5, 16 ℃ without induction; 6, 16 ℃ strain IPTG induction.

FIG. 6 detection of target protein PEP-CSN3 by Western hybridization.

FIG. 7 is a comparison of SDS-PAGE electrophoresis results of the expression of target proteins of BL21-PEP-LALBA strain.

FIG. 8Western blot detection of target protein PEP-LALBA.

Description of sequence listing

SEQ ID NO.1 is the nucleotide sequence of polypeptide PEP;

SEQ ID NO.2 is the protein amino acid sequence of polypeptide PEP;

Detailed Description

In order to prove that the PEP polypeptide improves the expression efficiency of eukaryotic proteins, the nucleotide sequence of the PEP polypeptide and eukaryotic source target protein genes are expressed together in a fusion expression mode, and the expression quantity of the target protein is detected through SDS-PAGE electrophoresis, so that the application of the PEP polypeptide in realizing the high-efficiency synthesis of the target protein is proved.

The plasmids and strains shown in the following examples are only used to further illustrate the present invention, and do not limit the essence of the present invention. Unless otherwise specified, the experimental conditions are generally determined according to conventional conditions well known to those skilled in the art or as recommended by the manufacturer. The plasmids and strains mentioned in the examples were derived from:

prokaryotic expression vector pET-28 a: is a commercially available product from holo-gold;

coli expression strain BL21(DE 3): is a commercial product of Gentle Corp.

Example 1 sequence analysis of PEP Polypeptides

Amino acid sequence analysis shows that the polypeptide PEP (the amino acid sequence of which is shown in SEQ ID NO. 2) consists of 114 amino acids coded by 342 nucleotides, and the molecular weight of the polypeptide PEP is 11.38 kDa. The nucleotide sequence is shown as SEQ ID NO. 1; the compound has 8 negative charge amino acids and 5 positive charge amino acids, the predicted isoelectric point is 4.51, the polar amino acid residues account for 23 percent, and the hydrophobic amino acid residues account for 20 percent. Secondary structure analysis showed that it contained a random coil, 4 beta folds and 2 alpha helices.

Example 2 PEP Polypeptides enhancing the high expression of α S2-Casein

First, experimental material

Prokaryotic expression vector pET-28 a: is a product sold in the whole gold company;

Coli expression strain BL21(DE 3): is a product sold by the company Takara Shuzo.

Second, Experimental methods

1. And (5) constructing a vector. The PCR specific primer is added by taking the recombinant plasmid pUC-CSN1S2 preserved in the laboratory as a template to amplify the complete nucleotide sequence of the alpha S2-casein gene CSN1S2, the genomic DNA of the deinococcus gobius is taken as a template to amplify the DNA of the PEP polypeptide, and the PCR product is subjected to gel recovery. The vector pET28a and the target gene fragment containing the complementary sequence of the adjacent fragments were subjected to homologous recombination ligation by using Nco I/Xho I double-restriction enzyme.

2. And obtaining the expression strain. The expression vector is transformed into escherichia coli BL21, the correct insertion sequence is verified through PCR, enzyme digestion and sequencing, and the strain is named as BL21-PEP-CSN1S 2. Meanwhile, CSN1S2 without fusion fragment PEP polypeptide is connected with pET28a vector in the same way, the expression vector is transformed into escherichia coli BL21, the correct insertion sequence is verified through PCR, enzyme digestion and sequencing, and the strain is named as BL21-CSN1S 2. And selecting a single colony for PCR verification, extracting recombinant plasmid for enzyme digestion, sequencing and verifying.

3. The target protein induces expression. The recombinant strain was inoculated in LB liquid medium containing antibiotics at an inoculum size of 1%, and cultured overnight in a shaker at 37 ℃. Inoculating the strain into 500mL LB liquid medium added with kanamycin at an initial concentration of 0.1 OD600, and culturing at 37 ℃ until the concentration of the strain liquid is 0.6-0.8. IPTG (final concentration of 0.5 mu mol/L) is added to carry out protein induction expression at the conditions of 16 ℃, 22 ℃ and 30 ℃, cells are broken by ultrasonic waves, and the expression condition of the target protein is detected by SDS-PAGE.

4. And (4) analyzing gene expression. And (3) adopting an innuPREP RNA Mini Kit to extract total RNA of the target IPTG-induced recombinant expression strain, removing genome DNA, and performing reverse transcription to synthesize cDNA. Specific qRT-PCR primers are designed according to the sequence characteristics of target genes, and an internal reference gene is a 16SrDNA gene. The expression of the target gene in the strain was determined by fluorescent quantitative PCR reaction, 3 replicates were set for each sample.

Western blot. Collecting the strain, breaking the cell by ultrasonic wave, and performing SDS-PAGE electrophoresis. Transferring the protein on the gel to a PVDF membrane, carrying out antibody incubation after sealing treatment for 3 hours, carrying out color reaction according to the label of a second antibody, and detecting the expression condition of the target protein.

Third, experimental results

SDS-PAGE shows that the recombinant strain BL21-CSN1S2 does not express the target protein under different induction conditions, as shown in

lanes

3 and 4 of FIG. 1. Research results show that the alpha S2-casein CSN1S2 gene of eukaryotic origin is influenced during recombinant expression and cannot be induced to express. The fluorescent real-time quantitative PCR result shows (figure 2), in the recombinant strain BL21-CSN1S2, the CSN1S2 gene is transcribed, which indicates that the mRNA translation process of the CSN1S2 in the strain is blocked, and the target alpha S2-casein cannot be synthesized. In contrast, a remarkable protein-induced expression band can be seen in the recombinant strain BL21-PEP-CSN1S2 in which the PEP polypeptide is expressed in a fusion mode. FIG. 3 shows that proteins are expressed at 16 deg.C, 22 deg.C, and 30 deg.C, with 22 deg.C and 30 deg.C being the best. The Western hybridization result shows that the protein band for inducing expression is the target protein PEP-CSN1S 2.

Fourth, conclusion of experiment

Eukaryotic alpha S2-casein gene csn1S2 cannot be subjected to in vitro recombinant expression; the fusion expression of the PEP polypeptide and the target csn1S2 gene can complete the high-efficiency expression of eukaryotic alpha S2-casein csn1S2 gene.

Example 3 PEP Polypeptides enhancing high expression of kappa-Casein

First, experimental material

Second, Experimental methods

1. And (5) constructing a vector. The PCR specific primer is added by taking the recombinant plasmid pUC-CSN3 preserved in the laboratory as a template to amplify the complete nucleotide sequence of the kappa-casein gene CSN3, and the DNA of the gene group of the deinococcus gobius is taken as a template to amplify the DNA of the PEP polypeptide, and the PCR product is subjected to gel recovery. The vector pET28a and the target gene fragment containing the complementary sequence of the adjacent fragments were subjected to homologous recombination ligation by using Nco I/Xho I double-restriction enzyme.

2. And obtaining the expression strain. The expression vector is transformed into escherichia coli BL21, the correct insertion sequence is verified through PCR, enzyme digestion and sequencing, and the strain is named as BL21-PEP-CSN 3. Meanwhile, CSN3 without fusion fragment PEP polypeptide is connected with pET28a vector, the expression vector is transformed into escherichia coli BL21, PCR, enzyme digestion and sequencing are carried out to verify that the insertion sequence is correct, and the strain is named as BL21-CSN 3. And selecting a single colony for PCR verification, extracting recombinant plasmid for enzyme digestion, sequencing and verifying.

3. The target protein induces expression. The recombinant strain was inoculated in LB liquid medium with 1% inoculum size and cultured overnight in a shaker at 37 ℃. Inoculating the strain into 500mL LB liquid culture medium added with kanamycin at an initial concentration of 0.1 OD600, and culturing at 37 ℃ until the concentration of the strain liquid is 0.6-0.8. IPTG (final concentration 0.5 mu mol/L) is added to carry out protein induction expression at the conditions of 16 ℃, 22 ℃ and 30 ℃, cells are broken by ultrasonic waves, and the expression condition of a target kappa-casein is detected by SDS-PAGE.

4. And (4) analyzing gene expression. And (3) adopting an innuPREP RNA Mini Kit to extract total RNA of the target IPTG-induced recombinant expression strain, removing genome DNA, and performing reverse transcription to synthesize cDNA. Specific qRT-PCR primers are designed according to the sequence characteristics of a target gene, and an internal reference gene is a 16SrDNA gene. The expression of the target gene in the strain was determined by fluorescent quantitative PCR reaction, with 3 replicates per sample set.

Western blot. Collecting the strain, breaking the cell by ultrasonic wave, and performing SDS-PAGE electrophoresis. Transferring the protein on the gel to a PVDF membrane, carrying out antibody incubation after sealing treatment for 3 hours, carrying out color reaction according to a second antibody label, and detecting the expression condition of the target protein.

Third, experimental results

SDS-PAGE electrophoresis results show that the recombinant strain BL21-CSN3 does not express target protein under different induction conditions, such as

lanes

5 and 6 in figure 1, and research results show that the eukaryotic kappa-casein gene CSN3 is influenced during recombinant expression and cannot be induced to express. FIG. 2 shows that the CSN3 gene in the recombinant strain BL21-CSN3 can be normally transcribed and up-regulated by more than 100 times after induced expression, which indicates that the mRNA translation process of CSN3 in the strain is blocked, and the target kappa-casein cannot be synthesized. In contrast, a significant protein-induced expression band can be seen in the recombinant strain BL21-PEP-CSN3 in which the PEP polypeptide is expressed by fusion, as shown in FIG. 5. The expression quantity of the target protein PEP-LALBA is similar under different induction conditions of 16 ℃, 22 ℃ and 30 ℃. Western blot results FIG. 6 shows that the protein band induced to be expressed is the target protein PEP-CSN3, and the expressed protein is almost all present in the cell disruption supernatant and is soluble protein.

Fourth, conclusion of experiment

Eukaryotic kappa-casein gene csn3 could not be expressed recombinantly in vitro; the fusion of PEP polypeptide and target csn3 gene can complete the high-efficiency expression of eukaryotic kappa-casein.

Example 4 PEP Polypeptides enhancing the high expression of alpha-lactalbumin

First, experimental material

Second, Experimental methods

1. And (5) constructing a vector. The recombinant plasmid pUC-LALBA stored in the laboratory is taken as a template, a PCR specific primer is added to amplify the nucleotide sequence of the complete alpha-lactalbumin gene LALBA, the genome DNA of the deinococcus gobius is taken as a template to amplify the DNA of PEP polypeptide, and the PCR product is subjected to gel recovery. The vector pET28a and the target gene fragment containing the complementary sequence of the adjacent fragments were subjected to homologous recombination ligation by using Nco I/Xho I double-restriction enzyme.

2. And obtaining the expression strain. The expression vector is transformed into escherichia coli BL21, the insertion sequence is verified to be correct through PCR, enzyme digestion and sequencing, and the strain is named as BL 21-PEP-LALBA. Meanwhile, LALBA not containing fusion fragment PEP polypeptide is also connected with pET28a vector, the expression vector is transformed into Escherichia coli BL21, the insertion sequence is verified to be correct through PCR, enzyme digestion and sequencing, and the strain is named as BL 21-LALBA. And selecting a single colony for PCR verification, extracting recombinant plasmid for enzyme digestion, sequencing and verifying.

3. The target protein induces expression. The recombinant strain was inoculated in LB liquid medium supplemented with antibiotics at an inoculum size of 1%, and cultured overnight in a shaker at 37 ℃. Inoculating the strain into 500mL LB liquid medium added with kanamycin at an initial concentration of 0.1 OD600, and culturing at 37 ℃ until the concentration of the strain liquid is 0.6-0.8. IPTG (final concentration 0.5 mu mol/L) is added to carry out protein induction expression at the conditions of 16 ℃, 22 ℃ and 30 ℃, cells are broken by ultrasonic waves, and the expression condition of a target kappa-casein is detected by SDS-PAGE.

4. And (4) analyzing gene expression. And (3) adopting an innuPREP RNA Mini Kit to extract total RNA of the target IPTG induced recombinant expression strain, removing genomic DNA, and performing reverse transcription to synthesize cDNA. Specific qRT-PCR primers are designed according to the sequence characteristics of a target gene, and an internal reference gene is a 16SrDNA gene. The expression of the target gene in the strain was determined by fluorescent quantitative PCR reaction, with 3 replicates per sample set.

Third, experimental results

SDS-PAGE electrophoresis results show that the recombinant strain BL 21-does not express the target protein under different induction conditions of 16 ℃, 22 ℃ and 30 ℃, and research results show that the alpha-lactalbumin gene LALBA from eukaryotic sources is influenced during recombinant expression and cannot be induced to express, such as

lanes

7 and 8 in figure 1. The fluorescent real-time quantitative PCR result shows (fig. 2) that in the recombinant strain BL 21-lala, the α -lactalbumin gene lala can be transcribed normally, and the gene expression is increased by 22 times after induction, indicating that the mRNA translation process of the lala in the strain is blocked, resulting in the failure to synthesize α -lactalbumin. In the recombinant strain BL21-PEP-LALBA expressed by fusion of PEP polypeptide, a clear protein-induced expression band can be seen, as shown in FIG. 7. The expression quantity of the target protein PEP-LALBA is similar under different induction conditions of 16 ℃, 22 ℃ and 30 ℃. FIG. 8 shows that the protein band induced to be expressed is target protein PEP-LALBA, and most of the protein band is soluble protein.

Fourth, conclusion of experiment

The eukaryotic casein alpha-lactalbumin gene LALBA can not be subjected to in vitro recombinant expression; the fusion of the PEP polypeptide and the target LALBA gene can complete the high-efficiency expression of eukaryotic alpha-lactalbumin.

The above three different protein experiments show that: PEP polypeptide can promote eukaryotic protein gene to express in vitro, so that the protein gene which cannot be expressed in vitro originally can obtain the function of in vitro recombination expression.

Sequence listing

<110> institute of biotechnology of Chinese academy of agricultural sciences

<120> amino acid sequence for improving heterologous expression efficiency of recombinant milk protein

<160> 2

<170> PatentIn version 3.1

<210> 1

<211> 342

<212> DNA

<213> Deinococcus gobius (Deinococcus gobiensis)

<400> 1

tgcggaacct cgactacccc catggcccag acgccggccc agggcagcgc cccggccgcc 60

gacagcggtc tggcccccct gcgcggcacc gacaacccca gcgccatcgc cgggcagtac 120

atcgtggtcc tcaaggaagg cacccagagc gccctgagcg cccagagcgc cggcggcctg 180

atcggcagcc tgggtctgga cccgcagggc atcacggtcc tgagcgtgta cggtcaggcc 240

attgagggct tcgccgccaa gctcagcgcc cagaacctgg agaaggtgcg cgccaacgcg 300

aacgtcgcct acgtcgagca ggacggcatg atgtacgcct cc 342

<210> 2

<211> 114

<212> PRT

<213> Deinococcus gobius (Deinococcus gobiensis)

<400> 2

Cys Gly Thr Ser Thr Thr Pro MET Ala Gln Thr Pro Ala Gln Gly Ser

1 5 10 15

Ala Pro Ala Ala Asp Ser Gly Leu Ala Pro Leu Arg Gly Thr Asp Asn

20 25 30

Pro Ser Ala Ile Ala Gly Gln Tyr Ile Val Val Leu Lys Glu Gly Thr

35 40 45

Gln Ser Ala Leu Ser Ala Gln Ser Ala Gly Gly Leu Ile Gly Ser Leu

50 55 60

Gly Leu Asp Pro Gln Gly Ile Thr Val Leu Ser Val Tyr Gly Gln Ala

65 70 75 80

Ile Glu Gly Phe Ala Ala Lys Leu Ser Ala Gln Asn Leu Glu Lys Val

85 90 95

Arg Ala Asn Ala Asn Val Ala Tyr Val Glu Gln Asp Gly MET MET Tyr

100 105 110

Ala Ser

Claims

1. The application of the polypeptide with the amino acid sequence shown as SEQ ID NO.2 in promoting the high-efficiency expression of escherichia coli heterologous protein is realized by coexpressing the gene of the polypeptide and the target protein gene in a fusion expression mode.

2. The use of claim 1, wherein the nucleotide sequence of the gene encoding the polypeptide is shown in SEQ ID No. 1.

3. The use of claim 1, wherein the high expression efficiency means that the polypeptide can promote expression of a protein gene which cannot be heterologously expressed in escherichia coli originally, or improve the expression efficiency of the protein.

4. The application of the plasmid containing the coding gene of the polypeptide with the amino acid sequence shown as SEQ ID NO.2 in promoting the heterologous expression of recombinant protein of escherichia coli is realized by coexpression of the gene of the polypeptide and the target protein gene in a fusion expression mode.

5. A method for promoting escherichia coli to express heterologous protein efficiently is characterized in that a gene of polypeptide with an amino acid sequence shown as SEQ ID NO.2 and a target protein gene are co-expressed in a fusion expression mode.