CN104250288B - Amphiphilicα-helix self-assembling peptides and its application - Google Patents

Abstract

The present invention relates to derived from SEQ ID NO:1 amphiphilicα-helix self-assembling peptides and its application in protein production and purifying.During with destination protein amalgamation and expression, the amphiphilicα-helix self-assembling peptides can induced fusion albumen form active aggregation in intracellular, and with SEQ ID NO:1 polypeptide is compared, and the spatial distribution of the active aggregation of formation in cell is changed, and is improved host cell growth state, is improved the yield of fusion protein.

Description

Amphiphilicα-helix self-assembling peptides and its application

Technical field

The present invention relates to genetic engineering field.Specifically, the present invention relates to new amphiphilicα-helix self-assembling peptides and its Application in protein production and purifying.

Background technology

As people increasingly increase the demand of the albumen such as enzyme preparation, protein drug and polypeptide product, protokaryon system is utilized Application of the system expression recombinant protein in industrial biotechnology becomes more and more extensive.The expression alien gene in prokaryotic system When, destination protein is easily with insoluble protein masses（Also referred to as inclusion body）Form exist [1].Traditional view is thought, wraps Contain body and refer to the insoluble precipitation [2] without biological activity that polypeptide false folding is formed.However, albumen is with inclusion bodies Expression has many good qualities, for example expressing quantity is big, purity is higher, lock out operation is easy, avoids proteasome degradation etc., and fits For producing the foreign protein or polypeptide poisonous to cell.But the albumen of inclusion bodies expression need to answer by external change Property and purification process can just obtain the functional protein with bioactivity.At present albumen refolding strategy technology still suffer from cost it is high, The problems such as yield is low, technical sophistication [3], it significantly limit application of the inclusion body expression-form in actual production.

The discovery of active aggregation changes conventional wisdom [4-6] of the people to inclusion body.Research shows, by adjusting table Up to condition or amalgamation and expression the construction unit of aggregation can be caused to obtain that there are amount of activated protein masses.It is this not Protein masses that are molten, having bioactivity inherit the advantage of inclusion body expressing protein, while the change that it also avoid complexity is answered Property operation.

The previous research of the present inventor finds that some amphipathic self-assembled short peptides can be with when with destination protein amalgamation and expression Induced fusion albumen forms activated protein aggregation [7-9] in Escherichia coli.Such amphipathic small peptide two level knot of self assembly Structure is simple, and the aggregation specific activity of formation is high, and prepares very convenient.

The present invention is transformed existing amphipathic self-assembled short peptide so that can change active aggregation in the cell Distribution, improve the growth conditions of host cell, while improve the yield of destination protein.Such improvement has been expanded amphipathic Application of the self-assembled short peptide in terms of protein production and purifying.

Summary of the invention

In a first aspect, the invention provides a kind of polypeptide, it includes and is derived from SEQ ID NO:1 amino acid sequence, The amino acid sequence and SEQ ID NO:1 compare contain selected from K4E/E1K, K4E/E8K, K9E/E12K, K15E/E12K and The double mutation of one or more of K13E/E16K, wherein when the polypeptide is thin in host as the fusion protein formed with destination protein When being expressed in born of the same parents, the fusion protein can form active aggregation in the host cell.

In some embodiments, polypeptide of the invention includes and is selected from SEQ ID NO:2、SEQ ID NO:3、SEQ ID NO:4、SEQ ID NO:5、SEQ ID NO:6、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO:9、SEQ ID NO: 10、SEQ ID NO:11、SEQ ID NO:12、SEQ ID NO:13、SEQ ID NO:14、SEQ ID NO:15、SEQ ID NO:16、SEQ ID NO:17、SEQ ID NO:18、SEQ ID NO:19 and SEQ ID NO:20 amino acid sequence.

In some embodiments, when the polypeptide as the fusion protein formed with destination protein the table in host cell Up to when, the protein yield of the fusion protein with by SEQ ID NO:The fusion protein that 1 polypeptide is formed with the destination protein Protein yield compared to being increased.

In second aspect, the present invention provides a kind of polynucleotides of separation, and it encodes the above-mentioned polypeptide of the present invention.

In the third aspect, the invention provides a kind of fusion protein, and it includes destination protein and above-mentioned polypeptide of the invention. In some embodiments, the destination protein is connected with the polypeptide by joint.In some embodiments, the joint Include SEQ ID NO:40 sequence.In other embodiments, the joint includes cleavage site.The cleavage site can With selected from chemical cleavage site, enzyme process cleavage site and Self cleavage site, preferably Self cleavage site.In some embodiments In, the Self cleavage site is intein, such as sequence is shown in SEQ ID NO:41 intein.

In fourth aspect, the invention provides the polynucleotides of separation, and it includes the core of the fusion protein of the coding present invention Nucleotide sequence.

At the 5th aspect, the invention provides expression construct, and it includes the above-mentioned polynucleotides of the present invention.

At the 6th aspect, the invention provides host cell, and it includes the above-mentioned polynucleotides of the present invention or with the present invention Above-mentioned expression construct conversion, wherein the host cell can express the fusion protein.

At the 7th aspect, the invention provides the method for producing and purifying destination protein, the described method comprises the following steps：

(a) the above-mentioned host cell of the culture present invention, so as to express the fusion protein；

(b) host cell is cracked, the soluble fraction of cell lysate is then removed, reclaims insoluble part.

The present invention is produced and purified in some embodiments of method of destination protein, if in wherein described fusion protein Destination protein with the present invention polypeptide be connected by the joint containing cleavage site, methods described can also include following step Suddenly：

(c) solvable destination protein is discharged from the insoluble part that step (b) obtains by cutting the cleavage site；

(d) the insoluble part in removal step (c), soluble fraction of the recovery containing the destination protein.

Brief description of the drawings

Fig. 1 show amphiphilicα-helix self-assembling peptides structure and its with phosphatide interact schematic diagram.A：18A helical wheel Figure；B：18Arev helical wheel figure；C：" Snorkel " model structure schematic diagram.

Fig. 2 show the 18A lysine of the 4th from N-terminal（K）With the glutamic acid of the 1st（E）The stabilization formed The amino acid pair of spiral.

Fig. 3 show the fusion protein expression vector collection of illustrative plates based on 18A variants.A：Without cleavage site；B：Band Self cleavage position Point.

Fig. 4 show the expressing fusion protein analysis comprising 18A or 18A variants and LipA.A：Cell growth state；B： LipA enzyme activity determination results.

Fig. 5 show the result that the GFP fusion protein aggregations comprising 18A or 18A variants are distributed in Escherichia coli.

Fig. 6 show 18Av3,18Av4,18Av6 and 18Av8 compared with the intracellular distribution of GFP and LipA fusion protein.

Fig. 7 show that 18Arev and 18Av8 is used for the result for producing and purifying LipA.

Fig. 8 show that 18Arev and 18Av8 is used for the result for producing and purifying AMA.

Detailed description of the invention

The previous research of the present inventor finds that sequence is shown in SEQ ID NO:1 amphiphilicα-helix self-assembling peptides 18A with Destination protein forms fusion protein and energy mediated fusion albumen forms insoluble activity in cell after host cell expression Aggregation.Active aggregation refers to the destination protein in aggregation although insoluble but still can keep all or part of life Thing activity." amphiphilicα-helix self-assembling peptides 18A " with " self-assembling peptides 18A " and " 18A " have used interchangeably herein Identical meanings.

Amphiphilicα-helix self-assembling peptides 18A（Such as Figure 1A）With following features：1）Have compared with common alpha helical peptides only Special hydrophilic, hydrophobic amino acid arrangement so that in the side of the αhelix of formation it is mainly hydrophilic amino acid, and another Side is mainly hydrophobic amino acid.2）According to " Snorkel " model [10]（Such as Fig. 1 C）, amphiphilicα-helix self-assembling peptides 18A It can be combined with phosphatide or grease.This combination be mainly by the lysine of the upper positively charged of hydrophobic interaction and 18A and The electrostatic interaction of electronegative phospholipid molecule.

The inventors discovered that the water-wet side in 18A spiral has 5 pairs by the adjacent positively charged amino acid in locus With negatively charged amino acids formed amino acid pair：The lysine of the 4th from N-terminal（K）With the glutamic acid of the 1st（E）；From N-terminal plays the lysine of the 4th（K）With the glutamic acid of the 8th（E）；The lysine of the 9th from N-terminal（K）With the paddy ammonia of the 12nd Acid（E）；The lysine of N-terminal the 15th from（K）With the glutamic acid of the 12nd（E）；The lysine of the 13rd from N-terminal（K）With The glutamic acid of the 16th（E）.

Herein, term " amino acid to " refers to the amino acid that positive and negative charge is complementary in α spirals, locus is adjacent It is right.Herein, adjacent two amino acid residues for referring to form amino acid pair in locus are spatially positioned at adjacent bung flange, Interval is no more than 5 amino acid residues between the two amino acid residues on primary sequence.Fig. 2 illustrates 18A's The lysine of the 4th from N-terminal（K）With the glutamic acid of the 1st（E）The amino acid pair formed.Not by any theoretical Limitation, thus it is speculated that can form salt bridge between two residues of amino acid centering, further stablize αhelix.

In our current research, the present inventor is surprisingly found out that amphiphilicα-helix self-assembling peptides 18A 5 amino acid To respectively or combination be mutated the variant polypeptide of acquisition and forming fusion protein and in host cell expression with destination protein Afterwards, equally can induced fusion albumen active aggregation is formed in cell, while the active aggregation formed is in cell In spatial distribution be changed, improve the growth conditions of host cell, improve the yield of fusion protein.

Therefore, the invention provides a kind of polypeptide, it includes and is derived from SEQ ID NO:1（18A）Amino acid sequence, institute State amino acid sequence and SEQ ID NO:1 compare contain selected from K4E/E1K, K4E/E8K, K9E/E12K, K15E/E12K and The double mutation of one or more of K13E/E16K.Such polypeptide is also referred to as " 18A variants " herein.When the 18A variants of the present invention During as being expressed with the fusion protein that destination protein is formed in host cell, the fusion protein can be thin in the host Intracellular forms active aggregation.

The present invention also provides the polynucleotides of the separation comprising the nucleotide sequence for encoding 18A variants of the present invention.

Herein, double mutation K4E/E1K represent SEQ ID NO:1 from N-terminal the lysine of the 4th（K）Sport paddy Propylhomoserin（E）, and SEQ ID NO:1 from N-terminal the glutamic acid of the 1st（E）Sport lysine（K）；K4E/E8K represents SEQ ID NO:1 from N-terminal the lysine of the 4th（K）Sport glutamic acid（E）, and SEQ ID NO:1 from N-terminal the glutamic acid of the 8th （E）Sport lysine（K）；K9E/E12K represents SEQ ID NO:1 from N-terminal the lysine of the 9th（K）Sport glutamic acid （E）, and SEQ ID NO:1 from N-terminal the glutamic acid of the 12nd（E）Sport lysine（K）；K15E/E12K represents SEQ ID NO:1 from N-terminal the lysine of the 15th（K）Sport glutamic acid（E）, and SEQ ID NO:1 from N-terminal the paddy ammonia of the 12nd Acid（E）Sport lysine（K）；K13E/E16K represents SEQ ID NO:1 from N-terminal the lysine of the 13rd（K）Sport paddy Propylhomoserin（E）And SEQ ID NO:1 from N-terminal the glutamic acid of the 16th（E）Sport lysine（K）.

In some instances, the mutation and amino acid sequence and nucleotide sequence that 18A variants provided by the invention include As shown in table 1.

The exemplary 18A variants of table 1

Lysine positively charged on 18A is changed into electronegative glutamic acid by the mutation in the 18A variants of the present invention, simultaneously The glutamic acid that salt bridge is formed with lysine is changed into the lysine of positively charged, to keep the stabilization of salt bridge.Not by any Theoretical limitation, charge reversal can produce effective repulsive interaction, decrease or even eliminate 18A variants and phospholipid molecule phase interaction With.Therefore, compared with 18A, 18A variants of the invention can occur distribution of its fusion protein aggregation in host cell Change.

Shown by following article embodiment, usually, LYS-GLU is more to the number of change, 18A variants and cell The interaction of membrane phospholipid molecule is weaker, and fusion protein aggregation more tends to be distributed in cytoplasm.For example, when change one Amino acid to when, 4/5 18A variant fusion proteins are distributed on film；When simultaneously change two amino acid to when, 3/ 6 18A variant fusion proteins are distributed in cytoplasm；When simultaneously change three and with upper amino acid to when, 5/8 18A Variant fusion proteins are distributed in cytoplasm.In addition, distribution of the α spirals stability of itself to fusion protein in host cell Also have a certain impact.Such as include mutation K4E/E1K（No. 1 position of N-terminal is the lysine of positive charge）, it is unfavorable for whole The stabilization of spiral（The effect of dipole）, this kind of 18A variants（Such as 18Av6,18Av7）Fusion protein aggregation tends to be distributed in carefully Around after birth；For another example, for variant 18Av8, two pairs of salt bridges are destroyed on its spiral（K4E/E1K、K15E/E12K）, compare 18Av9 and 18Av11（Only destroy a pair of salt bridges）, 18Av8 stability is relatively low, the fusion protein aggregation comprising the variant Show feature distribution of the part around cell membrane.

Inventor is also surprisingly found that compared with 18A 18A variants of the invention can improve expression, and it merges egg The growth conditions of white host cell." growth conditions for improving host cell " refer to initial amount identical host cell in phase The amount increase of the host cell obtained after the identical time is cultivated under same condition of culture.

Without being bound by theory, the change of the growth conditions of host cell is probably due to fusion protein aggregation It is changed in the distribution of intracellular.Shown by following article embodiment, on the whole, cell membrane is distributed in for can induce fusion protein The 18A variants of surrounding, the growth conditions for expressing the host cell of corresponding fusion protein aggregation are worse than expression 18A fusion proteins The host cell of aggregation；And the 18A variants being distributed in for can induce fusion protein in cytoplasm, express corresponding fusion egg The host cell growth state of white aggregation is better than the host cell of expression 18A fusion protein aggregations.Several examples but be present Outside, 18Av4,18Av6,18Av8,18Av13, the host cell growth state comprising these four variant fusion proteins aggregations are good Completely or partially divide in the fusion protein aggregation of the host cell for including 18A fusion protein aggregations, but four kinds of variant inductions Cloth is around cell membrane.

Compared with expressing the host cell of 18A fusion proteins, the growth of the host cell of the fusion protein of 18A variants is expressed State is improved.And according to Fig. 4 A and 4B contrast, wherein melting in the 12 kinds of 18A that can improve host cell growth state variants In hop protein, there is the total enzyme activity of 11 kinds of fusion proteins higher than 18A fusion protein；Host cell growth state is caused to compare 18A at 7 kinds In the 18A variant fusion proteins of host cell difference, there is the total enzyme activity of 6 kinds of fusion proteins lower than 18A fusion protein.This means with The improvement of cell growth state, the total enzyme activity of fusion protein improves.According to the data [7] before inventor, LipA melts with 18A Merging does not significantly change LipA enzyme activity itself, therefore the raising of fusion protein total enzyme activity means carrying for fusion protein total output It is high.That is, host cell growth condition improvement, be advantageous to improve the total output of fusion protein.Correspondingly, can also improve The yield of destination protein is (see such as embodiment 6 and 7).

Another aspect provides a kind of fusion protein, and it includes destination protein and 18A variants, wherein described 18A variants include and are derived from SEQ ID NO:1 amino acid sequence, the amino acid sequence and SEQ ID NO:1 compared to containing One or more double mutation selected from K4E/E1K, K4E/E8K, K9E/E12K, K15E/E12K and K13E/E16K.The fusion egg Active aggregation can be formed after host cell inner expression in vain, and its protein yield is formed higher than the destination protein with 18A Fusion protein protein yield.

Herein, term " polypeptide ", " peptide " and " albumen " is used interchangeably.Herein, " destination protein " refer to can Expressed with the 18A variant fusions with the present invention and form any more peptide or proteins of active aggregation.Destination protein can come from Any source, including microbe-derived polypeptide, mammal source polypeptide and artificial proteins（Such as chimeric protein or mutation Protein）Etc..

In some embodiments, connected between the destination protein and 18A variants in fusion protein of the invention by joint Connect.As used herein, " joint " refers to have the amino acid by low hydrophobicity and low charge effect of certain length to form more It peptide, can be sufficiently spread out connected each several part when it is used for fusion protein, fully be folded into respective day without interfering with each other Right conformation.Such joint commonly used in the art is included for example, being rich in glycine（G）And serine（S）Flexible GS type joints； Pro-rich（P）And threonine（T）Rigid PT type joints.Have more because PT type joints are commonly angled relative to GS type joints Good protease tolerance, thus PT type joints are preferably used in the present invention.In some specific embodiments, institute of the present invention The joint used includes sequence PTPPTTPTPPTTPTPT（SEQ ID NO:40）.

In some embodiments, the joint in fusion protein of the invention can also include cleavage site.Pass through cutting The cleavage site, destination protein can be separated from aggregation.Suitable cleavage site includes can be with chemical cleavage, enzyme process Cutting or the cleavage site of Self cleavage, or other any cleavage sites well known by persons skilled in the art.It is preferable in the present invention Cleavage site can carry out Self cleavage, for example, its include can Self cleavage intein amino acid sequence.Because based on interior Cutting method containing peptide does not need external enzyme or using hydrogen bromide harmful substance used in such as chemical method, and needs only to change Becoming the buffer environment residing for aggregation just can simply induce cutting [11,12].A variety of Self cleavage inteins known in the art, example Such as a series of inteins with different Self cleavage characteristics of NEB companies.In a specific embodiment, the intein SEQ ID NO are shown in for sequence:41 MxeGyrA, by adding proper amount of dithiothreitol (DTT) in buffer system（DTT）Just It can induce Self cleavage of the intein in its c-terminus.

On the other hand, the present invention also provides a kind of polynucleotides of separation, and it includes the fusion protein of the coding present invention Nucleotide sequence.The present invention also provides the expression construct for including such polynucleotides.

As used herein, " polynucleotides " refer to that multiple nucleotides are divided greatly by what 3 ' -5 '-phosphodiester bond was formed by connecting Son, wherein the nucleotides includes ribonucleotide and deoxyribonucleotide.The sequence of the polynucleotides of the present invention can be with pin To different host cells（Such as Escherichia coli）Codon optimization is carried out, so as to improve the expression of fusion protein.Carry out codon The method of optimization is known in the art.

In the expression construct of the present invention, the sequence and expression control sequence of the polynucleotides of fusion protein of the present invention are encoded Row are operably connected to be transcribed desired by progress and the fusion protein is finally produced in host cell.Suitable expression control Sequence processed includes but is not limited to promoter, enhancer, ribosomes action site such as ribosome bind site, polyadenylation position Point, the sequence for transcribing montage sequence, transcription terminator and stable mRNA etc..

Carrier for the expression construct of the present invention includes the carrier of those autonomous replications in host cell, such as plasmid Carrier；Also include the carrier that can be incorporated into host cell DNA and be replicated together with host cell DNA.It is commercially available to be permitted The carriers for being suitable to the present invention more.In a specific embodiment, expression construct of the invention is derived from Novagen companies pET30a(+)。

On the other hand, the present invention provides a kind of host cell, and it contains the core of the fusion protein comprising the coding present invention The polynucleotides of nucleotide sequence or with comprising the expression construct of the polynucleotides convert, wherein the host cell being capable of table Up to the fusion protein of the present invention.

Host cell for expressing fusion protein of the present invention includes prokaryotes, yeast and higher eucaryotic cells.Example The prokaryotic hosts of property include Escherichia（Escherichia）, bacillus（Bacillus）, Salmonella （Salmonella）And pseudomonas（Pseudomonas）And streptomyces（Streptomyces）Bacterium.Preferred Embodiment in, host cell is Escherichia cells, preferably Escherichia coli.In the specific embodiment party of the present invention In case, used host cell is e. coli bl21 (DE3) strain cell（Novagen）.

The recombinant expression construct body of the present invention can be imported into host cell by one of many well known technologies, so Technology include but is not limited to：Heat-shock transformed, electroporation, DEAE- glucans transfect, microinjection, and liposome connects turning for mediation Dye, calcium phosphate precipitation, Protoplast fusion, microparticle bombardment, virus Transformation and similar techniques.

On the other hand, the invention provides the method for producing and purifying destination protein, the described method comprises the following steps：

(a) the above-mentioned host cell of the culture present invention, so as to express melting comprising 18A variants and destination protein of the invention Hop protein；

(b) host cell is cracked, the soluble fraction of cell lysate is then removed, reclaims insoluble part.

In other embodiments, if destination protein and the 18A variants in wherein described fusion protein pass through Joint containing cleavage site is connected, and methods described can also comprise the following steps：

(c) solvable destination protein is discharged from the insoluble part that step (b) obtains by cutting the cleavage site；

(d) the insoluble part in removal step (c), reclaims the soluble fraction containing destination protein.

The 18A with the ability in purification process, with the formation of induced activity aggregation is produced in purpose of the present invention albumen As fusion protein after host cell inner expression, the fusion protein of expression forms insoluble aggregation for variant and destination protein Body.The formation of aggregation can avoid degraded of the intracellular protein enzyme to fusion protein.Cell cracking after, can simply by from The heart precipitates or insoluble aggregation is collected from cell lysate the methods of filtering, removes solvable impurity, realizes to merging egg White preliminary purification.Afterwards, by cutting the cleavage in the joint between the 18A variants and destination protein of the present invention Point so that the solvable part comprising destination protein never soluble fraction（Precipitation）Discharge, be distributed in supernatant, again Simply by can remove insoluble impurity the methods of centrifugation or filtering, solvable destination protein is harvested.By so The production albumen of the method based on the 18A variants that can cause aggregation can simplify purification procedures, avoid using expensive pure Change post, significantly decrease production cost.The inventors discovered that compared with 18A, can significantly be carried using the 18A variants of the present invention The yield of high destination protein.

Embodiment

The present invention will be further illustrated by way of embodiment below, but is not therefore limited the present invention to described Scope of embodiments in.Method therefor is conventional method unless otherwise instructed in following examples, and specific steps can be found in, example Such as,《Molecular Cloning:A Laboratory Manual》（Sambrook, J., Russell, David W., Molecular Cloning:A Laboratory Manual, 3rd edition, 2001, NY, Cold Spring Harbor）.The primer is by handsome biology（Invitrogen）Synthesis.

Embodiment 1：With Bacillus subtillis lipase A（LipA）And green fluorescent protein（GFP）Built for purpose albumen Fusion protein expression vector

1.1 amplification 18A variants（19 kinds）Polynucleotide passage

It is first utilized in the nucleotide sequence of the Line tool DNAworks design PT type joints and 18A variants.Pass through DNAWorks Design [13] simultaneously synthesizes oligonucleotide primer as shown in table 2, and then passes through over-lap PCR（overlapping PCR）Method obtains To coding 18A variants（Hind III- joint -18A variant-Xho I）Complete polynucleotide sequence.

Table 2 is used for the list of primers for expanding 18A variants

^aPrimer underscore part is respectively restriction enzyme Hind III and Xho I recognition site.

Below by taking one of which variant 18Av1 as an example, illustrate the specific method of polynucleotide passage amplification for encoding 18A variants.

DNAWorks is exported for synthesizing 4 overlapped oligonucleotide fragments of PT joint 18Av1 genes（Including A set of primer of a set of primer and 18Av1 of PT joints, sequence are as shown in table 2）.These oligonucleotide fragments are by handsome biology （Invitrogen）Company synthesizes.

By the oligonucleotide fragment of synthesis 10mM Tris-HCl, pH8.0 buffer solution, prepared according to table 3 PCR reaction solutions, then enter performing PCR reaction according to the response procedures of table 4.

Table 3：PCR reaction systems

Reactant	Volume
		Oligonucleotide fragment mixture	Per bar segment final concentration 625nM
5 × fast pfu buffer solutions	10μL
		Fast pfu	1μL
dNTPs	5μL
		Dual distilled water	32μL
Amount to	50μL

Table 4：PCR response procedures

Step	Temperature	Time
			1	95℃	5min

2	95℃	20s
			3	59℃	20s
4	72℃	15s
			5	Return to step 2	24 times
6	72℃	5min
			7	4℃	For a long time
8	Terminate

Using PCR primer obtained above as template, using following forward primer and reverse primer, conventionally enter Performing PCR amplification obtains PT joint -18Av1 nucleotide fragments：- the ATGAA of sense primer 5 'AAGCTTCCGACCCCACCGACCAC- 3’（SEQ ID NO:42, band underscore base is restriction enzyme Hind III recognition sites）, and anti-sense primer 5 '-CTCGAGTCAGAACAGTTCTTTCAGTTTCTCCAGGACCTTTTCGTAGAACGCTTC-3’（SEQ ID NO:43, with lower stroke Line base is restriction enzyme Xho I recognition sites）.PCR reaction solutions are prepared according to table 5 below, then according to the reaction of table 6 below Program expands PT joint -18Av1 genes.

Table 5：The PCR reaction systems expanded

Reactant	Addition
		Template (assembling product)	2μL
5 × fast pfu buffer solutions	20μL
		dNTPs	8μL
Sense primer (20 μM)	4μL
		Anti-sense primer (20 μM)	4μL
Fast pfu	2μL
		Dual distilled water	60μL
Amount to	100μL

Table 6：Expand PCR response procedures

Step	Temperature	Time
			1	95℃	5min
2	95℃	20s
			3	59℃	20s
4	72℃	15s
			5	Return to step 2	29 times
6	72℃	5min
			7	4℃	For a long time
8	Terminate

After reaction terminates, 1% agarose gel electrophoresis detection is carried out to pcr amplification product, as a result PCR is amplified with being expected The correct band being consistent.

1.2 structure LipA/GFP and 18A/18A variants fusion protein expression vector, and non-fused LipA/GFP albumen Expression vector

Expression vector pET-30a (+)-LipA-18A/18A variants and pET-30a used in the embodiment of the present application The building process process of (+)-GFP-18A/18A variants：Overlapping PCR products restriction enzyme will be obtained by embodiment 1.1 Hind III and Xho I carry out double digestion after respectively with plasmid pET-30a (+)-LipA-ELK16 through same enzyme double digestion and pET-30a(+)-GFP-ELK16（This two kinds of carriers be inventor built but it is undocumented be used for LipA, GFP and another kind can Cause the carrier of self-assembled short peptide ELK16 labels [8] amalgamation and expression of aggregation, the full length sequence of the plasmid is found in SEQ ID NO:82 and SEQ ID NO:83）It is attached, substitutes ELK16 labels with 18A/18A variants, so as to obtain fusion expression vector.

Below by taking pET-30a (+)-LipA-18Av1 and pET-30a (+)-GFP-18Av1 as an example, illustrate specific structure Method：

First build pET-30a (+)-LipA-18Av1 carriers, the carrier structure as shown in Figure 3A, wherein " Target Protein " sequences are bacillus subtilis lipase A（LipA）Sequence（SEQ ID NO:84）.Above-mentioned steps are obtained After overlapping PCR products Hind III- joint -18Av1-Xho I carry out double digestion with restriction enzyme Hind III and Xho I With plasmid pET-30a (+)-LipA-ELK16 through same enzyme double digestion（SEQ ID NO:82）It is attached, by connection product It is transformed into e. coli bl21 (DE3)（Novagen）Competent cell, by transformed cells be coated on added with 50 μ g/mL cards that Screening positive clone on the LB flat boards of mycin, plasmid is extracted, it is sequenced, sequencing result shows cloned pET-30a (+)-LipA-18Av1 sequences are correct.

Likewise, for pET-30a (+)-GFP-18Av1 carriers, the carrier structure as shown in Figure 3A, wherein " Target Protein " sequences are green fluorescent protein（GFP）Sequence（SEQ ID NO:85）.The over-lap PCR that above-mentioned steps are obtained produces Thing Hind III- joint -18Av1-Xho I are same with warp after carrying out double digestion with restriction enzyme Hind III and Xho I Plasmid pET-30a (+)-GFP-ELK16 of enzyme double digestion（SEQ ID NO:83）It is attached, connection product is transformed into large intestine Bacillus BL21 (DE3)（Novagen）Competent cell, transformed cells are coated on the LB added with 50 μ g/mL kanamycins and put down Screening positive clone on plate, plasmid is extracted, it is sequenced, sequencing result shows cloned pET-30a (+)-GFP- 18Av1 sequences are correct.

In addition, expression vector pET-30a (+)-LipA-18A and pET-30a (+) used in the embodiment of the present application- GFP-18A, and expression vector pET-30a (+)-LipA-native and pET-30a (+)-GFP-native of reference protein are equal Built for the present inventor but undocumented plasmid, its full length sequence are respectively SEQ ID NO:86、SEQ ID NO:87、SEQ ID NO:88 and SEQ ID NO:89.Those skilled in the art can easily prepare these plasmids.

Embodiment 2：With Bacillus subtillis lipase A（LipA）Expression, cell as the fusion protein of destination protein Growth conditions and enzyme activity determination

The induced expression of 2.1 fusion proteins

The bacterial strain that will be built in embodiment 1（Containing plasmid pET-30a (+)-LipA-native, pET-30a (+)- GFP-18A and pET-30a (+)-GFP-18A variants）It is inoculated into the LB fluid nutrient mediums containing 50 μ g/mL kanamycins, and Cultivated in 37 DEG C of shaking tables to logarithmic phase（OD₆₀₀=0.4-0.6）, 0.2mM IPTG are added, are induced 6 hours at 30 DEG C, harvest is thin Born of the same parents, and measure bacteria concentration OD₆₀₀（Below by 1mL OD₆₀₀It is referred to as 1OD for 1 cell concentration）.

2.2 cell growth state

The cell growth state of LipA-18A variant fusion proteins is expressed as shown in table 7 below and Fig. 4 A.

Table 7 expresses the OD of LipA-18A and LipA-18A variant fusion proteins cells₆₀₀Value

18A variants	LipA-18A	LipA-18Arev	LipA-18Av1	LipA-18Av2	LipA-18Av3
						OD	1.57	2.82	0.98	1.05	1.49
18A variants	LipA-18Av4	LipA-18Av5	LipA-18Av6	LipA-18Av7	LipA-18Av8
						OD	2.18	1.1	1.81	1.2	2.39
18A variants	LipA-18Av9	LipA-18Av10	LipA-18Av11	LipA-18Av12	LipA-18Av13
						OD	2.1	1.64	2.34	0.67	1.88
18A variants	LipA-18Av14	LipA-18Av15	LipA-18Av16	LipA-18Av17	LipA-18Av18
						OD	2.77	2.85	2.58	2.83	0.88

The cell growth state of wherein 12 kinds LipA-18A variant fusion proteins of expression is than expression LipA-18A fusion proteins Cell it is good（Show as OD₆₀₀Value is high）：LipA-18Av9、LipA-18Av10、LipA-18Av11、LipA-18Av14、LipA- 18Av15, LipA-18Av16, LipA-18Av17, LipA-18Arev and LipA-18Av4, LipA-18Av6, LipA-18Av8, LipA-18Av13.The cell growth state and LipA-18A of wherein 7 kinds LipA-18A variant fusion proteins of expression are quite or ratio LipA-18A growth conditions are poor：LipA-18Av1、LipA-18Av2、LipA-18Av3、LipA-18Av5、LipA-18Av7、 LipA-18Av12、LipA-18Av18。

2.3 enzyme activity determination

Harvest unit volume（1 liter of LB fluid nutrient medium）Cell lysis buffer after above-mentioned induction（50mM Tris- HCl, 50mM NaCl, 5% glycerine, pH7.2）It is resuspended.Pass through sonicated cells on ice（Broken condition is：Power 200W, surpass Sound time 3s, interval time 3s, ultrasonic number 99 times）.After the completion of ultrasound, by the supernatant for centrifuging carefully separating and cracking liquid Precipitation.In order to be eliminated as much as the soluble ingredient mixed in precipitation, the washing of precipitate that will be obtained with isometric lysis buffer Twice.Supernatant and precipitation re-suspension liquid are directly used in corresponding enzyme activity determination.Wherein, the method for quantitatively determining of lipase active is such as Under：

LipA is determined to palmitic acid p-nitrophenyl fat（P-nitrophenyl palmitate, pNPP）Activity.To pNPP Measuring method refers to document（Winkler, U.K., M.Stuckmann, Glycogen, Hyaluronate, and Some Other Polysaccharides Greatly Enhance the Formation of Exolipase by Serratia Marcescens, JOURNAL OF BACTERIOLOGY, 1979,138 (3):663-670）.Activity definition is：In condition determination Under, the p-nitrophenol that above-mentioned substrate produces 1nmol is hydrolyzed in 1 minute（p-nitrophenol）Or aliphatic acid（fatty acid）Required enzyme amount is defined as 1 active unit.

The enzyme-activity data of fusion protein is as shown in Figure 4 B.It is the same with LipA-18A fusion proteins, for most variant For fusion protein, 70-97% enzyme activity is distributed in precipitation（LipA-18Av11 and LipA-18Av16 exceptions, both fusions The ratio that enzyme activity of the albumen in precipitation accounts for total enzyme activity is respectively 65% and 54%）.In these variant fusion proteins, there are 12 kinds to melt The total enzyme activity of hop protein is higher than LipA-18A, and they are respectively（Activity is from high to low）：LipA-18Av8、LipA-18Av15、 LipA-18Av17、LipA-18Arev、LipA-18Av6、LipA-18Av14、LipA-18Av9、LipA-18Av11、LipA- 18Av16、LipA-18Av4、LipA-18Av3、LipA-18Av10.Wherein LipA-18Av8 total enzyme activitys are about LipA-18A total 2.9 times of enzyme activity.

As a result illustrate that 19 kinds of 18A variants can cause the aggregation of LipA albumen, and the aggregation formed can be not With the activity for retaining LipA albumen in degree, wherein 12 kinds of variants are advantageous to express total enzyme activity in the bacteriums of LipA fusion proteins Improve.

Comprehensive cell growth state data and total enzyme activity data（Fig. 4 A and B）, wherein host cell life can be improved at 12 kinds In the 18A variant fusion proteins of long status, there is the total enzyme activity of 11 kinds of fusion proteins higher than 18A fusion protein；Cause host at 7 kinds In 18A variant fusion proteins of the cell growth state than 18A host cell difference, the total enzyme activity for having 6 kinds of fusion proteins merges than 18A Albumen is low.This means the total enzyme activity with the improvement of cell growth state, fusion protein improves.

Embodiment 3：With green fluorescent protein（GFP）Expression as the fusion protein of destination protein and its point in intracellular Cloth

By the inoculation built in embodiment 1 into the LB fluid nutrient mediums containing 50 μ g/mL kanamycins, and 37 Culture is to logarithmic phase in DEG C shaking table（OD₆₀₀=0.4-0.6）, 0.2mM IPTG are added, are induced 22 hours at 23 DEG C, harvest is thin Born of the same parents.

The cell of harvest is handled into 1h with 4% paraformaldehyde at 4 DEG C.The fluorescent confocal microscopic observation of GFP cells exists Zeiss710 inverted confocal microscopes（Zeiss LSM710confocal microscope）Upper completion, excitation wavelength are 488nm。

The distribution situation of the active enzyme aggregate of 18A and 18A variants induction in the cell is as shown in Figure 5.From fluorescence photo It will be clear that：The fluorescence for expressing the cell of GFP-18A fusion proteins is mainly distributed on the inner side of cell membrane.Illustrate GFP- 18A fusion protein aggregations are distributed in the inner side of cell membrane.Cell for expressing the fusion protein comprising 18A variants, there is 8 kinds The distribution of 18A variant fusion proteins aggregations is similar with GFP-18A（It is distributed on the inside of cell membrane）, they are GFP- respectively 18Av1、GFP-18Av2、GFP-18Av4、GFP-18Av5、GFP-18Av6、GFP-18Av7、GFP-18Av12、GFP- 18Av13.Also there is the distribution of 8 kinds of 18A variant fusion proteins aggregations entirely different with GFP-18A（It is distributed in cell matter In, can be similar with typical inclusion body positioned at one end or both ends of cell, shape）, they be respectively GFP-18Av9, GFP-18Av10, GFP-18Av11, GFP-18Av14, GFP-18Av15, GFP-18Av16, GFP-18Av17 and GFP- 18Arev.There are three kinds of 18A variant fusion proteins aggregations to show point of the part on the inside of cell membrane, partly in cytoplasm Cloth, they are GFP-18Av3, GFP-18Av8, GFP-18Av18 respectively.

Result above shows that above-mentioned 18A variants, which can induce green fluorescent protein GFP to be formed in intracellular, has bioactivity Protein masses, and distributions of the GFP in Escherichia coli can be changed.

With reference to the result that GFP-18A variant fusion proteins are distributed in intracellular and express LipA-18A variant fusion proteins Cell growth state understand：The 18A variants being distributed in for can induce fusion protein around cell membrane（LipA-18Av1、 LipA-18Av2、LipA-18Av3、LipA-18Av5、LipA-18Av7、LipA-18Av12、LipA-18Av18）, expression is accordingly LipA fusion proteins host cell growth conditions than express LipA-18A host cell it is poor；And for can induce fusion Albumen is distributed in the 18A variants in cytoplasm（LipA-18Av9、LipA-18Av10、 LipA-18Av11、LipA-18Av14、 LipA-18Av15、LipA-18Av16、LipA-18Av17、LipA-18Arev）, the host that expresses corresponding LipA fusion proteins Cell growth state is better than the host cell for expressing LipA-18A.Only several exceptions, LipA-18Av4, LipA-18Av6, LipA-18Av8, LipA-18Av13, the host cell growth state comprising these four fusion proteins is better than comprising LipA-18A, But the GFP fusion proteins of these four variants induction are completely or partially distributed in around cell membrane.As a result show, when the activity of expression When aggregation is distributed in cytoplasm, be advantageous to cell growth.

Embodiment 4：Compare the intracellular distribution of LipA-18A variant fusion proteins and GFP-18A variant fusion proteins

In order to confirm distribution of the LipA-18A variant fusion proteins in intracellular point of GFP fusion proteins in intracellular with corresponding to The uniformity of cloth, inventor pick 4 kinds and express LipA-18Av3, LipA-18Av4, LipA-18Av6 and LipA-18Av8 The Escherichia coli of fusion protein carry out ultra-thin cell section, with transmission electron microscope observation LipA fusion proteins intracellular point Cloth and compared with distribution results of the corresponding GFP fusion proteins in intracellular.

Experimental method is as follows：

The bacterial strain that accordingly will be built in embodiment 1（Contain plasmid pET-30a (+)-LipA-18Av3, pET-30a (+)-LipA-18Av4、pET-30a(+)-LipA-18Av6、pET-30a(+)-LipA-18Av8）Be inoculated into card containing 50g/mL that In the LB fluid nutrient mediums of mycin, and cultivated in 37 DEG C of shaking tables to logarithmic phase（OD₆₀₀=0.4-0.6）, 0.2mM IPTG are added, Induced 6 hours at 30 DEG C, harvesting.

Sequentially add 2.5% glutaraldehyde solution and 2% osmium tetroxide（osmium tetraoxide）Solution is consolidated to cell Fixed processing.Fixed cell is through a series of gradient concentrations（30%, 50%, 70%, 90%, 100%）Ethanol dehydration step after, use ring Oxygen resin embedding.Utilize ultramicrotome（Lecia EM UC6）Ultra-thin cell section is obtained, then with acetic acid uranium（uranyl acetate）Solution and lead citrate（lead citrate）Solution dyes certain time, in Hitachi H-7650B transmission-types Electron microscope observation, electron accelerating voltage 80kV.

Experimental result is as follows：

For 18Av3, the distribution of its fusion protein is as shown in Figure 6A.TEM result（It is left）It has been shown that, part LipA-18Av3 Fusion protein forms aggregation on the inside of cell membrane（Arrows fusion protein aggregation）, part LipA-18Av3 fusion eggs In vain aggregation is formd in cytoplasm（Arrows fusion protein aggregation）.The result and GFP fusion proteins（It is right）In intracellular Distribution results be consistent.

For 18Av4, the distribution of its fusion protein is as shown in Figure 6B.TEM result（It is left）It has been shown that, LipA-18Av4 fusions Albumen forms a strata collective on the inside of cell membrane（Arrows fusion protein aggregation）.The result and GFP fusion proteins （It is right）It is consistent in the distribution results of intracellular.

The distribution of 18Av6 fusion proteins is similar with the distribution of 18Av4 fusion proteins, as shown in Figure 6 C.TEM result（It is left） It has been shown that, LipA-18Av6 fusion proteins also form a strata collective on the inside of cell membrane（Arrows fusion protein is assembled Body）.The result and GFP fusion proteins（It is right）It is consistent in the distribution results of intracellular.

For 18Av8, the distribution of its fusion protein is as shown in Figure 6 D.TEM result（It is left）It has been shown that, part LipA-18Av8 Fusion protein forms aggregation on the inside of cell membrane（Arrows fusion protein aggregation）, part LipA-18Av8 fusion eggs In vain aggregation is formd in one end of cell（Arrows fusion protein aggregation）.The result and GFP fusion proteins（It is right） The distribution results of intracellular are consistent.

The above results illustrate that the result that the experiment of GFP fusion proteins obtains can represent merging for 18A variants and other albumen Distribution of the albumen in cell.

Embodiment 5：Structure is with Bacillus subtillis lipase A（LipA）With aspergillus fumigatus Amadoriase II（AMA）For mesh Albumen the fusion protein expression vector for including cleavage site

Bacillus subtillis lipase A is selected respectively（LipA）With aspergillus fumigatus Amadoriase II（AMA）As purpose egg In vain, respectively with the extra fusion for including Mxe GyrA Self cleavages site of two 18A variants 18Arev and 18Av8 structure of the present invention Albumen, and destination protein production and purifying are carried out by the method for the present invention.

Expression vector pET-30a (+)-LipA-Mxe-18Arev/18Av8 and pET- used in the embodiment of the present application 30a (+)-AMA-Mxe-18Arev/18Av8 building process：Overlapping PCR products restriction enzyme will be obtained by embodiment 1 Enzyme Hind III and Xho I carry out double digestion after with plasmid pET-30a (+)-LipA-Mxe-ELK16 through same enzyme double digestion With pET-30a (+)-AMA-Mxe-ELK16（Inventor has built but undocumented plasmid, full length sequence SEQ ID NO:90 Hes SEQ ID NO:91）It is attached, so as to obtain required fusion expression vector（Schematic diagram is shown in Fig. 3 B）.Connection product is transformed into E. coli bl21 (DE3)（Novagen）Competent cell, transformed cells are coated on added with 50 μ g/mL kanamycins Screening positive clone on LB flat boards, extract plasmid, it is sequenced, sequencing result show cloned pET-30a (+)- LipA-Mxe-18Arev/18Av8 and pET-30a (+)-AMA-Mxe-18A rev/18Av8 sequences are correct.

In addition, expression vector pET-30a (+)-LipA-Mxe- of the reference protein used in the embodiment of the present application ELK16, pET-30a (+)-AMA-Mxe-ELK16, pET-30a (+)-LipA-Mxe-18A and pET-30a (+)-AMA-Mxe- 18A is that the present inventor has built but undocumented plasmid, its full length sequence are respectively SEQ ID NO:90、SEQ ID NO: 91、SEQ ID NO:92 and SEQ ID NO:93.Those skilled in the art can easily prepare these plasmids.

Embodiment 6：Fusion protein LipA-Mxe-18Arev and LipA-Mxe-18Av8 expression and purification

Expression vector pET-30a (+)-LipA-Mxe-18Arev/18Av8 and pET-30a (+)-LipA- will be contained respectively Mxe-ELK16/18A inoculation adds 0.2mM IPTG into the LB fluid nutrient mediums containing 50 μ g/mL kanamycins, Induced 6 hours at 30 DEG C, harvesting.And measure bacteria concentration OD₆₀₀（Below by 1mL OD₆₀₀It is referred to as 1OD for 1 cell concentration）.

By thalline lysis buffer B1（2.4g Tris, 29.22g NaCl, 0.37g Na₂EDTA·2H₂O dissolves In 800mL water, pH to 8.2 is adjusted, adds water to be settled to 1L）It is resuspended to 10OD/mL, ultrasonication.At 4 DEG C, 10000rpm bar 10min is centrifuged under part, collects supernatant sediment fraction respectively.After precipitation is washed into 2 times with lysis buffer, buffered using cutting Liquid（20mM Tris-HCl（pH8.0）, 500mM NaCl, 40mM dithiothreitol (DTT)s, 1mM EDTA）It is resuspended fully, is placed in 4 DEG C of mistakes Night 24h so that intein fully carries out Self cleavage.Suspension is centrifuged afterwards, before obtained supernatant precipitation and cutting Precipitation carry out SDS-PAGE detections together（The sediment fraction lysis buffer weight that step identical volume is resuspended with upper one It is outstanding）.As a result it is as shown in Figure 7.Respectively swimming lane 1：Cell lysate precipitation before cutting, it can detect clearly ternary fusion egg The enzyme aggregate being expressed as in vain；Swimming lane 2：The supernatant separated after cutting, it can detect clearly destination protein band；Swimming lane 3-5： Protein quantification standard items containing bovine serum albumin BSA, applied sample amount are followed successively by 6 μ g, 3 μ g, 1.5 μ g and 0.75 μ g.

According to protein quantification standard items, using the Quantity ONE quantitative gel analysis softwares of Bio-Rad companies to mesh Band carry out photodensitometry, can be calculated fusion protein formation aggregation yield, including peptide-mediated Self cleavage The purity of destination protein yield and desired polypeptides in supernatant being discharged into afterwards in supernatant, as a result as shown in table 8.

The expression quantity of table 8LipA fusion protein aggregations and the yield of destination protein

^aAggregation yield and^bThe destination protein yield after peptide-mediated Self cleavage is included (with bacteria concentration OD₆₀₀For 2 when, Bacillus coli cells in every milliliter of LB culture medium produce 2.66mg wet cell weights and calculated).

For LipA fusion proteins（SDS-PAGE results are shown in Fig. 7）, four kinds of self-assembled short peptides：ELK16,18A, 18Arev and 18Av8 is 24.4-34.1 μ g/mg wet cell weights with can largely form aggregation, expression quantity after LipA amalgamation and expressions；Through including For releasable destination protein LipA among supernatant, yield is 8.3-15.1 μ g/mg wet cell weights after peptide-mediated Self cleavage.Examine Consider the different self-assembled short peptide fusion protein of expression to have an impact the OD values of cell（Compared with 18A, 18Arev has with 18Av8 Beneficial to the growth of cell）, we calculate the yield of the aggregation that every liter of bacterium solution obtains and destination protein.For ELK16,18Arev With tri- kinds of fusion proteins of 18Av8, the expression quantity of aggregation is 96.7-98.1mg/L bacterium solutions, hence it is evident that is assembled higher than 18A fusion proteins The expression quantity of body（33.3mg/L bacterium solutions）；Through including peptide-mediated Self cleavage afterwards releasably to the destination protein LipA in supernatant Yield is 30.6-54mg/L bacterium solutions, and the wherein LipA of 18Av8 fusion proteins release yield is suitable with 18Arev, and its yield is 1.8 times of ELK16 fusion proteins, it is 5.4 times of 18A fusion proteins.

Embodiment 7：Fusion protein AMA-Mxe-18Arev and AMA-Mxe-18Av8 expression purify with cutting

Expression vector pET-30a (+)-AMA-Mxe-18Arev/18Av8 and pET-30a (+)-AMA- will be contained respectively Mxe-ELK16/18A inoculation adds 0.2mM IPTG into the LB fluid nutrient mediums containing 50 μ g/mL kanamycins, Induced 6 hours at 30 DEG C, harvesting.And measure bacteria concentration OD₆₀₀（Below by 1mL OD₆₀₀It is referred to as 1 cell concentration 1OD）.

By thalline lysis buffer B1（2.4g Tris, 29.22g NaCl, 0.37g Na₂EDTA·2H₂O dissolves In 800mL water, pH to 8.2 is adjusted, adds water to be settled to 1L）It is resuspended to 10OD/mL, ultrasonication.At 4 DEG C, 10000rpm bar 10min is centrifuged under part, collects supernatant sediment fraction respectively.After precipitation is washed into 2 times with lysis buffer, buffered using cutting Liquid（20mM Tris-HCl（pH8.0）, 500mM NaCl, 40mM dithiothreitol (DTT)s, 1mM EDTA）It is resuspended fully, is placed in 4 DEG C of mistakes Night 24h so that intein fully carries out Self cleavage.Suspension is centrifuged afterwards, before obtained supernatant precipitation and cutting Precipitation carry out SDS-PAGE detections together（The sediment fraction lysis buffer weight that step identical volume is resuspended with upper one It is outstanding）.As a result it is as shown in Figure 8.Respectively swimming lane 1：Cell lysate precipitation before cutting, can detect clearly fusion protein three The conjuncted enzyme aggregate being expressed as；Swimming lane 2：The supernatant separated after cutting, it can detect clearly destination protein band；Swimming lane 3- 5：Protein quantification standard items containing bovine serum albumin BSA, applied sample amount are followed successively by 6 μ g, 3 μ g, 1.5 μ g and 0.75 μ g.

According to protein quantification standard items, using the Quantity ONE quantitative gel analysis softwares of Bio-Rad companies to mesh Band carry out photodensitometry, can be calculated fusion protein formation aggregation yield, including peptide-mediated Self cleavage The purity of destination protein yield and desired polypeptides in supernatant being discharged into afterwards in supernatant, as a result as shown in table 9.

The expression quantity of table 9LipA fusion protein aggregations and the yield of destination protein

^aProtein masses yield and^bInclude the desired polypeptides yield after peptide-mediated Self cleavage（With in bacteria concentration OD₆₀₀For 2 When, the Bacillus coli cells in every milliliter of LB culture medium produce 2.66mg wet cell weights and calculated）.

For AMA fusion proteins（SDS-PAGE results are shown in Fig. 8）, four kinds of self-assembled short peptides：ELK16,18A, 18Arev and 18Av8 is 19.1-25.5 μ g/mg wet cell weights with can largely form aggregation, expression quantity after AMA amalgamation and expressions；Through including For releasable destination protein LipA among supernatant, yield is 4.0-7.9 μ g/mg wet cell weights after peptide-mediated Self cleavage.Examine Consider the different self-assembled short peptide fusion protein of expression has certain influence to the OD values of cell（Compared with 18A, 18Arev with 18Av8 is advantageous to the growth of cell）, we calculate the yield of the aggregation that every liter of bacterium solution obtains and destination protein.For Tri- kinds of fusion proteins of ELK16,18Arev and 18Av8, the expression quantity of aggregation is 65.3-86.9mg/L bacterium solutions, hence it is evident that higher than 18A The expression quantity of fusion protein aggregation（35.3mg/L bacterium solutions）；Through including after peptide-mediated Self cleavage releasably into supernatant Destination protein AMA yield is 8.2-24mg/L bacterium solutions, wherein the AMA of 18Av8 fusion proteins release yield highest, is ELK16 2.9 times of fusion protein burst size, it is 1.8 times of 18A fusion protein burst sizes.

Bibliography

1.Williams,D.C.,et al.,Cytoplasmic inclusion-bodies in escherichia- coli producing biosynthetic human insulin proteins.Science,1982.215(4533): p.687-689.

2.Bowden,G.A.,A.M.Paredes,and G.Georgiou,Structure and morphology of protein inclusion bodies in Escherichia coli.Nat.Biotechnol.,1991.9(8):p.725- 730.

3.von Maltzahn,G.,et al.,Positively charged surfactant-like peptides self-assemble into nanostructures.Langmuir,2003.19(10):p.4332-4337.

4.Garcia-Fruitos,E.,Inclusion bodies:a new concept.Microbial Cell Factories,2010.9:p.80.

5.Mitraki,A.,Protein aggregation:from inclusion bodies to amyloid and biomaterials,in Advances in Protein Chemistry and Structural Biology, Vol79.2010,Elsevier Academic Press Inc:San Diego.p.89-125.

6.Garcia-Fruitos,E.,et al.,Bacterial inclusion bodies:making gold from waste.Trends in Biotechnology,2012.30(2):p.65-70.

7.Wu,W.,Active enzyme aggregates induced by self-assembling peptides.2011,Tsinghua University.

8.Wu,W.,et al.,Active protein aggregates induced by terminally attached self-assembling peptide ELK16in Escherichia coli.Microbial CellFactories,2011.10.

9.Zhou,B.,et al.,Small surfactant-like peptides can drive soluble proteins into active aggregates.Microbial Cell Factories,2012.11.

10.Mishra,V.K.,et al.,Interactions of synthetic peptide analogs of the class a amphipathic helix with lipids-Evidence for the snorkel hypothesis.Journal of Biological Chemistry,1994.269(10):p.7185-7191.

11.Telenti,A.,et al.,The Mycobacterium xenopi GyrA protein splicing element:Characterization of a minimal.Journal of Bacteriology,1997.179(20): p.6378-6382.

12.Wu,H.,M.Q.Xu,and X.Q.Liu,Protein trans-splicing and functional mini-inteins of a cyanobacterial dnaB intein.Biochimica Et BiophysicaActa- Protein Structure and Molecular Enzymology,1998.1387(1-2):p.422-432.

13.Hoover,D.M.and J.Lubkowski,DNAWorks:an automated method for designing oligonucleotides for PCR-based gene synthesis.Nucleic Acids Research,2002.30(10)。

Claims (16)

1. a kind of polypeptide, it is by derived from SEQ ID NO:1 amino acid sequence composition, the amino acid sequence and SEQ ID NO:1 compares containing the double mutation of K4E/E1K, or containing K4E/E1K and selected from K4E/E8K, K9E/E12K, K15E/E12K and The double mutation of one or more of K13E/E16K, wherein when the polypeptide and the fusion protein of destination protein formation are in host cell During expression, the fusion protein can form active aggregation in the host cell.

2. the polypeptide of claim 1, it is by selected from SEQ ID NO:2、SEQ ID NO:7、SEQ ID NO:8、SEQ ID NO: 13、SEQ ID NO:14、SEQ ID NO:19 and SEQ ID NO:20 amino acid sequence composition.

3. the polypeptide of claim 1 or 2, wherein the table in host cell when the polypeptide and the fusion protein of destination protein formation Up to when, the protein yield of the fusion protein with by SEQ ID NO:The fusion protein that 1 polypeptide is formed with the destination protein Protein yield compared to being increased.

4. the polynucleotides of separation, it encodes any one of claim 1-3 polypeptide.

5. fusion protein, it includes any one of destination protein and claim 1-3 polypeptide.

6. the fusion protein of claim 5, wherein the destination protein is connected with the polypeptide by joint.

7. the fusion protein of claim 6, wherein the joint includes SEQ ID NO:40 sequence.

8. the fusion protein of claim 6, wherein the joint includes cleavage site.

9. the fusion protein of claim 8, wherein the cleavage site is selected from chemical cleavage site, enzyme process cleavage site and autotomyed Cut site.

10. the fusion protein of claim 9, wherein Self cleavage site are intein.

11. the fusion protein of claim 10, wherein the sequence of the intein is shown in SEQ ID NO:41.

12. the polynucleotides of separation, it includes the nucleotide sequence of any one of coding claim 5-11 fusion protein.

13. expression construct, it includes the polynucleotides of claim 12.

14. host cell, it is included the polynucleotides of claim 12 or converted with the expression construct of claim 13, wherein The host cell can express the fusion protein.

15. the method for producing and purifying destination protein, the described method comprises the following steps：

(a) host cell of claim 14 is cultivated, so as to express the fusion protein；With

(b) host cell is cracked, the soluble fraction of cell lysate is then removed, reclaims insoluble part.

16. the method for claim 15, wherein the destination protein and the polypeptide in the fusion protein are by containing cutting The joint for cutting site is connected, and methods described is further comprising the steps of：

(c) solvable destination protein is discharged from the insoluble part that step (b) obtains by cutting the cleavage site；

(d) the insoluble part in removal step (c), soluble fraction of the recovery containing the destination protein.