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

Abstract

The present invention provides a polypeptide that promotes high protein expression. The experimental results show that by co-expressing the gene of the polypeptide and the target protein gene in the form of fusion expression, the protein gene that cannot be expressed in vitro can obtain the function of in vitro recombinant expression, thereby realizing the efficient synthesis of the target protein.

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 a recombinant protein and its application.

Background technique

Soluble recombinant proteins with biological activity are the basis for the study of protein structure and function. However, the low level of soluble protein expression has become a major obstacle to study the structure and function of target proteins.

With the development of synthetic biology and genetic engineering technology, more and more researchers use DNA recombinant methods to produce useful proteins, which requires high activity and high yield protein expression systems. However, in conventional expression, heterologous recombinant proteins are difficult to express efficiently, and even cannot be correctly folded into a biologically active conformation, which has become a difficult problem in the development and production of basic scientific research, immunology, industrial and agricultural enzyme preparations and protein products.

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

SUMMARY OF THE INVENTION

The purpose of the present invention is to improve the in vitro expression efficiency of recombinant protein.

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

Therefore, the present invention provides a method for improving the high-efficiency expression of eukaryotic proteins, by co-expressing the gene encoding the polypeptide PEP and the target protein gene through fusion expression, so as to improve the translation protein of the target protein, especially the eukaryotic source gene. to achieve efficient synthesis of target proteins.

The present invention finds for the first time that the polypeptide PEP whose amino acid sequence is shown in SEQ ID NO.

The present invention selects three protein genes that are difficult to be recombinantly expressed in vitro: bovine αS2-casein gene csn1s2, bovine κ-casein gene csn3 and bovine α-lactalbumin gene LALBA as the research objects, and identifies the polypeptide PEP to improve the high-efficiency expression of recombinant milk protein in vitro Function, the specific invention content is as follows:

1. PEP polypeptide improves the high expression of αS2-casein

The αS2-casein gene csn1S2 was used as the target gene for recombinant expression in vitro. The csn1S2 was connected to the pET28a vector and transformed into E. coli BL21 to obtain the positive expression strain BL21-CSN1S2. The induced expression was performed at 16°C, 22°C, and 30°C, respectively, and the expression of the target protein was detected by SDS-PAGE.

The results showed that αS2-casein could not be expressed, and the in vitro expression failed. At the same time, the polypeptide PEP was fused and expressed with the αS2-casein gene csn1S2, and the expression was induced under different conditions using the same expression system. The SDS-PAGE detection showed that the target protein was highly expressed in the recombinant strain BL21-PEP-CSN1S2. The results of Western blotting confirmed that the highly expressed protein band was the target protein recombinant αS2-casein, most of which were soluble proteins (see Example 2 for details).

2. PEP polypeptide enhances the high expression of κ-casein

The κ-casein gene csn3 was used as the target gene for recombinant expression in vitro. The expression system was constructed by using the vector pET28a and the expression strain BL21, and the positive expression strain BL21-CSN3 was obtained. Attempt to induce expression under different conditions of 16°C, 22°C, and 30°C, respectively.

The results showed that κ-casein could not be expressed either. At the same time, the polypeptide PEP and the κ-casein gene csn3 were fused and expressed, and the expression strain was constructed using the same expression system, and the expression was induced under different conditions. The SDS-PAGE detection showed that the target protein in the recombinant strain BL21-PEP-CSN3 was highly expressed. . The results of Western blotting confirmed that the highly expressed protein bands were the target protein recombinant κ-casein, and all were soluble proteins (see Example 3 for details).

3. PEP polypeptide improves the high expression of α-lactalbumin

The same in vitro recombinant expression of α-lactalbumin gene LALBA was transformed. The polypeptide PEP and α-lactalbumin gene LALBA were fused and expressed, and the expression system was constructed by using the vector pET28a and the expression strain BL21, and the positive expression strain BL21-PEP-LALBA was obtained.

The results showed that after induction and expression under different conditions, SDS-PAGE showed that α-lactalbumin could not be recombinantly expressed in vitro, but the target protein was highly expressed in recombinant strains. The results of Western blotting also confirmed that the highly expressed protein bands were the target protein recombinant α-lactalbumin, and almost all of them were soluble proteins (see Example 4 for details).

The above comparative study with three protein genes confirmed that,

The polypeptide PEP whose amino acid sequence is shown in SEQ ID NO. 2 can promote the in vitro expression of eukaryotic protein genes, and endow the function of in vitro recombinant expression to protein genes that could not be expressed in vitro.

It can significantly enhance the expression of the target protein, especially the eukaryotic source protein, and realize the efficient synthesis of the target protein.

Polypeptide PEP can be used for the construction of high-efficiency protein heterologous expression system, and it can be used in the fields of basic scientific research, immunomedicine, industrial and agricultural enzyme preparations and protein products.

Description of drawings:

Fig. 1 Comparison of SDS-PAGE electrophoresis results of heterologous expression of eukaryotic genes csn1s2, csn3 and LALBA.

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

Figure 2. Transcriptional level analysis of the expression of genes csn1s2, csn3 and LALBA after induction.

Fig. 3 Comparison of SDS-PAGE electrophoresis results of target protein expression of BL21-PEP-CSN1S2 strain.

In the figure, M, marker; 1, the strain at 30°C is not induced; 2, the strain at 30°C is induced by IPTG; 3, the strain at 22°C is not induced; 4, the strain at 22°C is induced by IPTG; 5, the strain at 16°C is not induced; 6, 16 ℃ strain IPTG induction.

Figure 4 Western blot detection of the target protein PEP-CSN1S2.

Fig. 5 Comparison of SDS-PAGE electrophoresis results of expression of target protein of BL21-PEP-CSN3 strain.

In the figure, M, marker; 1, the strain at 30°C is not induced; 2, the strain at 30°C is induced by IPTG; 3, the strain at 22°C is not induced; 4, the strain at 22°C is induced by IPTG; 5, the strain at 16°C is not induced; 6, 16 ℃ strain IPTG induction.

Figure 6 Western blot detection of the target protein PEP-CSN3.

Fig. 7 Comparison of SDS-PAGE electrophoresis results of expression of target protein of BL21-PEP-LALBA strain.

In the figure, M, marker; 1, the strain at 30°C is not induced; 2, the strain at 30°C is induced by IPTG; 3, the strain at 22°C is not induced; 4, the strain at 22°C is induced by IPTG; 5, the strain at 16°C is not induced; 6, 16 ℃ strain IPTG induction.

Figure 8 Western blot detection of the target protein PEP-LALBA.

Sequence Listing Description

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

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

Detailed ways

In order to prove that the PEP polypeptide improves the expression efficiency of eukaryotic proteins, the present invention expresses the nucleotide sequence encoding the polypeptide PEP together with the eukaryotic target protein gene by fusion expression, and detects the expression of the target protein by SDS-PAGE electrophoresis. In order to prove the application of PEP polypeptide to achieve efficient synthesis of target protein.

The plasmids and strains listed in the following examples are only used to further describe the present invention in detail, and do not limit the essential content of the present invention. Where the specific experimental conditions are not indicated, all are in accordance with the conventional conditions well known to those skilled in the art or in accordance with the conditions suggested by the manufacturer. Plasmids, bacterial strain sources cited in the embodiment are as follows:

Prokaryotic expression vector pET-28a: it is a commercially available product of Quanxingjin Company;

Escherichia coli expression strain BL21(DE3): It is a commercial product of Quanshijin Company.

Example 1 Sequence analysis of PEP polypeptides

Analysis of the amino acid sequence showed that the polypeptide PEP (the amino acid sequence of which is shown in SEQ ID NO. 2) was composed of 114 amino acids encoded by 342 nucleotides, and the molecular weight was 11.38 kDa. The nucleotide sequence is shown in SEQ ID NO.1; it contains 8 negatively charged amino acids and 5 positively charged amino acids, with a predicted isoelectric point of 4.51, 23% of polar amino acid residues, and 20% of hydrophobic amino acid residues. Secondary structure analysis showed that it contained a random coil, 4 β sheets and 2 α helices.

Example 2 PEP polypeptide improves the high-efficiency expression of αS2-casein

1. Experimental materials

Prokaryotic expression vector pET-28a: it is a commercially available product of Quanxingjin Company;

Escherichia coli expression strain BL21(DE3): It is a commercial product of Quanshijin Company.

2. Experimental method

1. Vector construction. Using the recombinant plasmid pUC-CSN1S2 preserved in our laboratory as the template, adding PCR-specific primers to amplify the complete nucleotide sequence of the αS2-casein gene CSN1S2, and using the genomic DNA of Deinococcus gobi as the template to amplify the nucleotide sequence of the PEP polypeptide. DNA, PCR products were recovered by gel. The pET28a vector was digested with Nco I/Xho I and the target gene fragment containing the complementary sequence of the adjacent fragment was used for homologous recombination ligation.

2. Obtaining expression strains. The expression vector was transformed into Escherichia coli BL21, and the inserted sequence was verified by PCR, enzyme digestion and sequencing, and the strain was named BL21-PEP-CSN1S2. At the same time, the CSN1S2 without the fusion fragment PEP polypeptide was also connected to the pET28a vector, and the expression vector was transformed into Escherichia coli BL21. After PCR, enzyme digestion, and sequencing, it was verified that the inserted sequence was correct, and the strain was named BL21-CSN1S2. A single colony was picked for PCR verification, the recombinant plasmid was extracted for restriction digestion, and sequenced for verification.

3. Induced expression of target protein. Recombinant strains were inoculated into LB liquid medium containing antibiotics at 1% inoculum, and incubated overnight at 37°C on a shaker. With the initial concentration of OD600 of 0.1, it was received into 500 mL of LB liquid medium supplemented with kanamycin, and cultured at 37 °C to a bacterial concentration of 0.6 to 0.8. IPTG (final concentration 0.5 μmol/L) was added to induce protein expression at 16°C, 22°C, and 30°C, respectively. The cells were disrupted by ultrasonic waves, and the expression of the target protein was detected by SDS-PAGE.

4. Gene expression analysis. The innuPREP RNA Mini Kit was used to extract total RNA from the recombinant expression strains induced by IPTG. After removing the genomic DNA, reverse transcription to synthesize cDNA. Specific qRT-PCR primers were designed according to the sequence characteristics of the target gene, and the internal reference gene was the 16SrDNA gene. The expression of the target gene in the strain was determined by fluorescence quantitative PCR reaction, and each sample was set in 3 parallels.

5. Western blotting. The strains were collected, the cells were disrupted by sonication, and subjected to SDS-PAGE electrophoresis. The protein on the gel was transferred to PVDF membrane, and the antibody was incubated after blocking for 3 hours. The color reaction was carried out according to the labeling of the secondary antibody, and the expression of the target protein was detected.

3. Experimental results

The results of SDS-PAGE electrophoresis showed that the recombinant strain BL21-CSN1S2 did not express the target protein under different induction conditions, as shown in lanes 3 and 4 in Figure 1. The results showed that the eukaryotic αS2-casein CSN1S2 gene was affected during recombinant expression and could not be induced to express. The results of real-time quantitative PCR (Figure 2) showed that in the recombinant strain BL21-CSN1S2, the csn1S2 gene was transcribed, indicating that the mRNA translation process of csn1S2 in the strain was blocked, resulting in the inability to synthesize the target αS2-casein. In contrast, in the recombinant strain BL21-PEP-CSN1S2 in which the PEP polypeptide was fused and expressed, an obvious protein-induced expression band could be seen. Figure 3 shows that the proteins were expressed at 16°C, 22°C, and 30°C, and the best conditions were at 22°C and 30°C. The results of Western blotting showed that the induced protein band was the target protein PEP-CSN1S2.

Fourth, the experimental conclusion

The eukaryotic αS2-casein gene csn1S2 could not be recombinantly expressed in vitro; the fusion expression of the PEP polypeptide and the target csn1S2 gene could complete the high-efficiency expression of the eukaryotic αS2-casein csn1S2 gene.

Example 3 PEP polypeptide improves the high-efficiency expression of κ-casein

1. Experimental materials

Prokaryotic expression vector pET-28a: it is a commercially available product of Quanxingjin Company;

Escherichia coli expression strain BL21(DE3): It is a commercial product of Quanshijin Company.

2. Experimental method

1. Vector construction. Using the recombinant plasmid pUC-CSN3 stored in our laboratory as the template, adding PCR-specific primers to amplify the nucleotide sequence of the complete kappa-casein gene CSN3, and using the genomic DNA of Deinococcus gobi as the template to amplify the nucleotide sequence of the PEP polypeptide. DNA, PCR products were recovered by gel. The pET28a vector was digested with Nco I/Xho I and the target gene fragment containing the complementary sequence of the adjacent fragment was used for homologous recombination ligation.

2. Obtaining expression strains. The expression vector was transformed into Escherichia coli BL21, and the inserted sequence was verified by PCR, enzyme digestion and sequencing, and the strain was named BL21-PEP-CSN3. At the same time, the CSN3 without the fusion fragment PEP polypeptide was also connected to the pET28a vector, and the expression vector was transformed into Escherichia coli BL21. After PCR, enzyme digestion, and sequencing, the inserted sequence was verified to be correct, and the strain was named BL21-CSN3. A single colony was picked for PCR verification, the recombinant plasmid was extracted for restriction digestion, and sequenced for verification.

3. Induced expression of target protein. The recombinant strains were inoculated into LB liquid medium supplemented with antibiotics at 1% inoculation amount, and cultured at 37°C on a shaker overnight. With the initial concentration of OD600 of 0.1, it was received into 500 mL of LB liquid medium supplemented with kanamycin, and cultured at 37 °C to a bacterial concentration of 0.6 to 0.8. IPTG (final concentration 0.5μmol/L) was added to induce protein expression at 16°C, 22°C, and 30°C, respectively. The cells were disrupted by ultrasonic waves, and the expression of target kappa-casein was detected by SDS-PAGE.

4. Gene expression analysis. The innuPREP RNA Mini Kit was used to extract total RNA from the recombinant expression strains induced by IPTG. After removing the genomic DNA, reverse transcription to synthesize cDNA. Specific qRT-PCR primers were designed according to the sequence characteristics of the target gene, and the internal reference gene was the 16SrDNA gene. The expression of the target gene in the strain was determined by fluorescence quantitative PCR reaction, and each sample was set in 3 parallels.

5. Western blotting. The strains were collected, the cells were disrupted by sonication, and subjected to SDS-PAGE electrophoresis. The protein on the gel was transferred to PVDF membrane, and the antibody was incubated for 3 hours after blocking, and the color reaction was carried out according to the secondary antibody labeling to detect the expression of the target protein.

3. Experimental results

The results of SDS-PAGE electrophoresis showed that the recombinant strain BL21-CSN3 did not express the target protein under different induction conditions, as shown in lanes 5 and 6 in Figure 1. The results showed that the eukaryotic κ-casein gene csn3 was not expressed during recombinant expression. When the effect is reached, it cannot be induced to express. Figure 2. The results of real-time quantitative PCR showed that in the recombinant strain BL21-CSN3, the csn3 gene could be transcribed normally, and its expression was upregulated by more than 100 times, indicating that the csn3 mRNA translation process in the strain was blocked, resulting in the inability to synthesize the target κ-casein . In contrast, in the recombinant strain BL21-PEP-CSN3 in which the PEP polypeptide is fused and expressed, an obvious protein-induced expression band can be seen, as shown in Figure 5. The expression levels of the target protein PEP-LALBA were similar under different induction conditions at 16°C, 22°C and 30°C. Western blotting results Figure 6 shows that the protein band induced to express is the target protein PEP-CSN3, and almost all of the expressed proteins are present in the cell disrupted supernatant and are soluble proteins.

Fourth, the experimental conclusion

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

Example 4 PEP polypeptide improves the high-efficiency expression of α-lactalbumin

1. Experimental materials

Prokaryotic expression vector pET-28a: it is a commercially available product of Quanxingjin Company;

Escherichia coli expression strain BL21(DE3): It is a commercial product of Quanshijin Company.

2. Experimental method

1. Vector construction. Using the recombinant plasmid pUC-LALBA stored in our laboratory as a template, adding PCR-specific primers to amplify the nucleotide sequence of the complete α-lactalbumin gene LALBA, and using the genomic DNA of Deinococcus gobi as a template to amplify the nucleotide sequence of the PEP polypeptide. DNA, PCR products were recovered by gel. The pET28a vector was digested with Nco I/Xho I and the target gene fragment containing the complementary sequence of the adjacent fragment was used for homologous recombination ligation.

2. Obtaining expression strains. The expression vector was transformed into Escherichia coli BL21, and the inserted sequence was verified by PCR, enzyme digestion and sequencing, and the strain was named BL21-PEP-LALBA. At the same time, LALBA without the fusion fragment PEP polypeptide was also connected to the pET28a vector, and the expression vector was transformed into Escherichia coli BL21. After PCR, enzyme digestion, and sequencing, the inserted sequence was verified to be correct, and the strain was named BL21-LALBA. A single colony was picked for PCR verification, the recombinant plasmid was extracted for restriction digestion, and sequenced for verification.

3. Induced expression of target protein. The recombinant strains were inoculated into LB liquid medium supplemented with antibiotics at 1% inoculation amount, and cultured at 37°C on a shaker overnight. With the initial concentration of OD600 of 0.1, it was received into 500 mL of LB liquid medium supplemented with kanamycin, and cultured at 37 °C to a bacterial concentration of 0.6 to 0.8. IPTG (final concentration 0.5μmol/L) was added to induce protein expression at 16°C, 22°C, and 30°C, respectively. The cells were disrupted by ultrasonic waves, and the expression of target kappa-casein was detected by SDS-PAGE.

4. Gene expression analysis. The innuPREP RNA Mini Kit was used to extract total RNA from the recombinant expression strains induced by IPTG. After removing the genomic DNA, reverse transcription to synthesize cDNA. Specific qRT-PCR primers were designed according to the sequence characteristics of the target gene, and the internal reference gene was the 16SrDNA gene. The expression of the target gene in the strain was determined by fluorescence quantitative PCR reaction, and each sample was set in 3 parallels.

5. Western blotting. The strains were collected, the cells were disrupted by sonication, and subjected to SDS-PAGE electrophoresis. The protein on the gel was transferred to PVDF membrane, and the antibody was incubated for 3 hours after blocking, and the color reaction was carried out according to the secondary antibody labeling to detect the expression of the target protein.

3. Experimental results

The results of SDS-PAGE electrophoresis showed that the recombinant strain BL21- did not express the target protein under different induction conditions at 16°C, 22°C and 30°C. When it is affected, it cannot be induced to express, as shown in lanes 7 and 8 in Figure 1. The results of real-time quantitative PCR (Figure 2) showed that in the recombinant strain BL21-LALBA, the α-lactalbumin gene LALBA could be transcribed normally, and the gene expression was increased by 22 times after induction, indicating that the mRNA translation and protein process of LALBA in the strain was blocked, resulting in Alpha-lactalbumin cannot be synthesized. In the recombinant strain BL21-PEP-LALBA in which the PEP polypeptide was fused and expressed, an obvious protein-induced expression band could be seen, as shown in Figure 7 . The expression levels of the target protein PEP-LALBA were similar under different induction conditions at 16°C, 22°C and 30°C. The results of Western blotting in Figure 8 show that the protein bands induced to express are the target protein PEP-LALBA, and most of them are soluble proteins.

Fourth, the experimental conclusion

The eukaryotic case α-lactalbumin gene LALBA cannot be recombinantly expressed in vitro; the fusion of PEP polypeptide and the target LALBA gene can complete the high-efficiency expression of eukaryotic α-lactalbumin.

The experiments of the above three different proteins show that: PEP polypeptide can promote the expression of eukaryotic protein genes in vitro, so that the original protein genes that cannot be expressed in vitro can obtain the function of in vitro recombinant expression.

sequence listing

<110> Institute of Biotechnology, 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 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 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 (5)

1. The application of the polypeptide whose amino acid sequence is shown in SEQ ID NO. 2 in promoting the efficient expression of E. coli heterologous proteins, the method is to co-express the gene of the polypeptide and the gene of the target protein by fusion expression. 2. The application of claim 1, wherein the nucleotide sequence of the polypeptide encoding gene is shown in SEQ ID NO.1. 3. The application according to claim 1, the high-efficiency expression means that the polypeptide can promote the expression of the protein gene of E. coli heterologous expression that could not be carried out originally, or improve the expression efficiency of the protein. 4. the plasmid comprising the coding gene of the polypeptide shown in SEQ ID NO.2 in promoting the application of Escherichia coli heterologous expression recombinant protein, the method is that the gene of the polypeptide and the target protein gene are combined with the mode of fusion expression. Express. 5. A method for promoting high-efficiency expression of heterologous proteins in Escherichia coli, characterized in that the gene of the polypeptide whose amino acid sequence is shown in SEQ ID NO. 2 and the target protein gene are co-expressed by fusion expression.

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