阅读说明：本技术 Pab蛋白在构建具有类伴侣样蛋白作用的融合蛋白表达载体中的应用 (Application of the PAB albumen in the fusion protein expression vector that building has the albumen effect of class companion sample ) 是由 李乾 于 2019-07-29 设计创作，主要内容包括：本发明公开一种PAB蛋白在构建具有类伴侣样蛋白作用的融合蛋白表达载体中的应用,利用PAB蛋白成功构建获得具有类伴侣样蛋白作用的融合蛋白表达载体。本发明通过构建含类伴侣样蛋白的pET-PAB1表达载体、测试目标蛋白融合蛋白表达载体以及测试含目标蛋白的pET对照载体,经过不同重组体中目标蛋白诱导表达的对比测试,发现PA的B结构域具有提高目标蛋白可溶性表达效率的伴侣样蛋白新功能,从而为蛋白质表达科学研究和工业化生产提供了一项有用的工具。(The present invention discloses a kind of application of PAB albumen in the fusion protein expression vector that building has the albumen effect of class companion sample, and fusion protein expression vector of the acquisition with the albumen effect of class companion sample successfully construct using PAB albumen.The present invention passes through the pET-PAB1 expression vector of the building sample of companion containing class albumen, the pET control vector of test target fusion protein expression vector and test containing target protein, by the contrast test of target protein inducing expression in different recombinants, it was found that the B structure domain of PA has the companion's sample albumen new function for improving target protein solubility expression efficiency, to provide a useful tool for protein expression scientific research and industrialized production.)

1. the B structure domain segment or collating sequence of the albumin A as shown in any in NO:1~5 SEQ ID have class companion in building Application in the fusion protein expression vector of companion's sample albumen effect.

2. application as described in claim 1, which is characterized in that will such as SEQ ID NO:6, it is optimized shown in 7 after PAB1 Albumen coded sequence is inserted into class companion's sample albumen in fusion protein expression vector as expression cassette upstream.

3. a kind of fusion protein expression vector, which is characterized in that the clone area upstream of the fusion protein expression vector includes to compile Code it is as claimed in claim 2 it is optimized after PAB1 albumen nucleic acid sequence, the DNA sequence dna of the fusion protein expression vector As shown in SEQ ID NO:26.

4. fusion protein expression vector as claimed in claim 3, which is characterized in that select the commercialization empty carrier for transformation For original parent carrier, the original parent carrier includes pET system expression carrier, Yeast system expression vector, insect cell Any one in system expression carrier and mammalian cell system expression vector.

5. fusion protein expression vector as claimed in claim 3, which is characterized in that it is described it is optimized after PAB1 albumen sequence Upstream addition is arranged just like transcription initiation aptamer sequence shown in SEQ ID NO:8,9；And/or

It is described it is optimized after the Sequences upstream of PAB1 albumen be added with commercial separation tags albumen；And/or

It is described it is optimized after PAB1 albumen sequence downstream include one section of flexible joint area, protease cleavage site cog region and For being inserted into the polyclonal area of downstream targets albumen.

6. fusion protein expression vector as claimed in claim 5, which is characterized in that it is described commercialization separation tags albumen include Any one in 5 '-HisTag and 5 '-Strept II-Tag；And/or

The protease cleavage site cog region includes Factor Xa protease restriction enzyme site cog region, fibrin ferment restriction enzyme site In cog region, enterokinase cleavage site cog region, tobacco etch virus protease restriction enzyme site cog region and hydroxylamine cleavage site Any one.

7. fusion protein expression vector as claimed in claim 6, which is characterized in that the sequence such as SEQ in the flexible joint area ID NO:10, shown in 11, the sequence of the protease cleavage site cog region such as SEQ ID NO:12 is described to be used for shown in 13 The sequence in the polyclonal area of downstream targets albumen is inserted into as shown in SEQ ID NO:14,15.

8. fusion protein expression vector as claimed in claim 3, which is characterized in that it is described it is optimized after PAB1 albumen it is same Source property and the homology of natural PAB1 albumen are not less than 85%.

9. the fusion protein expression vector as described in claim 3 to 8 any one is being expressed as new parental expression vector Application in target protein.

10. a kind of method of fusion protein of the expression comprising class companion sample albumen, which comprises the following steps:

Desired protein coding sequences are inserted into PAB1 in the fusion protein expression vector as described in claim 6 to 8 any one The polyclonal area in downstream obtains the fusion protein recombinant expression carrier containing desired protein coding sequences；

By the fusion protein recombinant expression carrier transfection host cell, host cell expression fusion protein is cultivated.

Technical field

The present invention relates to genetic engineering and protein engineering field, in particular to protein amalgamation and expression technology is led A kind of domain, and in particular to the application of PAB albumen in the fusion protein expression vector that building has the albumen effect of class companion sample.

Background technique

Protein folding is listed in the important topic of " biophysics of 21 century ", it is molecular biology center The still unsolved critical biological problem of rule.And in protein expression (production) engineering, natural structure albumen is obtained, is The prerequisite of protein solubility expression, and guarantee the basis of target protein physiological function, even more protein industry metaplasia Produce the link of procedure section cost-saving.

The function of native protein depends on the physiology conformation of protein.Molecular biochemistry science thinks that protein divides The three-dimensional structure of son depends entirely on the amino acid sequence of protein molecule.But numerous studies data in the past 30 years shows very The folding of more organism protein and the participation for being equipped with other albumen or enzyme, especially the constitutive protein of higher organism is natural The formation of structure, wherein protein molecule companion is exactly most important, is also a most studied albuminoid.Molecular chaperones are Very conservative protein in one kind evolution, the polypeptide chain for the albumen that can be different from structure, size, positioning and final function Non-specific binding is catalyzed the formation of the specific conformation of mediating protein, participates in the folding, assembly and transhipment of vivo protein.Newly It is just active that the polypeptide chain of synthesis must first form specific three-dimensional structure after folding and assembly.In polypeptide chain folding process In, it often generates and folds paraprotein, or because unfolded or do not fold entirely, hydrophobic region in protein molecule is caused mutually to be inhaled Draw, form congeries, expresses field, referred to as inclusion body (Inclusion) in engineered protein.Inclusion body is insoluble , do not have functional " dead albumen ".In the presence of with the protein molecule of companion's sample effect, it can effectively regulate and control other polypeptides The correct folding of chain, to avoid the formation of inclusion body.

The concept of protein molecule companion is to be proposed first by Dr.Laskey et al. in 1978.They are non-in research When the formation of continent Xenopus laevis nucleosome, it was found that a kind of acid nuclear protein (Nucleoplasmin).Experiment shows it in DNA and group egg It is white be assembled into nucleosome during be required.Under physiological ionic strength, DNA and histone are mixed in vitro, It is unable to self assembly, forms precipitating.But if histone is mixed with excess Nucleoplasmin albumen, DNA is added, then Nucleosomal structure can be formed, and does not include Nucleoplasmin molecule in finally formed nucleosome.It is presently believed that The effect of Nucleoplasmin may be to avoid strong electrostatic attraction between electronegative DNA and positively charged histone and formed The insoluble polymer of non-specific binding.

1987, the folding of Dr.Ikemura discovery subtilin (Subtilisin) needed propetide (Propeptide) Help.This kind of propetide is frequently located between signal peptide and mature polypeptide, the protein mediated in protein building-up process with it Polypeptide chain is one in front and one in back synthesized, and is connected with covalent bond, is necessary to mature polypeptide correctly folds, maturation is more Peptide is completed to be detached from by hydrolysis and propetide after folding.This kind of propetide is known as intramolecular chaperone by Shinde and Inouye (Intramolecular Chaperones)。

1993, E11is did more exact definition to molecular chaperones: i.e. molecular chaperones are a kind of related between each other The albumen of system, can combine and stablize the unstable conformation of another protein, their function is to aid in other containing polypeptide The substance of structure carries out correctly non-covalent assembling, controlled combination and release in vivo, promote nascent polypeptide folding, The assembly or degradation of polymer and the transdermal delivery of organelle albumen etc., and not the protein being completed is playing it just Component part when normal biological function.

The classification of protein molecule companion: the albumen (also referred to as auxilin) and albumen for helping nascent peptide to fold are currently known The protein molecule companion at least three categories of body assembling:

The first kind, the molecular chaperones of universal significance help correct folding, prevent and correct incorrect folding.

Second class, the molecular chaperones with enzyme activity, also known as folding enzymes.So far there are 2 folding enzymes: first is that two sulphur of protein Key isomerase (Proteindisulfideisomerase, PDI).Second is that peptidyl prolyl cis-trans isomerism (Peptidylprodylcis rans-isomerase, PPI).

Third class, Intramolecular chaperone are some studies have shown that many precursor forms synthesis containing leader peptide (Pro peptide) Protein folding must have the presence of leader peptide with maturation and could complete, not fully abide by Anfinsen rule.It is this kind of Leader peptide is known as Intramolecular chaperone (Intramolecular Chaperone, IMC).

As described above, protein partner is one group of protein being widely present from bacterium to people, noncovalently with nascent peptide The protein peptide chain combination of chain and unfolding, and help they fold and transhipment, be usually not involved in target protein physiological function and Subunit is constituted.

In short, there are two common features by the protein molecule companion of the above sort research: 1) natural sex, oneself exists；2) Homology, i.e., companion and by chaperone in same organism interior coding and expression.

However, people seem to have ignored another kind of chaperone with the research of genetic engineering and protein expression group Presence, this kind of chaperone or companion's sample albumen, into expression vector, form protein expression tool by artificial recombination. Tentatively for this kind of still unclassified companion's sample albumen, herein it is known as " albuminoid companion " or " chaperone sample albumen ", it is this kind of The characteristics of spatial position of chaperone is similar " intramolecular chaperone ", uses in the carrier is as follows:

1) artificial recombination is in expression vector, some have been commercialized nearly 40 years of sale, such as Pharmacia company PGEX serial carrier and it includes GST albumen, the pMAL serial carrier of NEB company and its use maltose-binding protein (Maltose Binding Protein, MBP).It was verified that BST and MBP have increases fusion protein solubility in various degree The effect of expression and expression quantity, and the original intention for researching and developing producer is " label " albumen being used as convenient for isolating and purifying.

2) amino acid sequence of natural or non-native protein sequence, i.e., not mutated modification or mutation modification.Stricti jurise For upper, belong to Non natural proteins sequence because or take its partial sequence, such as the C of HSP the segment GroE, GST of bacterium Hold the proteolytic cleavage recognition site of addition and polyclonal area (MCS) sequence of all fusion protein expression vectors manually added Column or " flexible joint " area (Flexible Linker), these are equivalent to end insertion mutation.

3) homologous or heterologous, in class companion's sample protein expression vector system, homology and heterologous concept include two A aspect, first is that homology and heterologous of the class companion's sample albumen of expression vector carrying to expressive host, as Escherichia coli come The GST and MBP in source are the albumen of homology for expressive host Escherichia coli；And the yeast on coli expression carrier Or the SUMO (small ubiquitin sample modifies albumen) or FABP6 (mankind's free-fat acid binding protein -6) class companion's sample egg of human origin It is white for expressive host Escherichia coli, be exactly the albumen of heterologous.In fusion protein expression vector, fusion protein itself Two albumen of upstream and downstream are all heterologous in most cases, and most of these carriers are used to express mankind's egg after all It is white, because a big chunk eukaryocyte albumen is expressed as inclusion body in prokaryotic cell, need by upstream class companion's sample egg White auxiliary increases amount of soluble expression and auxiliary divides autofolding.

4) amalgamation and expression or non-fusion expression.In natural situation there are molecule protein interior constitute companion, at It has cut off or has retained when ripe peptide.Autofolding high dissolubility is presented in self or heterologous express in some protein, as DNA is poly- Synthase, FABP.There is extremely strong runback sexuality, such as RNase A, Taq enzyme etc., these albumen after some protein denaturation in vitro There may be constitute row chaperone for intramolecule.Artificial constructed class companion's sample fusion protein expression vector is similar to Intramolecular chaperone can induce downstream albumen and improve correct folding efficiency.Some commercialization expression vectors use double expression frame (Two Operons), one of expression cassette expresses certain nonspecific proteins companion, another expression cassette expresses target protein (such as The chaperone series commercial vectors of the precious biology in the Dalian Takara-).

5) height autofolding and nontoxic to host.Height autofolding formed high dissolubility protein expression, to host without Poison makes the expression of middle and high or superelevation may.

Since not only there is this kind of class companion sample protein expression vector important scientific research value (to facilitate different albumen tests can Dissolubility expression and functional study), while there is important industrial value (producing for Rumen protein fermentation).But existing class companion Companion's sample protein expression vector is very limited, and has that certain, mechanism is unknown to the folding assisted effect of different target albumen Difference, it is therefore necessary to further increase the washability of class companion's sample protein expression vector.

Summary of the invention

The main object of the present invention is to propose that a kind of PAB albumen has the fusion protein of class companion sample albumen effect in building A kind of application in expression vector, it is desirable to provide fusion protein expression vector with class chaperone effect.

To achieve the above object, the present invention proposes the B structure domain piece of the albumin A as shown in any in NO:1~5 SEQ ID The application of section or collating sequence in the fusion protein expression vector that building has the albumen effect of class companion sample.

Optionally, will such as SEQ ID NO:6, it is optimized shown in 7 after the PAB1 albumen coded sequence be inserted into fusion Class companion's sample albumen in protein expression vector as expression cassette upstream.

The present invention also proposes a kind of fusion protein expression vector, and the clone area upstream of the fusion protein expression vector includes Coding it is as described above it is optimized after PAB1 albumen nucleic acid sequence, the DNA sequence dna of the fusion protein expression vector such as SEQ Shown in ID NO:26.

Optionally, selecting the commercialization empty carrier for transformation is original parent carrier, and the original parent carrier includes PET system expression carrier, Yeast system expression vector, insect cell system expression vector and mammalian cell system expression carry Any one in body.

Optionally, the Sequences upstream of the PAB1 albumen is added just like SEQ ID NO:8, the adaptation of transcription initiation shown in 9 Sequence；And/or

It is described it is optimized after the Sequences upstream of PAB1 albumen be added with commercial separation tags albumen；And/or

It is described it is optimized after PAB1 albumen sequence downstream include one section of flexible joint area, protease cleavage site identify Area and polyclonal area for being inserted into downstream targets albumen.

Optionally, the commercial separation tags albumen includes any one in 5 '-HisTag and 5 '-Strept II-Tag Kind；And/or

The protease cleavage site cog region includes Factor Xa protease restriction enzyme site cog region, fibrin ferment digestion Site cog region, enterokinase cleavage site cog region, tobacco etch virus protease restriction enzyme site cog region and hydroxylamine cleavage position Point in any one.

Optionally, the sequence in the flexible joint area such as SEQ ID NO:10, shown in 11, the protease cleavage site is known The sequence in other area such as SEQ ID NO:12, it is described for being inserted into the sequence such as SEQ in the polyclonal area of downstream targets albumen shown in 13 Shown in ID NO:14,15.

Optionally, it is described it is optimized after the homology of PAB1 albumen and the homology of natural PAB1 albumen be not less than 85%.

The present invention also proposes fusion protein expression vector as described above as new parental expression vector in expression target Application in albumen.

Further, a kind of method that the present invention also proposes fusion protein of the expression comprising class companion sample albumen, including Following steps:

Desired protein coding sequences are inserted into the polyclonal area in the downstream PAB1 in fusion protein expression vector as described above, Obtain the fusion protein recombinant expression carrier containing desired protein coding sequences；

By the fusion protein recombinant expression carrier transfection host cell, host cell expression fusion protein is cultivated.

In technical solution provided by the invention, B structure domain (the Protein A B-domain of newfound albumin A is utilized 1 to 5, PAB1~5) the effect of class companion's sample albumen, the fusion protein expression vector constructed can promote or induce quilt The folding of the downstream targets albumen of fusion has the characteristic of similar chaperone.

Detailed description of the invention

In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with Other relevant attached drawings are obtained according to these attached drawings.

Fig. 1 is PABx expression cassette schematic diagram in an embodiment of fusion protein expression vector provided by the invention；

Fig. 2 is pET28-PAB1 expression vector physical map obtained in embodiment 1；

Fig. 3 is pET28-PAB1-hTFF1 fusion protein expression vector physical map obtained in embodiment 2；

Fig. 4 is pET28-hTFF1 expression vector physical map obtained in embodiment 2.

The embodiments will be further described with reference to the accompanying drawings for the realization, the function and the advantages of the object of the present invention.

Specific embodiment

It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase Product.

Not only there is class companion's sample protein expression vector important scientific research value (to facilitate different albumen test solubility expressions And functional study), while there is important industrial value (producing for Rumen protein fermentation).But existing class companion sample albumen Expression vector is extremely limited, and there is difference certain, that mechanism is unknown to the folding assisted effect of different target albumen, Therefore, in practical application, as fruit companion sample protein fusion expression carrier library is bigger, selectivity is just bigger, to proteinology It is also more significant that the expression efficiency of scientific research and the big technique production of protein improves, promotes energy conservation and reduce cost.

In order to increase the washability of class companion's sample protein expression vector, the present invention proposes a kind of as in NO:1~5 SEQ ID The B structure domain segment or collating sequence of albumin A shown in any have the fusion protein table of class companion sample albumen effect in building Up to the application in carrier.

Albumin A (Protein A) is from staphylococcus aureus (Staphylococcus aureus) cell wall point From obtained bacterioprotein, 419 amino acid residue of molecule overall length can be with the immunoglobulin (antibody) of many mammals weight The Fc section of chain combines, or even can be in conjunction with the Fab of human antibodies VH3 family.SPA is without any intramolecular disulfide bond or by two sulphur The subunit that key maintains, theoretical pI/Mw:9.05/46716.32.The claimed present invention is newfound golden yellow in practice B structure domain (1 to 5 of Staphylococcus aureus Protein A B-domain, the abbreviation of staphylococcal protein A SPA-B1~B5 or PA-B1~B5 is abbreviated as PAB1~B5 in this paper the following contents) there is type companion's sample albumen Activity belongs to a kind of new features of albumin A, before this not it has been proposed that the B structure domain of albumin A have this characteristic, the present invention with This is built into the fusion protein expression vector comprising class companion's sample albumen, there is scientific research and commercial carrier to be worth, in original The higher organism protein molecule of not foldable is expressed in core biology (such as Escherichia coli).For ease of description, herein will be as The B structure domain segment of albumin A shown in NO:1~5 SEQ ID respectively correspond be named as PAB1, PAB2, PAB3, PAB4 and PAB5 can be in application provided by the invention and any one section of sequence in above-mentioned five segments is applied to building have Class companion's sample albumen effect fusion protein expression vector in, be also possible to by above-mentioned five segments two of them or two kinds Above collating sequence, which is applied to building, to be had in the fusion protein expression vector of class companion sample albumen effect.

In technical solution provided by the invention, acted on using class companion's sample albumen of B structure domain B1~B5 of albumin A, structure The fusion protein expression vector built can promote or induce the folding for the downstream targets albumen being fused, and have similar companion There is the characteristic of albumen scientific research and commercial carrier to be worth, and can be applied to express not in prokaryotes (such as Escherichia coli) The higher organism protein molecule of foldable.

Further, any structure domain of B1~B5 of the albumin A is consolidating as the fusion protein expression vector Determine element, can be inserted into variation element of the target protein as fusion expression vector downstream, formed with the fixing element of upstream It is merged with frame, between the two without termination codon.It is provided in a preferred Application Example in the present invention, to select PAB1 described in For the fixing element of fusion protein expression vector, it is inserted into fusion protein expression vector after being optimized to the PAB1, The fusion protein expression vector with similar chaperone characteristic is obtained, it specially will be such as SEQ ID NO:6, through excellent shown in 7 Class companion's sample albumen in PAB1 albumen coded sequence insertion fusion protein expression vector after change as expression cassette upstream, In, SEQ ID NO:6 show the amino acid sequence of the PAB1 after optimization, and SEQ ID NO:7 show the PAB1's after optimization DNA sequence dna.

The present invention also proposes a kind of fusion protein expression vector, including it is as described above it is optimized after PAB1 sequence.In In one embodiment of fusion protein expression vector provided by the invention, the PAB1 after sequence optimisation is linked into Escherichia coli In expression vector, the coli expression carrier (recombination as target protein expression vector comprising class companion sample albumen is obtained Parent vector), the clone area upstream of the specially described fusion protein expression vector include coding it is as described above it is optimized after The nucleic acid sequence of PAB1 albumen (sequence such as SEQ ID NO:6, shown in 7), the DNA sequence dna of the fusion protein expression vector is such as Shown in SEQ ID NO:26.It is understood that it is described it is optimized after PAB1 sequence be not limited to this sequence, with PAB1 egg White matter amino acid sequence homology is equal to or more than 85% and is limited.

For it is above-mentioned comprising the coli expression carrier of class companion sample albumen for, select for transformation commercialization Empty carrier is original parent carrier, and the original parent carrier can select pET system expression carrier, Yeast system expression Any one in carrier, insect cell system expression vector and mammalian cell system expression vector, preferably pET system Expression vector, more preferably pET28 (are said so that the original parent carrier selects pET28 as an example in following embodiments herein It is bright), the fusion protein expression vector of acquisition is named as pET28-PAB1, which is " the fusion for carrying class companion's sample albumen Protein expression vector " has the function of class companion's sample albumen to the target protein for being inserted in downstream, but can also regard one as Kind " empty carrier (empty vector) " for for target protein recombinant expression carrier, and is considered parent vector, can (as commercialization " empty carrier ") is sold using commercialization as expression vector, to construction of fusion protein.And the carrier is being used as Carrier is for that after being inserted into desired protein coding sequences downstream, can correspond to acquisition inserted with fusion when expressing target protein The fusion protein expression vector of albumen.

Preferably, in the present embodiment, it is described it is optimized after the Sequences upstream of PAB1 albumen add just like SEQ ID NO:8, transcription initiation aptamer sequence shown in 9, wherein SEQ ID NO:8 show the amino of the transcription initiation aptamer sequence Acid sequence, SEQ ID NO:9 show the DNA sequence dna of the transcription initiation aptamer sequence.

Further, in the present embodiment, it is described it is optimized after PAB1 albumen Sequences upstream being also an option that property Added with commercial separation tags albumen, it to be used for labelled protein.Specifically, the commercial separation tags albumen can be selected for example Any one the commercial separation tags albumen such as 5 '-HisTag and 5 '-Strept II-Tag.

Further, in the present embodiment, it is described it is optimized after PAB1 albumen sequence downstream include one section of flexibility Connector area, protease cleavage site cog region and the polyclonal area for being inserted into downstream targets albumen, the PAB1 expression constituted Frame schematic diagram is as shown in Figure 1, in Fig. 1: RBS indicates ribosome bind site or the transcription initiation region mRNA；PABx indicates albumin A B structure domain (B1~B5), x indicates the combination in wherein any one B structure domain or multiple B structure domains, in the present embodiment for PAB1；FL indicates flexible joint (Flexible Linker, FL)；PRS indicates protease site (Proteinase Recognition Site, PRS)；MCS indicates polyclonal insertion point area (Multiple Cloning Site, MCS)； 6xHis indicates the nickel column separation tags (6x Histidine Tag) of 6 histidine parallel connections.

The protease cleavage site cog region include factor Xa (abbreviation FXa) protease cleavage site cog region, Fibrin ferment restriction enzyme site cog region, enterokinase cleavage site cog region, tobacco etch virus protease restriction enzyme site cog region and Any one in hydroxylamine cleavage site, so that going needing to carry out protease digestion to the fusion protein expression vector When except class companion's sample albumen, coagulation factor protein enzyme, fibrin ferment, enterokinase or tobacco etch virus protease either hydroxyl are selected Any one in amine cleavage site.

It is illustrated so that the protease cleavage site cog region in the present embodiment is factor Xa as an example below, it is right Ying Di, the sequence in the flexible joint area such as SEQ ID NO:10, (SEQ ID NO:10 show the flexible joint shown in 11 The amino acid sequence in area, SEQ ID NO:11 show the DNA sequence dna in the flexible joint area), the protease cleavage site (SEQ ID NO:12 show the protease cleavage site cog region to the sequence of cog region as shown in SEQ ID NO:12,13 Amino acid sequence, SEQ ID NO:13 show the DNA sequence dna of the protease cleavage site cog region), it is described for inserting The sequence for entering the polyclonal area of downstream targets albumen is DNA restriction endonuclease recognition sequence, such as SEQ ID NO:14, shown in 15 (SEQ ID NO:14 show the amino acid sequence of polyclonal area's DNA encoding, and SEQ ID NO:15 show described for being inserted into The DNA sequence dna in the polyclonal area of downstream targets albumen).

Optionally, it is described it is optimized after the homology of PAB1 albumen and the homology of natural PAB1 albumen be not less than 85%, to be conducive to expand the application range of the fusion protein expression vector using PAB1 building, avoid existing because of protein Homology difference and the problem for causing application range limited.

The present invention also proposes the fusion protein expression vector as described above for including class companion sample albumen PAB1 as new Parental expression vector expression target protein in application, the fusion protein expression vector for express target protein when, Have the advantages that the raising of solubility expression efficiency, for protein expression scientific research and industrialized production provide one it is useful Tool.

The present invention uses institute as above it is further proposed that a kind of method of fusion protein of the expression comprising class companion sample albumen The fusion protein expression vector stated carries out.In the method that expression provided by the invention includes the fusion protein of class companion sample albumen In one embodiment, it is described expression comprising class companion sample albumen fusion protein method the following steps are included:

By desired protein coding sequences insertion, the expressing fusion protein comprising class companion sample albumen PAB1 is carried as described above In body the downstream PAB1 polyclonal area (namely it is described it is optimized after PAB1 downstream setting for being inserted into downstream targets albumen Polyclonal area, sequence such as SEQ ID NO:14, shown in 15), obtain containing desired protein coding sequences fusion protein recombination Expression vector；

By the fusion protein recombinant expression carrier transfection host cell, host cell expression fusion protein is cultivated.

Make the fusion protein recombinant expression carrier transfection host cell and cultivates host cell expression fusion protein Concrete operation method can refer to prior art progress, and this will not be repeated here.

Technical solution of the present invention is described in further detail below in conjunction with specific embodiments and the drawings, it should be understood that Following embodiment is only used to explain the present invention, is not intended to limit the present invention.