CN106636165B - It is a kind of based on the dual-function dna element of RbsB signal peptide and its application - Google Patents
It is a kind of based on the dual-function dna element of RbsB signal peptide and its application Download PDFInfo
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- CN106636165B CN106636165B CN201710060002.3A CN201710060002A CN106636165B CN 106636165 B CN106636165 B CN 106636165B CN 201710060002 A CN201710060002 A CN 201710060002A CN 106636165 B CN106636165 B CN 106636165B
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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- C12N2830/00—Vector systems having a special element relevant for transcription
- C12N2830/001—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination
- C12N2830/002—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor
- C12N2830/003—Vector systems having a special element relevant for transcription controllable enhancer/promoter combination inducible enhancer/promoter combination, e.g. hypoxia, iron, transcription factor tet inducible
Abstract
The present invention relates to a kind of based on the dual-function dna element of RbsB signal peptide and its application, and sequence is as shown in SEQ ID NO:1, or is the same sense mutation body of the sequence, and 16-36 nucleotide of the same sense mutation body do not mutate.The invention further relates to double promoter DNA elements and protein expression system including the dual-function dna element.By the inducible promoter for disposing an Escherichia coli in dual-function dna components upstream of the invention, and the exogenous gene sequence that need to be expressed is connected in the member downstream, can be in expression in escherichia coli foreign protein, and can be used different inductive conditions respectively by the protein expression in cytoplasm or periplasmic space.The DNA construct enables researcher by constructing a kind of expression vector, without constructing cytoplasm and periplasmic space expression vector respectively, can compare the superiority and inferiority of two kinds of expression ways.
Description
Technical field
The present invention relates to molecular biology fields, relate more particularly to a kind of dual-function dna member based on RbsB signal peptide
Part and its application, and double promoter DNA element and protein expression system including the dual-function dna element.
Background technique
Escherichia coli are one of the common bacterium of engineered protein expression.Escherichia coli have the growth rate and height being exceedingly fast
The transcription of effect, translation ability can synthesize a large amount of foreign proteins in a short time.Majority engineered protein expression system at present
All be by exogenous protein expression in the cytoplasm of Escherichia coli.If expressed protein has good soluble and steady
Qualitative, common engineering bacterium can produce and the comparable foreign protein of their own Tot Prot within several hours.
Although exogenous protein expression can be obtained high yield in the cytoplasm of bacterium, there is also lack for this method
Point.One problem is must to be just folded into that conformation is correct, dissolution after foreign protein synthesis under the assistance of the chaperone of bacterium
Property good, the great-hearted albumen of tool, and the chaperone in cytoplasm is limited, so once expressed foreign protein is not allowed
Foldable, and when bacterium cannot provide enough chaperones and be assisted, those fold incorrect albumen and will coalesce
Together, insoluble inclusion body is formed in cytoplasm.Another problem is the ring that the cytoplasm of bacterium is a reproducibility
Border is unfavorable for the formation of disulfide bond, therefore success rate is very low when foreign protein of the expression containing disulfide bond.
In addition to cytoplasm, also there is expression system can be at the end N- of foreign protein plus signal peptide, by newly synthesized egg at present
In the white gap being transported between Bacillus coli cells wall and cell membrane, it is then folded into the great-hearted albumen of tool.This gap,
Claim periplasmic space (Periplasm), be the stronger environment of oxidisability, the formation of disulfide bond can be promoted, to containing disulfide bond
Exogenous protein expression than advantageous.On the other hand, the mechanism of bacterium also restricted periplasmic space albumen aggregate velocity, can be substantially
Degree reduces the foreign protein with signal peptide and synthesizes, and avoids the foreign protein that can not correctly fold from being formed in periplasmic space insoluble
Inclusion body.Therefore, it although protein level expressed by periplasmic space expression system is lower, is easier to form correct three
Tie up conformation.
Since cytoplasm and periplasmic space expression system cut both ways, people often require point when expressing foreign protein
It does not attempt, then determines which approach used.The plasmid vector of both expression systems and incompatible at present.With Merck
For the pET series expression vector of Sigma company (former EMD Millipore company), wherein pET23, pET28 are cytoplasm tables
Up to carrier, and pET22 is periplasmic space expression vector.In these carriers, control exogenous gene expression is all T7 promoter.It will
Clone has the expression vector of foreign gene to be transformed into containing T7 RNA polymerase (T7 RNA Polymerase, abbreviation T7RNP) base
The bacterial strain of cause, if BL21 (DE3) is inner, the expression that T7RNP can be induced by the way that IPTG is added in bacterium solution, to activate in carrier
T7 promoter, lead to the expression of foreign gene.These carriers the difference is that volume is contained in the foreign gene upstream of pET22 carrier
The signal peptide sequence of code bacterium pelB albumen, and do not have in pET23, pET28 carrier.This difference determines expressed external source egg
White is eventually positioned at periplasmic space or cytoplasm.
Although establishing a kind of both the bacterium table of periplasmic space can also be transported to by exogenous protein expression in cytoplasm
The workload of protein expression conditional optimizing phase can be substantially reduced up to carrier, but there is presently no successful examples.Its difficulty
It is that periplasmic space expression vector must contain signal coding sequence, and the sequence must be designed in the upstream of foreign gene
It can just work, but this arrangement also makes the expression vector be difficult to be used to express to accumulate without containing signal peptide, in cytoplasm
Foreign protein.
Summary of the invention
Inventor in the course of the research, passes through mutant Escherichia coli ribose binding protein (Ribose-binding
Protein, abbreviation RbsB) gene signal peptide coding region domain, obtain the chimeric signal coding sequence containing T7 promoter
(abbreviation T7SP).This special T7SP sequence can not only encode the signal peptide with normal function, while be also T7 promoter,
It can be identified by T7 RNA polymerase (T7RNP), activate the transcriptional expression of downstream gene.
Based on the above research, the present invention provides a kind of dual-function dna elements based on RbsB signal peptide, and sequence is such as
Shown in SEQ ID NO:1 or its sequence is the same sense mutation body of sequence shown in SEQ ID NO:1, and the same sense mutation
16-36 nucleotide of body do not mutate.
The present invention also provides application of the above-mentioned dual-function dna element in promotor gene transcription.
The present invention also provides application of the above-mentioned dual-function dna element in protein of the coding with signal peptide.
The present invention also provides a kind of double promoter DNA elements comprising above-mentioned dual-function dna element and is connected to institute
State the escherichia coli promoter of dual-function dna components upstream.
Preferably, the escherichia coli promoter is inducible promoters.
Preferably, the inducible promoters are non-IPTG inducible promoters.
Preferably, the non-IPTG inducible promoters are tetracycline promoter, and the sequence of the tetracycline promoter is such as
Shown in SEQ ID NO:2.
The present invention also provides a kind of protein expression systems comprising above-mentioned double promoter DNA element and encoding histone sequence
Column.
Preferably, the albumen coded sequence is that the protein for whether needing to be folded in periplasmic space do not known.
By the way that dual-function dna element of the invention to be connect with foreign gene coded sequence to be studied, fusion base is formed
Cause, and by the inducible promoter of one Escherichia coli of the arranged upstream of the fusion, such as tetracycline promoter, it can pass through
The promoter is activated, transcription generates the foreign gene mRNA containing overall length signal peptide sequence, so that expression is with the outer of signal peptide
Source protein matter is accumulated in the periplasmic space of Escherichia coli.And on the other hand, started by the T7 in activation T7SP coded sequence
Son, the DNA construct can transcribe the foreign gene mRNA for generating and containing only 3 ' terminal sequence of T7SP again, and with T7SP sequence end
AUG codon as translation initiation site, expression is free of the exogenous proteins of signal peptide, is accumulated in the cell of Escherichia coli
Matter.The DNA construct enables researcher by constructing a kind of expression vector, empty without constructing cytoplasm and pericentral siphon respectively
Between expression vector, the superiority and inferiority of two kinds of expression ways can be compared.
Detailed description of the invention
Fig. 1 is that T7SP is fitted into signal peptide compared with the DNA sequence dna and amino acid sequence of wild type RbsB signal peptide；
Fig. 2 is by T7SP and tetracycline promoter PTetB, double-promoter albumen composed by GFP nano antibody gene order
Expression system structural schematic diagram；
Fig. 3 is pACYC-TetR-PTetB- T7SP-GFPNb carrier and pACYC-TetR-PTetB- SP (WT)-GFPNb carrier
Protein electrophoresis after anhydrotetracycline (ATc) inducing expression analyzes result；
Fig. 4 is pACYC-TetR-PTetB- T7SP-GFPNb carrier and pACYC-TetR-PTetB- SP (WT)-GFPNb carrier
Protein electrophoresis after IPTG inducing expression analyzes result；
Fig. 5 is pACYC-TetR-PTetB- T7SP-GFPNb carrier and common cytoplasm, periplasmic space expression vector pass through
Protein electrophoresis after anhydrotetracycline (ATc) inducing expression analyzes result；
Fig. 6 is pACYC-TetR-PTetB- T7SP-GFPNb carrier and common cytoplasm, periplasmic space expression vector pass through
Protein electrophoresis after IPTG inducing expression analyzes result.
Specific embodiment
Principles and features of the present invention are described below in conjunction with example, the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
1. the building of dual function element T7SP
Work of the invention is based on the research to RbsB signal peptide sequence.As shown in Figure 1, inventor is by wild type RbsB
16-36 coding mutations in signal peptide sequence obtain dual function element T7SP, sequence is such as at T7 promoter sequence
Shown in SEQ ID NO:1.
2. the building of double-promoter protein expression system
The upstream of T7SP coded sequence is configured into an Escherichia coli inducible promoter, and is merging required table downstream
The foreign gene reached can form the protein expression system of a double-promoter.Here, it is opened so that T7SP and tetracycline is used in combination
Mover PTetBFor (SEQ ID NO:2) expresses the nano antibody (GFPNb) (SEQ ID NO:3) of GFP, illustrate double-promoter egg
The building process of white expression system.It is synthesized first by DNA, obtains the double chain DNA fragment containing T7SP sequence, be cloned into
Between EcoRI the and SpeI restriction enzyme site of pACYC-TetR carrier, pACYC-TetR-T7SP is obtained.Wherein TetR gene can table
Inhibit albumen (Tetracycline Repressor Protein, abbreviation TetR) up to tetracycline, inhibits tetracycline promoter
Expression.Then by PCR amplification GFPNb gene order, be cloned into the end of T7SP sequence 3 ' SpeI and HindIII restriction enzyme site it
Between, fusion is formed with T7SP.Finally by the tetracycline promoter (P in PCR amplification Escherichia coli TetB geneTetB) sequence
Column, are cloned between XbaI the and EcoRI restriction enzyme site of the upstream T7SP, obtain pACYC-TetR-PTetB- T7SP-GFPNb carrier
(Fig. 2).The carrier is transformed into the protein expression strain for carrying T7 rna polymerase gene, such as BL21 (DE3), Shuffle T7
In Express, Origami (DE3), TetR can be inhibited with tetracycline analogue ATc, activate PTetBPromoter, transcription, which generates, to be contained
There is the mRNA of overall length T7SP-GFPNb coded sequence, thus GFPNb albumen of the expression containing signal peptide, and it is transported to the week of bacterium
In matter space；And T7 RNA polymerase is synthesized with IPTG Induction of bacterial, and the T7 promoter in T7SP sequence can be started, it is generated
Although GFPNb coded sequence of the mRNA containing overall length, contains only 37-75 nucleotide sequences in T7SP, thus with
The AUG (corresponding to 70-72 nucleotide ATG in T7SP DNA sequence dna) of the end T7SP is used as translation initiation codon, table
Up to the GFPNb albumen for being free of signal peptide, it is distributed in cytoplasm.
3. result verification
1) T7SP is fitted into signal peptide sequence compared with the function of wild type RbsB signal peptide sequence
In order to verify the validity of the double-promoter expression system, inventor constructs pACYC-TetR-PTetB-T7SP-
GFPNb double-promoter protein expression vector, such as above-mentioned specific embodiment 2.
PACYC-TetR-P is replaced with the RbsB signal peptide sequence of wild typeTetBT7SP sequence in-T7SP-GFPNb, obtains
To pACYC-TetR-PTetB- SP (WT)-GFPNb carrier, as control.
Above two carrier is transformed into BL21 (DE3) bacterial strain respectively, picking single colonie, is inoculated into containing 34 μ g/ml chlorine
In the LB liquid medium of mycin, 37 DEG C of cultures.Work as OD600When reaching 0.6, protein expression induction is carried out with two kinds of distinct methods:
A. it uses 20ng/ml ATc inducing expression 12 hours, extracts periplasmic space albumen, then use nickel ion column purification GFPNb；B. it uses
1mM IPTG inducing expression 4 hours, then sonicated cells, extract cytoplasm protein, then use nickel ion column purification
GFPNb。
As a result as shown in Figures 3 and 4, when being induced with ATc, two kinds of carriers can express GFPNb, Er Qiebiao in periplasmic space
It is close up to level；And when being induced with IPTG, pACYC-TetR-PTetB- T7SP-GFPNb carrier can be in cytoplasmic expression
GFPNb, and pACYC-TetR-PTetB- SP (WT)-GFPNb carrier cannot.Therefore, T7SP DNA sequence dna not only has normally
Signal peptide encoding function, and can have T7 promoter function, it can be activated by T7 RNA polymerase, express downstream external source base
Cause.
2) compared with carrying out the result of GFPNb expression, purifying with common pET carrier with double-promoter expression system.
By GFPNb gene cloning to currently used periplasmic space expression vector pET22b and cytoplasmic expression carrier
In pET28b, pET22b-GFPNb and pET28b-GFPNb carrier is respectively obtained；By them and pACYC-TetR-PTetB-T7SP-
GFPNb carrier is transformed into respectively together in protein expression strain BL21 (DE3).
Picking pACYC-TetR-PTetB- T7SP-GFPNb clone, is inoculated into the LB culture medium containing 34 μ g/ml chloramphenicol
In；Picking pET22b-GFPNb, pET28b-GFPNb clone, are inoculated into the LB culture medium containing 50 μ g/ml kanamycins.It will
Each bacterial strain is placed in 37 DEG C of cultures, works as OD600It when reaching 0.6, is induced with two kinds of distinct methods: A. 20ng/ml ATc induction
Periplasmic space albumen is extracted in expression 12 hours；B. it uses 1mM IPTG inducing expression 4 hours, then sonicated cells, are extracted thin
Cytoplasmic protein.GFPNb expression is detected with SDS-PAGE separation and coomassie brilliant blue staining respectively.
As a result as it can be seen in figures 5 and 6, when being induced with ATc, contain pACYC-TetR-PTetBThe bacterium of-T7SP-GFPNb exists
Periplasmic space expresses GFPNb, and the bacterium containing pET22b-GFPNb and pET28b-GFPNb does not express GFPNb.When with IPTG
When induction, contain pACYC-TetR-PTetBThe bacterium of-T7SP-GFPNb can express GFPNb in cytoplasm, and contain
The bacterium of pET22b-GFPNb and pET28b-GFPNb is respectively in periplasmic space and cytoplasmic expression GFPNb.Therefore by using
Different inducers, pACYC-TetR-PTetB- T7SP-GFPNb can be respectively in bacteria periplasm space and cytoplasmic expression GFPNb, function
Substitution pET22b-GFPNb and pET28b-GFPNb expression vector can be gone up.
Although using tetracycline promoter and GFP nano antibody gene as example in above embodiments, guarantor of the invention
Shield range should not be so limited to this.Skilled person will appreciate that road, T7SP coded sequence and any other Escherichia coli are induced
Other gene associations other than type promoter and GFP nano antibody gene use, and can construct the double function to form gene used
It can expression system.Therefore all within the spirits and principles of the present invention, any modification, equivalent replacement, improvement and so on should all
It is included within protection scope of the present invention.
Sequence table
<110>Chengdu molecule pulse condition Biotechnology Co., Ltd
<120>a kind of based on the dual-function dna element of RbsB signal peptide and its application
<130> 1
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 75
<212> DNA
<213>artificial sequence
<400> 1
atgaacatga aaaaattaat acgactcact ataggagttg cgctaagcgc caccgtcagt 60
gcgaatgcga tggct 75
<210> 2
<211> 113
<212> DNA
<213>artificial sequence
<400> 2
atgattaatt cctaattttt gttgacactc tatcattgat agagttattt taccactccc 60
tatcagtgat agagaaaagt gaaatgaata gttcgacaag aaggagatat acc 113
<210> 3
<211> 390
<212> DNA
<213>artificial sequence
<400> 3
gcgcaggttc aactggtcga atcgggtggt gccctggttc agccgggtgg ctctctgcgt 60
ctgagctgcg cggcgagctc gagcgatttt tcgcgtaatg ccattggttg gtttcgccag 120
gccccgggta aagaacgcga aggtgtgtcg tgtattaact ggaccggttc tggctacgcg 180
tatgcagact ccgtgaaggg tcgctttacc atttcgcgtg atgacgcacg caacacggtg 240
tacctgcaac tgaatagtct gaaaccggaa gataccgcgg tgtactattg ccatggctcg 300
accgtttttg cggaaattac gggtctggca ggttatcaat cgggtagcta tgagatctgg 360
ggccagggca cccaggtcac ggtgtcgtcc 390
Claims (9)
1. a kind of dual-function dna element based on RbsB signal peptide, which is characterized in that sequence is as shown in SEQ ID NO:1.
2. application of the dual-function dna element described in claim 1 in promotor gene transcription.
3. application of the dual-function dna element described in claim 1 in protein of the coding with E. coli signal peptides.
4. a kind of double promoter DNA element, which is characterized in that including dual-function dna element described in claim 1 and connection
In the escherichia coli promoter of the dual-function dna components upstream.
5. double promoter DNA element according to claim 4, which is characterized in that the escherichia coli promoter is that can lure
Lead promoter.
6. double promoter DNA element according to claim 5, which is characterized in that the inducible promoters are non-IPTG
Inducible promoters.
7. double promoter DNA element according to claim 6, which is characterized in that the non-IPTG inducible promoters are
Tetracycline inducible promoters, the sequence of the tetracycline inducible promoters is as shown in SEQ ID NO:2.
8. a kind of protein expression system, which is characterized in that including the member of double promoter DNA described in any one of claim 4-7
Part and the albumen coded sequence for being connected to the double promoter DNA member downstream.
9. protein expression system according to claim 8, which is characterized in that the protein of the albumen coded sequence coding
Not know the protein for whether needing to be folded in periplasmic space.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908175A (en) * | 2006-08-22 | 2007-02-07 | 中山大学 | Prokaryotic secretion expression carrier and application thereof |
| CN103797122A (en) * | 2011-04-08 | 2014-05-14 | 安瑟生物科技私人有限公司 | Novel expression and secretion vector systems for heterologous protein production in escherichia coli |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1908175A (en) * | 2006-08-22 | 2007-02-07 | 中山大学 | Prokaryotic secretion expression carrier and application thereof |
| CN103797122A (en) * | 2011-04-08 | 2014-05-14 | 安瑟生物科技私人有限公司 | Novel expression and secretion vector systems for heterologous protein production in escherichia coli |
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