CN1189565C - Efficient prokaryotic expression carrier - Google Patents
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Abstract
The present invention utilizes a molecular companion thioredoxin (TRX) of colibacillus as a combining companion body to structure an efficient karyogamy expression carrier which comprises a T7 strong promoter, a TRX companion body protein, connecting regions (comprising a flexibility region, 6 groups of amino acid purification sites, an enterokinase cutting site and a polycloning site). The carrier can efficiently express exogenous genes in a fused protein form. The expression product has good solubility and easy purification. Furthermore, most of the product has the natural bioactivity of exogenous protein. Thereby, the expression carrier not only is suitable for mass production of recombination protein, but also is suitable for researching the relation between gene function and conformation.
Description
The invention belongs to the prokaryotic expression system in the genetically engineered.
Engineered expression system has prokaryotic expression system and eukaryotic expression system two big classes.The expression of foreign gene in prokaryotic cell prokaryocyte is that foreign gene mode with fermentation in prokaryotic cell prokaryocyte of order clone is synthesized the exogenous gene expression product fast and efficiently.Be that understanding is the most deep at present, practical application is expression system the most widely.
In prokaryotic cell prokaryocyte during expression alien gene, because the difference of experimental design generally speaking can produce pattern of fusion and non-pattern of fusion recombinant protein.When with non-amalgamation mode (aminoacid sequence that is the gained recombinant protein is consistent with the purpose product) expression alien gene, by the bacterioprotein enzyme liberating, expression product exists with insoluble inclusion body form usually for fear of expression product.The formation of inclusion body is very beneficial for purifying, but because of expression product does not wherein have biologic activity, thereby need become the renaturation processing.Proteic renaturation is an extremely complicated process, and different proteic renaturation conditions are different, and renaturation yield often is difficult to improve.This is the main restricting factor that limits its application.Adopt the secreting, expressing mode can partly overcome this problem, but expression amount is on the low side, practical application also has big difficulty.In addition, when expressing the albumen that number molecular weight is less, disulfide linkage is more, the non-fusion expression system is difficult to obtain the albumen of biologic activity.Another unfavorable factor of non-fusion expression is exactly that expression amount is widely different.During with the different foreign protein of identical expression vector expression, expression amount has the difference of 10 times even 100 times.A large amount of studies show that translation initiation sequence (TIR) secondary structure or higher structure are one of key factor that influences genetic expression (Thanaraj TA, PanditMW.Nucleic Acids Res, 1989; 17 (8): 2973).Some foreign genes have only just can reach by the secondary structure of optimizing TIR and efficiently express (Calvez HL et al.Gene, 1996; 170:51).This has strengthened the difficulty and the workload of experiment undoubtedly.In order to overcome these difficulties, the expressing fusion protein system arises at the historic moment.Promptly utilize some specific fusion companion bodys to make it to merge expressed fusion protein in prokaryotic cell prokaryocyte with foreign protein.These merge the companion body and comprise GST, lacZ, and trpE, DsBA etc. (Smith etal, Gene, 1988,67:31-40).The amalgamation and expression system has all adopted the TIR that optimizes, efficient stable aspect expression amount.Because the fusion companion body that adopts mostly is the molecular chaperones from bacterium, both can protect foreign protein to avoid the degraded of bacteria protease, can assist the correct folding of foreign protein again.Therefore, the solvability of the fusion rotein of expression is good, and the conformation of foreign protein more approaches native protein, for guaranteeing that its biologic activity has very important effect, is very suitable for the functional study of gene and the mass production of recombinant protein.Certainly, as seek out the foreign protein monomer, just must will merge companion body excision.The companion body is merged in excision can adopt enzymolysis and chemical cracking method.The alternative enzyme of enzymolysis has zymoplasm, protease 3 C, trypsinase, Xa factor etc.; Chemical cracking often adopts bromize fluoride crack.
The amalgamation and expression system advantage is obvious, but the place that demands perfection urgently is also arranged.As aspect the purifying, need purifying measure at the simple and efficient of fusion rotein; Aspect the selection of merging the companion body, need the molecular chaperones that to assist target protein folding better; Merge in cutting on the method for the companion body, need more efficient, cutting mild condition, cutting reagent cheaply.
The objective of the invention is design construction and go out a kind of new and effective fusion expression vector, be used for efficiently expressing exogenous gene.Make expression fusion rotein can with easy method purifying it.
The present invention adopts colibacillary Trx, and (Thioredoxin is TRX) as merging the companion body.The TRX gene is the method by PCR, from e. coli k12 the amplification and get.TRX is colibacillary a kind of molecular chaperones, can assist proteic correct folding in cell.Intestinal bacteria TRX molecular weight is about 12kD, and its conformation is tight, Heat stability is good, even can tolerate 80 ℃ high temperature.When TRX in born of the same parents during by overexpression, its content can account for total bacterial protein 40% and not by the bacterioprotein enzyme liberating.The people such as Mc Coy of U.S. Genetics Insitute company in 1993 find can realize solubility expression after the various kinds of cell factor such as human interleukin-13, human interleukin 11, mouse interleukin 4, mouse interleukin-15, murine leukemia supressor etc. and the intestinal bacteria TRX gene fusion in Bacillus coli cells, expressed fusion protein accounts for 10～20% (La Vallie et a, 1993) of cell soluble protein.Further studies show that, when TRX and above-mentioned cytokine with form coexpression in intestinal bacteria independently, cytokine is an insolubility, this shows that TRX assists target protein folding with the form of intramolecular chaperone.These characteristics of TRX are very suitable for the fusion companion body as foreign protein.Described promotor can be the T7 promotor, and it is a structural element of carrier, also available other strong promoter such as Tac, replacements such as Lac.
The present invention has designed a joining region at the carboxyl terminal that TRX merges the companion body.The joining region comprises flexible zone, purifying site, restriction enzyme site and multiple clone site.The purpose that flexible zone is set is in order to provide a transition between the fusion companion body and foreign protein, to reduce the two phase mutual interference on space structure and some rigid structures that may occur.By computer simulation, finally selected the GSGSG sequence as the joining region.Selecting this joining region to be on the one hand because the snappiness of GSGSG is good, is because G, S (glycine, Serine) molecular weight is little, sterically hindered little on the other hand, helps the cutting of enteropeptidase to fusion rotein.
The present invention has designed the purifying site of 6 Histidines in the joining region.Histidine can provide coordination electronics and some metal ions such as Ni
2+The chelating site.6 successive Histidines can make protein adsorption in containing metal Ni
2+Chromatographic stuffing on, thereby can utilize metal chelating and chromatography to come purified fusion protein, thereby simplify protein purification work.
The present invention has also designed the cleavage site of enteropeptidase after 6 Histidine sites.The problem that the expressing fusion protein system must be faced is how effectively to excise companion body albumen and obtain the target protein monomer.Enteropeptidase is a kind of serine protease, N-Asp-Asp-Asp-Asp-Lys-C five amino acid residue in its energy specific recognition peptide chain, and cut point is at the carboxyl terminal of Lys.With respect to other nickase, enteropeptidase has advantages such as identification specificity is stronger, cutting efficiency is higher, reaction conditions gentleness.Because the cleavage site of enteropeptidase is at the C-terminal of recognition site, thereby the foreign protein that cuts down do not have extra amino acid at aminoterminal, thereby makes its aminoacid sequence and native protein in full accord.U.S. FDA is exactly to utilize the enteropeptidase cleavage of fusion proteins and the monomer that obtains at the human interleukin 11 of approval listing in 1997.
In sum, the fusion rotein carrier pTRX of the present invention's structure can be applicable to the scale operation of recombinant protein.For example, the pTRX-IL10 expression vector that this laboratory applications pTRX makes up, the fusion rotein of expression is solvable more than 95%, accounts for 40% of the total soluble protein of bacterium, and the prokaryotic expression of IL-10 does not all reach this level both at home and abroad.
Fusion rotein carrier pTRX of the present invention also can be used for the research of gene function, for example, pTRX-PLA2 (sea snake Phospholipase A2) expression vector that this laboratory applications pTRX makes up and pTRX-NEU (the short neurotoxin of sea snake) expression vector, the fusion rotein TRX/PLA2 that expresses promptly has the biologic activity of sea snake Phospholipase A2, and fusion rotein TRX/NEU promptly has the biologic activity of sea snake neurotoxin.And report that both at home and abroad the sea snake Phospholipase A2 of prokaryotic expression and the most abiology activity of sea snake neurotoxin or activity are very low.Trace it to its cause, key is that can expressed recombinant protein correctly fold.
The clone method of carrier of the present invention:, press CaCl with reference to Sambrook (Sambrook, etal.1989.Molecular cloning.Cold spring Harbor Laboratory Press.USA) method
2Method transforms plasmid in E.coli DH5 α or E.coli JM101 bacterial strain, with the bacterium of the LB culture medium culturing that contains penbritin (50ug/mL) through transforming, alkaline process extracts plasmid.
Brief Description Of Drawings:
Fig. 1 is the sequence of carrier of the present invention from promotor to multiple clone site.
Fig. 2 is the pcr amplification product synoptic diagram of TRX gene, wherein 1,3rd, and be the PCR product of template gained with the e. coli k-12 genome, the 2nd, the 100bp Marker of Gibico company, the 4th, negative control.
Fig. 3 is that transfer vector pBSK-TRX makes up synoptic diagram.
Fig. 4 is the structure synoptic diagram of transfer vector pBSK-LINKER.
Fig. 5 is the structure synoptic diagram of fusion expression vector pTRX.
Fig. 6 is the physical map of maternal carrier pET22b.
Fig. 7 is that the enzyme of fusion expression vector pTRX is cut the evaluation synoptic diagram, and wherein 1 is the 100bp Marker of NEB company, and 4 is the 1kb Marker of Gibico company, and 2 is pTRX/NdeI+NotI, and 3 is pET22b/NdeI+NotI.
Fig. 8 utilizes pTRX to express the SDS-PAGE synoptic diagram of TRX/IL10 fusion rotein and purifying, and wherein 1 for containing empty carrier pET22b host's soluble protein contrast, and 2 for containing the soluble proteins behind pTRX-IL10 host's abduction delivering, and 3,4,5 for using Ni
2+The foreign protein of Chelating Sepharose affinity column wash-out, 6 for using Ni
2+The TRX/IL10 fusion rotein of Chelating Sepharose affinity column wash-out,
7 is albumen Marker.
Fig. 9 utilizes pTRX to express the SDS-PAGE synoptic diagram of TRX/PLA2 fusion rotein and purifying, and wherein 1 be albumen Marker, and 2 for containing empty carrier pET22b host's soluble protein contrast, and 3 is the soluble proteins that contains behind pTRX-PLA2 host's abduction delivering, and 4 for using Ni
2+The TRX/PLA2 fusion rotein of Chelating Sepharose affinity column wash-out, 5 is the TRX/PLA2 fusion rotein that is further purified, 6 is with enteropeptidase cutting TRX/PLA2 fusion rotein.
Figure 10 utilizes pTRX to express the SDS-PAGE synoptic diagram of TRX/NEU fusion rotein and purifying, wherein 1 for containing empty carrier pET22b host's soluble protein contrast, 2 for containing the total protein behind pTRX-NEU host's abduction delivering, and 3 for for containing soluble protein, 4 behind pTRX-NEU host's abduction delivering for using Ni
2+The TRX/NEU fusion rotein of Chelating Sepharose affinity column wash-out, 5 is the TRX/PLA2 fusion rotein that is further purified, 7 is albumen Marker.
The structure of embodiment one fusion expression vector pTRX
1.TRX the pcr amplification of gene
Colibacillary TRX gene announces in GeneBank, so we utilize PCR method, is template amplification TRX gene with the e. coli k12 genome.According to the analysis of computer software, having designed and synthesized a pair of primer to the TRX gene: upstream primer is 5 '-AAAgaattcatatgAGCGATAAAATTATTCACCTGAC-3 ', downstream primer is 5 '-AAAaagcttGGCCAGGTTAGCGTCGAGGA-3 '.For convenient clone, in the upstream and downstream primer, add EcoRI, HindIII restriction endonuclease recognition sequence respectively.The PCR reaction is carried out with reference to Sambrook (Sambrook, et al.1989.Molecular cloning.Cold spring Harbor LaboratoryPress.USA) method.
2. the structure that contains the transfer vector pBSK-TRX of TRX gene
The TRX gene of pcr amplification detect to reclaim through agarose gel electrophoresis, again through EcoRI and EcoRI and HindIII enzyme that HindIII is two to be inserted into metastasis transplanting physique grain pBSK after cutting cut window, be built into transfer vector pBSK-TRX, Fig. 2 between the plasmid construction process is detailed.
3. the structure that contains the transfer vector pBSK-LINKER of joining region
At first, according to synthetic two oligomerization nucleic acids of the complementary fragment of the sequence in Linker district:
LA：5′-AAAAGCTTGGTTCTGGTTCTGGTCATCAT?CA?TCATCATCATGGTACCGACGACGACGA
CAAAGAA-3′
LB：5′-AAAGCGGCCGCAAGCTTGGATCCAGAGATATCCTCGAGACCGAATTCTTTGTCGTCGT
CGTCGGTA-3′
Oligomerization nucleic acid fragment LA, LB through sex change, annealing, extend double-stranded Linker sequence, cut the NotI and the HindIII enzyme that are inserted into metastasis transplanting physique grain pBSK and cut window so that HindIII and NofI are two again, be built into transfer vector pBSK-LINKER, Fig. 4 between the plasmid construction process is detailed.
4. the structure of fusion expression vector pTRX
Downcut the TRX sequence with NdeI and HindIII from transfer vector pBSK-TRX, downcut the Linker sequence with HindIII and NotI from transfer vector pBSK-LINKER, the two is inserted into NdeI and the NotI enzyme of pET22b simultaneously and cuts window, be built into fusion expression vector pTRX, Fig. 5 between the plasmid construction process is detailed.
The application of embodiment two fusion expression vector pTRX
The KpnI and the NotI enzyme that people IL-10, the short neurotoxin (NEU) of sea snake, sea snake Phospholipase A2 (PLA2) gene are inserted into the pTRX carrier are respectively cut window, are built into expression vector pTRX-IL10, pTRX-NEU, pTRX-PLA2.PTRX-IL10, pTRX-NEU, pTRX-PLA2 are transformed into e. coli bl21 (DE3) respectively.The order bacterium colony is in 50ml ammonia Bian resistance LB substratum, 37 ℃, the 250rpm overnight incubation is got in the ammonia Bian resistance LB substratum that overnight culture 20ml is inoculated in 2L, 37 ℃, 250rpm is cultured to OD600=0.6, adds 100mM IPTG and 20% glucose to final concentration and is respectively 1mM and 0.2%, 26 ℃, centrifugal results thalline behind the 250rpm inducing culture 10h, the ultrasonication thalline, centrifugal collection supernatant is used Ni
2+Chelating Sepharose affinitive layer purification.PTRX-IL10 Expression of Fusion Protein purifying sees that Fig. 8, pTRX-PLA2 Expression of Fusion Protein purifying see that Fig. 9, pTRX-NEU Expression of Fusion Protein purifying see Figure 10.
Utilize the gel thin-layer scanner that the glued fruit of SDS-PAGE is analyzed, the expression amount of pTRX-IL10 accounts for more than 40% of total bacterial protein, the expression amount of pTRX-NEU account for total bacterial protein 25%, the expression amount of pTRX-PLA2 accounts for more than 20% of total bacterial protein, these fusion roteins are solvable more than 90%.
Claims (5)
1. fusion expression vector, it contains a kind of specific nucleotide fragment, this nucleotide fragment is made of gene, the joining region series connection of promotor, the colibacillary Trx of coding, the flexible zone of encoding serine and/or glycine is contained in wherein said joining region, 6 Histidine sites, enteropeptidase cleavage site and multiple clone site.
2. according to the described expression vector of claim 1, wherein, described promotor is the T7 promotor.
3. according to the described expression vector of claim 1, wherein, described flexible zone is the sequence of coding GSGSG amino-acid residue.
4. according to the described expression vector of claim 1, wherein, the sequence of described specific nucleotide fragment is:
CATATGAGCGATAAAATTATTCACCTGACTGACGACAGTTTTGACACGGATGTACTCAAAGCGGACGGGGCG
ATCCTCGTCGATTTCTGGGCAGAGTGGTGCGGTCCGTGCAAAATGATCGCCCCGATTCTGGATGAAATCGCT
GACGAATATCAGGGCAAACTGACCGTTGCAAAACTGAACATCGATCAAAACCCTGGCACTGCGCCGAAATAT
GGCATCCGTGGTATCCCGACTCTGCTGCTGTTCAAAAACGGTGAAGTGGCGTCGGCAACCAAAGTGGGTGCA
CTGTCTAAAGGTCAGTTGAAAGAGTTCCTCGACGCTAACCTGGCCAAGCTTGGTTCTGGTTCTGGTCATCAT
CATCATCATCATGGTACCGACGACGACGACAAAGAATTCGGTCTCGAGGATATCTCTGGATCCAAGCTTGCGGC
CGC
5. according to the described expression vector of claim 1, it also contains peace penicillin G resistant gene, the reproduction element of plasmid in intestinal bacteria.
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| CNB001248324A CN1189565C (en) | 2000-09-18 | 2000-09-18 | Efficient prokaryotic expression carrier |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007112677A1 (en) * | 2006-03-31 | 2007-10-11 | Shenzhen Watsin Genetech Ltd. | Method of preparing human parathyroid hormone 1-34 |
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| CN1313489C (en) * | 2004-12-29 | 2007-05-02 | 中山大学 | GP thymosin alpha 1 and preparation method |
| CN100484958C (en) * | 2006-03-31 | 2009-05-06 | 东莞太力生物工程有限公司 | Fusion protein containing haman parathyroxin 1-34 and its expression vector |
| CN100432231C (en) * | 2006-08-22 | 2008-11-12 | 中山大学 | Prokaryotic secretion expression carrier and application thereof |
| CN102533835A (en) * | 2010-08-31 | 2012-07-04 | 上海交通大学 | Plasmid for heterologous protein solubility expression and preparation and application method thereof |
| CN102477098A (en) * | 2010-11-29 | 2012-05-30 | 清华大学深圳研究生院 | Fusion protein and application thereof in preparation of human leukemia inhibitory factor |
| CN102154301B (en) * | 2010-12-31 | 2013-01-16 | 中山大学 | Preparation and application of conotoxin striatus S21a in South China Sea |
| CN105087725B (en) * | 2015-08-21 | 2019-04-02 | 江苏大学 | A method of improving 2 β expression of the soluble former filamentous actin of recombination in Escherichia coli |
| CN105296517B (en) * | 2015-11-05 | 2018-12-28 | 盘古基因生物工程(南京)股份有限公司 | A kind of fusion protein expression vector of companion's sample albumen |
| CN105483150B (en) * | 2015-12-22 | 2018-11-23 | 盘古基因生物工程(南京)股份有限公司 | A kind of human metallothionein -2a fusion protein expression vector |
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| CN105441472B (en) * | 2015-12-22 | 2018-11-23 | 盘古基因生物工程(南京)股份有限公司 | A kind of -4 fusion protein expression vector of human metallothionein |
| CN105543262B (en) * | 2015-12-22 | 2018-11-23 | 盘古基因生物工程(南京)股份有限公司 | A kind of -3 fusion protein expression vector of human metallothionein |
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2000
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2007112677A1 (en) * | 2006-03-31 | 2007-10-11 | Shenzhen Watsin Genetech Ltd. | Method of preparing human parathyroid hormone 1-34 |
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| CN1343789A (en) | 2002-04-10 |
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