CN110863002A - Method for modifying secondary translation initiation site to enable protein to be correctly expressed - Google Patents
Method for modifying secondary translation initiation site to enable protein to be correctly expressed Download PDFInfo
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Abstract
The invention belongs to the technical field of gene technology and microorganisms, and particularly relates to a method for modifying a secondary translation initiation site to ensure that a protein is correctly expressed. The method comprises the following steps: analyzing the gene sequence of the target protein, and judging that a secondary translation initiation site exists when a similar ribosome binding site sequence and a proper spacer sequence are positioned at the upstream of an initiation codon; carrying out synonymous mutation on the similar ribosome binding site sequence or directly removing the similar ribosome binding site sequence to obtain a new gene sequence; and constructing a new gene sequence into a pET expression vector to obtain correctly expressed protein. The method of the invention modifies the similar ribosome binding site sequence in the gene sequence containing the secondary translation initiation site, effectively avoids protein expression abnormality caused by the existence of the secondary translation initiation site, realizes normal expression of the protein with the abnormal expression of the target protein, and ensures that the molecular weight of the expressed target protein conforms to the expected size.
Description
Technical Field
The invention belongs to the technical field of gene technology and microorganisms, and particularly relates to a method for modifying a secondary translation initiation site to ensure that a protein is correctly expressed.
Background
Protein expression refers to the process of expressing a protein using genetic recombination techniques. There are many factors that affect protein expression, and sometimes the molecular weight of the expressed protein of interest is smaller than expected, and there may be two cases where degradation of the protein occurs and another case where a secondary translation initiation site exists in the gene sequence.
By analyzing the target gene sequence of the protein with abnormal expression, the existence of the secondary translation initiation site can be effectively avoided, so that the expressed protein can be effectively prevented from being truncated. Proteins will only exert their effect when correctly expressed in the correct conformation.
Disclosure of Invention
The present invention is directed to overcoming the deficiencies of the prior art by providing a method for modifying a secondary translational start site to allow for proper expression of a protein.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows:
a method of engineering a secondary translational start site for proper protein expression, comprising the steps of:
(1) analyzing the gene sequence of the target protein, and judging that a secondary translation initiation site exists in the gene sequence when a similar ribosome binding site sequence plus a proper spacer sequence is found to be positioned at the upstream of an initiation codon;
(2) performing synonymous mutation on the similar ribosome binding site sequence in the step (1) or directly removing the similar ribosome binding site sequence to obtain a new gene sequence;
(3) and (3) constructing the new gene sequence obtained in the step (2) into a pET expression vector, and then transforming the pET expression vector into escherichia coli for protein induction expression to obtain correctly expressed protein.
In the above scheme, the analogous ribosome binding site sequence is "AAGGAGG".
In the above embodiment, the appropriate spacer sequence is a 5 to 13 nucleotide sequence.
In the above scheme, the initiation codon is AUG or GUG.
In the above scheme, the synonymous mutation means mutation of the base sequence "CAGGAG" into the base sequence "CAAGAA".
The invention has the beneficial effects that: the method of the invention modifies the similar ribosome binding site sequence in the gene sequence containing the secondary translation initiation site, effectively avoids protein expression abnormality caused by the existence of the secondary translation initiation site, realizes normal expression of the protein with the abnormal expression of the target protein, and ensures that the molecular weight of the expressed target protein conforms to the expected size.
Drawings
FIG. 1 is an electrophoresis diagram of the expression abnormality of the recombinant plasmid pET32a-irak1 protein constructed from the original gene, wherein 1: marker, 2-4: pET32a-irak1 transformed E.coli BL21(DE3), 5: pET32a was transformed into E.coli BL21(DE 3).
FIG. 2 is an electrophoretogram of normal expression of recombinant plasmid pET32a-irak1 protein after modification, wherein 1: marker, 2-4: pET32a-irak1 transformed E.coli BL21(DE3), 5: pET32a was transformed into E.coli BL21(DE 3).
FIG. 3 is an electrophoresis diagram of the original gene constructed recombinant plasmid pET32a-NT3 protein expression abnormality, wherein 1: marker, 2: pET32a-NT3 transformed E.coli BL21(DE3), 3: pET32a was transformed into E.coli BL21(DE 3).
FIG. 4 is an electrophoresis diagram of the normal expression of recombinant plasmid pET32a-NT3 protein constructed after modification, wherein 1: marker, 2-4: pET32a-NT3 transformed E.coli BL21(DE3), 5: pET32a was transformed into E.coli BL21(DE 3).
Detailed Description
In order to better understand the present invention, the following examples are further provided to illustrate the present invention, but the present invention is not limited to the following examples.
Example 1
The gene sequence of Irak1 is shown in SEQ ID NO.1, when the murine IRAK1 protein is expressed in a prokaryotic expression system, the molecular weight of the protein expression is found to be obviously different from the molecular weight of the protein in a selected region (smaller than the expected molecular weight, as shown in figure 1), the sequencing result is checked to be completely matched with the original sequence and can be translated normally, but the protein expression is abnormal, and the protein expression is truncated.
The Irak1 gene sequence is analyzed to find that: the presence of the same sequence as the ribosome binding site sequence (AGGAG) in the pET vector and the methionine (Met) codon ATG 10bp downstream thereof in the gene sequence may constitute a secondary translation initiation site, and when the secondary translation initiation site is present in the gene sequence, the expression of the protein of interest may be truncated. It was preliminarily concluded that the protein Irak1, which has a protein sequence of about 166 amino acids (about 18 kD) and 8 amino acids (1.1kD) at the cleavage site, is translated from this secondary translation start site, and that the recombinant protein has about 20kD, which corresponds to the band on the electrophoretogram. DNMAN 2 DNMAN 1 identity 100.00% (5/5) gap 0.00% (0/5)
In the embodiment, two pairs of primers (F1/R1, Fm/Rm) are designed, synonymous mutation is carried out on similar ribosome binding site sequences in a gene sequence through an Overlap PCR technology, Gln Glu is encoded by bases (CAG GAG) at the similar ribosome binding sites in a target gene sequence, the base sequence is (CAA GAA) after the synonymous mutation is carried out, and then a mutated gene sequence fragment is built into a pET32a expression vector and is transformed into escherichia coli BL21(DE3) to carry out induced expression on protein.
The specific operation steps are as follows: (1) the original gene sequence of interleukin 1 receptor-associated kinase protein is shown as SEQID NO. 1;
(2) taking cDNA prepared by reverse transcription of RNA extracted from mouse liver tissues or lung tissues as a template, and carrying out synonymous mutation on a base sequence ' CAGGAG ' from ' 115 th site to ' 120 th site ' in an original gene sequence by using two pairs of primers F1/R1 and Fm/Rm through an Overlap PCR amplification technology to obtain a new gene sequence shown as SEQ ID NO. 2;
the sequences of the two pairs of primers F1/R1 and Fm/Rm are as follows:
the system of the PCR amplification reaction is
The PCR amplification reaction conditions are as follows:
(3) constructing a gene fragment with a sequence shown in SEQ ID NO.2 obtained in the step (2) into a pET32a expression vector, then transforming into escherichia coli BL21(DE3) for protein induction expression, and purifying to obtain a target protein, wherein the specific operation process of the construction-transformation-induction expression comprises the following steps of (1) taking cDNA prepared by reverse transcription of RNA extracted from liver tissues or lung tissues of a mouse as a template, and respectively carrying out PCR amplification by using primers F1 and Rm, Fm and R1 to obtain PCR products P1 and P2; (2) carrying out PCR amplification by using the PCR products P1 and P2 obtained in the step (1) as templates and using primers F1 and R1 to obtain a PCR product P3; (3) carrying out double enzyme digestion on the PCR products P3 and pET32a plasmids by using BamH I and Xho I respectively, and carrying out agarose gel electrophoresis detection and recovery on the enzyme digestion products; (4) Connecting the recovered product with T4 DNA ligase at 16 ℃ overnight, (5) transforming the connected product into Escherichia coli DH5 α competent cells, coating the cells on an LB solid plate with ampicillin (amp) resistance, inversely placing the cells in an incubator at 37 ℃ for culturing for 12-14 h, (6) carrying out colony PCR (using a general primer of a pET32a vector) on a single colony on the LB solid plate, detecting and screening out positive clones through agarose gel, sending the positive clones to a sequencing company for sequencing, comparing the sequencing result with an original sequence to confirm whether the sequencing result is correct and whether the site to be mutated is successful, (7) transforming a recombinant plasmid pET32a-irak1 with correct sequencing and successful mutation into Escherichia coli BL21(DE3) competent cells, coating the cells on an LB solid plate with ampicillin (amp) resistance, inversely placing the cells in an incubator at 37 ℃ for culturing for 12-14 h, (8) inoculating the single colony on the plate in the step (7) to 3mL of a liquid containing ampicillin (amp) in a shaking table, placing the liquid medium at a constant temperature, and culturing at 37 ℃ to 200rpm, and culturing at 37 ℃ to obtain a strain600When the concentration is about 0.6, IPTG is added to make the final concentration be 0.5mM/L, and the mixture is cultured and induced for 3 hours continuously at 200rpm in a constant temperature shaking table at 37 ℃; (9) centrifuging at 5000rpm for 3min, collecting thallus, adding 100 μ L1 xSDS-PAGE loading buffer boiling water bath for 10min, and detecting whether the target protein is expressed by SDS-PAGE electrophoresis; (10) purifying the expressed target protein.
The results of the comparison between the original nucleotide sequence and the mutated nucleotide sequence are shown below:
upper line IRAK1, from 7to 632 (shown in SEQ ID NO. 2)
Lower line DNAMAN1, from 1to 626 (original sequence, shown in SEQ ID NO. 1)
995G:DNAMAN1 identity＝99.68％(625/627)gap＝0.95％(6/633)
The front and back ends are enzyme cutting sites in the vector sequence, and the bold area is mutation sites.
The results of translating them into amino acids are as follows (shown in SEQ ID NO. 3):
Upper line:DNAMAN4,from 3to 210(Irak1)
Lower line:DNAMAN8,from 1to 208(Origin)
DNAMAN4:DNAMAN8 identity＝100.00％(209/209)gap＝0.95％(2/211)
the front and back ends are amino acids translated by the restriction enzyme sites in the vector sequence, 4 amino acids before and after removal, and the rest are completely matched.
The vector constructed in this example was sequenced and aligned with the original sequence (alignment results are as above), and the sequencing, alignment and analysis results show that: the remaining gene sequences, except for the mutation sites, matched perfectly to the original sequence, indicating that the analogous ribosome binding site present in the IRAK1 gene sequence was successfully synonymously mutated. Meanwhile, an original IRAK1 gene sequence is constructed into a pET32a expression vector and then is transformed into escherichia coli for protein induction expression; and constructing the gene sequence successfully subjected to the synonymous mutation into a pET32a expression vector, transforming the expression vector into escherichia coli for protein induction expression, and performing electrophoresis analysis on the protein subjected to the protein induction expression respectively, wherein the results are shown in fig. 1 and fig. 2, and it can be known from fig. 1 that: the molecular weight of the protein expressed by the original IRAK1 gene sequence is about 20-21 KDa, and the molecular weight is obviously different from that of IRAK1 protein; as can be seen from fig. 2: the molecular weight of the protein expressed by the synonymy mutated IRAK1 gene sequence is about 40KDa, which is in line with the expected molecular weight (40KDa) of the protein. This indicates that the secondary translation initiation site affecting the correct expression of the protein of interest (IRAK1) is eliminated by the synonymous mutation to the analogous ribosome binding site in the secondary translation initiation site present in the gene sequence of the protein of interest, allowing the correct expression of the protein of interest. After the secondary translation initiation site is modified by the method, the normal expression of the interleukin 1 receptor-related kinase protein is realized, the interleukin 1 receptor-related kinase protein can play a role only after being correctly expressed to form a correct conformation, the expression yield of the interleukin 1 receptor-related kinase protein is greatly improved, and the protein concentration can reach 5 mg/mL.
Example 2
The gene sequence of the NT3 protein is shown as SEQ ID NO.3, when the murine NT3 protein is expressed in a prokaryotic expression system, the molecular weight of the protein expression is found to be obviously different from the molecular weight of the protein in a selected region (smaller than the expected molecular weight, see figure 3), the check sequencing result is completely matched with the original sequence and can be translated normally, but the protein expression is abnormal, and the protein expression is truncated.
The NT-3 gene sequence analysis shows that the 24 th to 28 th positions of the gene sequence are 'AGGAG', the gene sequence is completely identical to a ribosome binding site sequence (AGGAG) on a used pET carrier, and a valine (Val) codon GUG (also can be used as an initiation codon) is arranged at the 8bp position downstream of the gene sequence, so that a secondary translation initiation site can be formed, the expressed protein is truncated, and the normal expression of the protein is influenced. As a preliminary judgment, the protein of interest will be translated from the secondary translation initiation site, and the translated protein sequence is shown below, about 107 amino acids, about 12.8KDa, and 8 amino acids (1.1KDa) at the cleavage site, and the recombinant protein is about 14KDa, which corresponds to the band on the electrophoretogram.
Upper line:DNAMAN2,from 1to 4
Lower line:DNAMAN1,from 24to 27
DNAMAN2:DNAMAN1 identity＝100.00％(5/5)gap＝82.14％(23/28)
Analyzing the existence of similar ribosome binding sites in the NT-3 gene sequence and the positions 24-28 (AGGAG) of the gene sequence, directly removing the first 30 bases of the target gene sequence, namely directly removing the similar ribosome binding sites to obtain the modified gene sequence shown in SEQ ID NO.4, then constructing the modified target gene fragment into a pET32a expression vector, and transforming the modified target gene fragment into escherichia coli BL21(DE3) for induced expression of protein.
The vector constructed in this example was sequenced and aligned with the original sequence (alignment results are shown below), and the sequencing, alignment and analysis results show that: the first 30 bases were removed and the remaining gene sequence was perfectly matched and read through to the original sequence.
Sequencing results
Upper line DNAMAN1, from 7to 332 (shown in SEQ ID NO. 5)
Lower line DNAMAN2, from 31to 356(Origin, shown in SEQ ID NO. 4)
DNAMAN1:DNAMAN2 identity＝100.00％(327/327)gap＝1.80％(6/333)
The front and back ends are enzyme cutting sites in the vector sequence;
the results of translating them into amino acids are as follows (shown in SEQ ID NO. 6):
Upper line:DNAMAN14,from 3to 110pET32a-NT3
Lower line:DNAMAN13,from11 to 108ORIGIN
DNAMAN14:DNAMAN13 identity＝100.00％(109/109)gap＝1.80％(2/111)
the front and back ends are amino acids translated by the restriction enzyme sites in the vector sequence, 4 amino acids before and after removal, and the rest are completely matched.
Meanwhile, an original NT-3 gene sequence is constructed into a pET32a expression vector and then is transformed into escherichia coli BL21(DE3) for protein induction expression; and constructing the gene sequence modified by the embodiment into a pET32a expression vector, then transforming the vector into escherichia coli BL21(DE3) for protein induction expression, and respectively carrying out electrophoresis analysis on the protein subjected to induction expression, wherein the results are shown in figures 3 and 4, and show that: the expression of the modified gene protein is about 35KDa, and the protein accords with the expected protein molecular weight (32 KDa). This suggests that the secondary translation initiation site affecting the correct expression of the protein was eliminated by removing the analogous ribosome binding site from the secondary translation initiation site present in the gene sequence of the protein of interest (NT-3), resulting in the correct expression of the protein of interest.
It is apparent that the above embodiments are only examples for clearly illustrating and do not limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications are therefore intended to be included within the scope of the invention as claimed.
Sequence listing
<110> Wuhan Severe Biotech Co., Ltd
<120> a method for modifying secondary translation initiation site to make protein correctly express
<160>6
<210>1
<211>627bp
<212>DNA
<213> original gene sequence of murine IRAK1 protein
<400>1
caccgtcggg ccaagaagag gccccccatg acccaggtat acaagagact agaagggctt 60
caggcagggc ctccctggga gctagaggtt gccggccatg gctccccttc cccacaggag 120
aactcctaca tgtctaccac tggcagtgcc cagagtgggg atgaaccatg gcagcctcta 180
gtagtgacca caagagcccc agcccaggct gcccagcaac tccagagaag tcccaaccag 240
ccagtggaaa gtgatgagag tgttcccggc ctctctgcta ccctgcattc ctggcacttg 300
actccaggtt cccacccaag cccagcgtcc ttcagagagg ctagctgtac ccagggaggc 360
actaccagag aatcaagtgt gaggagtagc ccaggcttcc agcctacaac catggaaggc 420
tcacccacgg gcagctcatc cctgctgtca tcagaaccac cacagatcat catcaaccca 480
gcccgacaga agatggtaca aaagctggct ctttatgaag aaggggtctt ggatagcctg 540
caactgctgt catcaggctt tttcccaggc ttggatttag aacctgaaaa gagccaggga 600
cctgaagaaa gtgatgaatt ccagagc627
<210>2
<211>639bp
<212>DNA
<213> murine IRAK1 protein modified gene sequence
<400>2
ggatcccacc gtcgggccaa gaagaggccc cccatgaccc aggtatacaa gagactagaa 60
gggcttcagg cagggcctcc ctgggagcta gaggttgccg gccatggctc cccttcccca 120
caagaaaact cctacatgtc taccactggc agtgcccaga gtggggatga accatggcag 180
cctctagtag tgaccacaag agccccagcc caggctgccc agcaactcca gagaagtccc 240
aaccagccag tggaaagtga tgagagtgtt cccggcctct ctgctaccct gcattcctgg 300
cacttgactc caggttccca cccaagccca gcgtccttca gagaggctag ctgtacccag 360
ggaggcacta ccagagaatc aagtgtgagg agtagcccag gcttccagcc tacaaccatg 420
gaaggctcac ccacgggcag ctcatccctg ctgtcatcag aaccaccaca gatcatcatc 480
aacccagccc gacagaagat ggtacaaaag ctggctcttt atgaagaagg ggtcttggat 540
agcctgcaac tgctgtcatc aggctttttc ccaggcttgg atttagaacc tgaaaagagc 600
cagggacctg aagaaagtga tgaattccag agcctcgag 639
<210>3
<211>209
<212>PRT
<213> murine IRAK1 protein
<400>3
His Arg Arg Ala Lys Lys Arg Pro Pro Met Thr Gln Val Tyr Lys Arg
1 5 10 15
Leu Glu Gly Leu Gln Ala Gly Pro Pro Trp Glu Leu Glu Val Ala Gly
20 25 30
His Gly Ser Pro Ser Pro Gln Glu Asn Ser Tyr Met Ser Thr Thr Gly
35 40 45
Ser Ala Gln Ser Gly Asp Glu Pro Trp Gln Pro Leu Val Val Thr Thr
50 55 60
Arg Ala Pro Ala Gln Ala Ala Gln Gln Leu Gln Arg Ser Pro Asn Gln
65 70 75 80
Pro Val Glu Ser Asp Glu Ser Val Pro Gly Leu Ser Ala Thr Leu His
85 90 95
Ser Trp His Leu Thr Pro Gly Ser His Pro Ser Pro Ala Ser Phe Arg
100 105 110
Glu Ala Ser Cys Thr Gln Gly Gly Thr Thr Arg Glu Ser Ser Val Arg
115 120 125
Ser Ser Pro Gly Phe Gln Pro Thr Thr Met Glu Gly Ser Pro Thr Gly
130 135 140
Ser Ser Ser Leu Leu Ser Ser Glu Pro Pro Gln Ile Ile Ile Asn Pro
145 150 155 160
Ala Arg Gln Lys Met Val Gln Lys Leu Ala Leu Tyr Glu Glu GlyVal
165 170 175
Leu Asp Ser Leu Gln Leu Leu Ser Ser Gly Phe Phe Pro Gly Leu Asp
180 185 190
Leu Glu Pro Glu Lys Ser Gln Gly Pro Glu Glu Ser Asp Glu Phe Gln
195 200 205
Ser
<210>4
<211>357bp
<212>DNA
<213> original gene sequence of murine NT-3 protein
<400>4
tatgcagaac ataagagtca ccgaggagag tactcagtgt gtgacagtga gagcctgtgg 60
gtgaccgaca agtcctcagc cattgacatt cggggacacc aggtcacagt gctgggggag 120
atcaaaaccg gtaactctcc tgtgaaacaa tatttttatg aaacgagatg taaagaagcc 180
aggccggtca aaaacggttg cagggggatt gatgacaaac actggaactc tcagtgcaaa 240
acttcgcaaa cctatgtccg agcactgact tcagaaaaca acaaactcgt aggctggcgc 300
tggatacgaa tagacacttc ctgtgtgtgt gccttgtcga gaaaaattgg aagaaca 357
<210>5
<211>339bp
<212>DNA
<213> murine NT-3 protein modified gene sequence
<400>5
gaattctact cagtgtgtga cagtgagagc ctgtgggtga ccgacaagtc ctcagccatt 60
gacattcggg gacaccaggt cacagtgctg ggggagatca aaaccggtaa ctctcctgtg 120
aaacaatatt tttatgaaac gagatgtaaa gaagccaggc cggtcaaaaa cggttgcagg 180
gggattgatg acaaacactg gaactctcag tgcaaaactt cgcaaaccta tgtccgagca 240
ctgacttcag aaaacaacaa actcgtaggc tggcgctgga tacgaataga cacttcctgt 300
gtgtgtgcct tgtcgagaaa aattggaaga acactcgag 339
<210>6
<211>109
<212>PRT
<213> murine NT-3 protein
<400>6
Tyr Ser Val Cys Asp Ser Glu Ser Leu Trp Val Thr Asp Lys Ser Ser
1 5 10 15
Ala Ile Asp Ile Arg Gly His Gln Val Thr Val Leu Gly Glu Ile Lys
20 25 30
Thr Gly Asn Ser Pro Val Lys Gln Tyr Phe Tyr Glu Thr Arg Cys Lys
35 40 45
Glu Ala Arg Pro Val Lys Asn Gly Cys Arg Gly Ile Asp Asp Lys His
50 55 60
Trp Asn Ser Gln Cys Lys Thr Ser Gln Thr Tyr Val Arg Ala Leu Thr
65 70 75 80
Ser Glu Asn Asn Lys Leu Val Gly Trp Arg Trp Ile Arg Ile Asp Thr
85 90 95
Ser Cys Val Cys Ala Leu Ser Arg Lys Ile Gly Arg Thr
100 105
Claims (5)
1. A method of engineering a secondary translational start site for proper protein expression comprising the steps of:
(1) analyzing the gene sequence of the target protein, and judging that a secondary translation initiation site exists in the gene sequence when a similar ribosome binding site sequence plus a proper spacer sequence is found to be positioned at the upstream of an initiation codon;
(2) performing synonymous mutation on the similar ribosome binding site sequence in the step (1) or directly removing the similar ribosome binding site sequence to obtain a new gene sequence;
(3) and (3) constructing the new gene sequence obtained in the step (2) into a pET expression vector, and then transforming the pET expression vector into escherichia coli for protein induction expression to obtain correctly expressed protein.
2. The method of modifying a secondary translational start site for proper protein expression of claim 1, wherein the analogous ribosome binding site sequence is "AAGGAGG".
3. The method of claim 1, wherein the suitable spacer sequence is a sequence of 5 to 13 bases.
4. The method of claim 1, wherein the initiation codon is AUG or GUG.
5. The method for modifying the secondary translational initiation site for correct expression of a protein according to claim 1, wherein the synonymous mutation is mutation of the base sequence "CAGGAG" to the base sequence "CAAGAA".
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101014705A (en) * | 2004-07-07 | 2007-08-08 | 帝斯曼知识产权资产管理有限公司 | Improved enzymes |
| WO2017031399A1 (en) * | 2015-08-20 | 2017-02-23 | Genomatica, Inc. | Compositions and multiplexed systems for coupled cell-free transcription-translation and protein synthesis and methods for using them |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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