WO2010063172A1 - An elp fusion protein and uses thereof - Google Patents

Abstract

Provided are a fusion protein comprising ELP and uses thereof, and ELP-tagged protease that can be used as recycling utilized enzyme with immobilized enzyme characteric and uses thereof. For example, the purification or seperation method using of phase transition characteristic of ELP comprises contacting the ELP fusion protein having protease cleavage site with ELP-tagged enzyme, then subjecting the cleavage mixture to phase transition mediated by changing temperature, pH or salt ionic concentration to recover the protein of interest from the cleavage mixture.

Description

 ELP fusion protein and application thereof

Technical equipment

 The invention relates to the field of biotechnology, and in particular to an ELP fusion protein and application thereof. Background technique

With the development and widespread application of large-scale sequencing technologies, the Human Genome Project and other genomic programs including sea urchins, zebrafish, and amphioxus have also been completed. The direct result of genome sequencing of these species is the production of thousands of genes, and the clarification of the function of these genes helps us understand the location of the species in the evolutionary process; research on genes that cause disease helps to understand the pathogenesis In search of therapeutic targets, development and treatment of corresponding therapeutic drugs; at the same time, a large number of drug protein genes have yet to be studied for their physiological activities, and targets, to provide a basis for the development of new therapeutic drugs. There are many methods for gene function research, including gene knockout technology, proteomics research, and RNA interference. One of the most direct research methods is to recombinantly express the gene under study, obtain the corresponding protein, conduct physiological and biochemical activity research and analyze its structural biology to clarify its function. Due to the differences in species genes, not all genes can be successfully expressed. Genes are not expressed, protein expression is low or the expressed protein is unstable to form insoluble inclusion bodies. These phenomena are frequently found in heterologous expression systems, particularly in prokaryotic expression systems. With the development of DNA recombination technology, the gene of interest can be fused with the gene expressing the affinity tag, and then cloned and expressed in the expression vector for fusion expression. These affinity tags can not only help the folded target protein to be folded, but also enhance the target protein. The solubility and expression amount, while the affinity tag can be combined with the corresponding affinity chromatography resin, can play a role in purifying the fusion protein in one step. Commonly used affinity purification tags include Glutathione-S-Transferase (GST), Maltose-binding Protein (MBP), His tag and the like. Finally, the fusion protein containing the corresponding protease cleavage site is cleaved by protease to separate the target protein from the affinity tagged protein, and the affinity is removed by further chromatography. The target protein is obtained by labeling and protease.

 Affinity chromatography is a very efficient separation method, and the purification protein can be purified in one step without knowing the physicochemical properties of the fusion protein. This purification technique is widely used. However, the use of affinity chromatography separation techniques requires expensive affinity resins, and corresponding purification equipment, which limits the simultaneous expression of multiple proteins for high-throughput structural biology studies and high-throughput drug protein screening. In 1999, a heat-sensitive purified label elastin-like polypeptide (ELP) was reported, which originated from the repeating pentapeptide "Val-Pro-Gly-Val-Gly" of elastin. ELP can undergo a reversible phase transition process called Inverse Temperature Transition. When the temperature is lower than the transition temperature (Tt), ELP is highly soluble in the liquid phase. However, when the temperature is higher than Tt, the hydrophilic ELP dehydrates and agglomerates, and the ELP fusion protein also has This property, but it should be pointed out that when the phase transition occurs, only the ELP is coagulated and the foreign protein to be fused does not undergo denaturation or precipitation. Using this property of ELP, the ELP tag can be conveniently and quickly utilized to purify the ELP fusion protein, and chromatographic separation is eliminated during the purification process, avoiding expensive affinity resins and purification equipment, while concentrating and replacing the ELP fusion protein. The buffer also became very simple. The aggregation of the ELP fusion protein is triggered by heating or increasing the salt ion concentration, and the ELP fusion protein is separated from the expression host protein by centrifugation, and the precipitated ELP fusion protein is re-dissolved with a low-salt low-temperature solution, and then centrifuged to remove insoluble The protein is obtained to obtain an ELP fusion protein, and a more pure ELP fusion protein can be obtained by repeatedly triggering the precipitation, centrifugation and re-dissolution steps. This purification process is called Inverse Transition Cycling (ITC).

Enzymes are highly efficient and specific biocatalysts. Various chemical reactions in living organisms are carried out under enzymatic catalysis, but free enzymes are very unstable in aqueous solution, and soluble enzymes generally only catalyze in one time. At the same time, enzymes are proteins to heat, high ion concentration, Strong acids, strong bases and some organic solvents are not stable enough to be easily deactivated and reduce their catalytic ability. These disadvantages greatly limit the wide application of enzymatic reactions. The Immobilized enzyme technology that emerged in the 1960s overcomes these shortcomings of free enzymes, and the enzymes can be recycled and reused, making it one of the most active research areas in biotechnology. The immobilization method of the enzyme (cell) can be broadly classified into four types: an adsorption method, a covalent coupling method, a crosslinking method, and an embedding method. Adsorption method refers to the secondary bond phase between the surface of the carrier and the surface of the enzyme. The method of interaction to achieve enzyme immobilization can be further divided into physical adsorption and ion exchange adsorption according to the characteristics of the adsorbent. The method has the advantages of operating the tube, mild conditions and repeated use of the adsorbent, but also has the disadvantages of weak adsorption force, easy to desorb off under unsuitable pH, high salt concentration, high substrate concentration and high temperature conditions. The covalent coupling method combines the active non-essential side chain group of the enzyme with the functional group of the carrier through a covalent bond, so that it exhibits good stability and is beneficial to the continuous use of the enzyme, and is currently the most active application and research. One type of enzyme immobilization method, but the covalent coupling reaction easily denatures the enzyme and inactivates it. The cross-linking method is a method of immobilizing an enzyme by cross-linking between enzyme molecules using a bifunctional or multifunctional group reagent, which is more likely to inactivate the enzyme. The embedding method includes grid embedding, micro-encapsulation and liposome entrapment. In the embedding method, the enzyme itself does not participate in the chemical binding reaction, so that high enzyme activity recovery can be obtained, and the disadvantage is that it is not applicable. It is used for mass transfer of high molecular weight substrates and for column reaction systems, and often has problems such as diffusion limitations. Moreover, the cost of the immobilized enzyme is too high, and the substrate of the macromolecule is not easy to use. The deficiencies exhibited by the various methods of immobilizing enzymes described above limit their widespread use. Summary of the invention

 Based on the above theory, it is an object of the present invention to provide a method for expression purification of a tag-free recombinant protein comprising the steps of:

 (1) a gene encoding a fusion protein of a target protein gene and an ELP-containing fusion partner, and expressing the fusion protein A, wherein the fusion protein A has at least a junction of the ELP and the fusion partner containing the ELP a protease cleavage site;

 (2) mixing the fusion protein A obtained in the step (1) with a fusion protein B containing ELP, and the fusion protease B can act on the restriction site of the fusion protein A, but does not act on itself;

 (3) allowing the mixture of step (2) to interact, wherein fusion protein B degrades fusion protein A into an ELP-containing fusion partner and a target protein;

 (4) changing at least one of the temperature, the salt ion concentration or the pH of the mixed solution of the step (3), and after the aggregate is present, it is centrifuged, and the supernatant solution is taken as the purified target protein.

 The nucleotide sequence of the above ELP monomer is shown in SEQ ID N0.1.

The above ELP is a tetramer, a pentamer, a hexamer, a heptamer, an octamer or a nine of the ELP monomers. Polymer.

 The above fusion protein B includes an ELP-3C or ELP-SUMO (small ubiquitin-related modifier) protease.

 A second object of the present invention is to provide a label-free recombinant protein obtained by the following method:

 (1) a gene encoding a fusion protein of a target protein gene and an ELP to be a fusion protein gene, and expressing the fusion protein C, wherein the fusion protein C has at least a junction of the ELP and the fusion partner containing the ELP a protease cleavage site;

 (2) mixing the fusion protein C obtained in the step (1) with a fusion protein D containing ELP, and the fusion protease D can act on the cleavage site of the fusion protein C, but does not act on itself;

 (3) allowing the mixture of step (2) to interact, wherein fusion protein D degrades fusion protein C into an ELP-containing fusion partner and a target protein;

 (4) changing at least one of the temperature, the salt ion concentration or the pH of the mixed solution of the step (3), and after the aggregate is present, it is centrifuged, and the supernatant solution is taken as the purified target protein.

 The fusion partner of ELP refers to an ELP-containing protein in addition to a target protein in a fusion protein. The nucleotide sequence of the above ELP monomer is shown in SEQ ID N0.1.

 The above ELP is a tetramer, a pentamer, a hexamer, a heptamer, an octamer or a hexamer of an ELP monomer.

 The above fusion protein D includes an ELP-3C or ELP-SUMO (small ubiquitin-related modifier) protease.

 A third object of the present invention is to provide an ELP-containing fusion protein comprising an elastin-like polypeptide (ELP) and at least one protein or polypeptide of interest, which can be formed with ELP. Expression in the form of a fusion protein capable of being in a dissolved or precipitated state at at least one different temperature, different salt ion concentration or different pH values, and the fusion protein can be adjusted by temperature , salt ion concentration or pH is worth purifying.

 The nucleotide sequence of the monomer of the above elastin-like polypeptide is shown in SEQ ID N0.1.

The above elastin-like polypeptide includes a tetramer, a pentamer, a hexamer, a heptamer, and a hexa Polymer or 9-mer.

 A fourth object of the present invention is to provide a method for expressing a purified fusion protein containing ELP, comprising the steps of:

 (1) a gene encoding a fusion protein of a target protein gene and an ELP-containing fusion partner, and expressing the fusion protein E;

 (2) changing at least one of the temperature, the salt ion concentration or the pH value of the fusion protein E obtained in the step (1), and after centrifugation, the obtained aggregate is an ELP-containing fusion protein.

 The nucleotide sequence of the monomer of the ELP is shown in SEQ ID N0.1.

 The ELP is a tetramer, a pentamer, a hexamer, a heptamer, an octamer or a hexamer of an ELP monomer.

 The fusion protease E includes an ELP-3C or ELP-SUMO (small ubiquitin-related modifier) protease.

 A fifth object of the present invention is to provide a kit for purification of protein expression, the kit comprising an ELP-containing plasmid for preparing a fusion protein F and a fusion protein G, the fusion The protein G contains a protein or polypeptide of interest, and the fusion protein F can be digested to obtain a protein or polypeptide of interest.

 The fusion protein F includes an ELP-3C or ELP-SUMO (small ubiquitin-related modifier) protease.

 The fusion protein F is capable of cleaving the fusion protein G into an ELP and a target protein.

 A sixth object of the present invention is to provide a recyclable enzyme having an immobilized enzyme property, which is a fusion protease comprising ELP, which can be in a dissolved or precipitated state depending on temperature or salt ion concentration.

 The nucleotide sequence of the monomer of the ELP is shown in SEQ ID N0.1.

 The ELP is a tetramer, a pentamer, a hexamer, a heptamer, an octamer or a hexamer of an ELP monomer.

The fusion protease includes an ELP-3C or ELP-SUMO (small ubiquitin-related modifier) protease. A seventh object of the present invention is to provide a method for performing cyclic enzymatic purification using a protease comprising ELP, comprising the steps of:

 (1) mixing the fusion protein with the substrate to carry out a digestion reaction;

 (2) adjusting the temperature or salt ion concentration of the solution after the digestion reaction, and centrifuging after the aggregate is present; (3) adding the aggregate obtained after centrifugation to the enzymatic hydrolysate, and after it is dissolved, adding the substrate to carry out the enzyme Cut the reaction and repeat step (2).

 The substrate in the step (1) is an ELP fusion protein containing a protein of interest.

 The monomer of the artificially synthesized ELP is the pentapeptide "Val-Pro-Gly-Xaa-Gly" (Xaa is an arbitrary amino acid other than the pro amino acid), and in the present invention, the ELP contains the tetramer of the ELP monomer, and the pentapolymer Various types, such as body, hexamer, heptamer, octamer, and 9-mer, change the conditions of ELP or ELP-containing fusion protein, including at least one of temperature, salt ion concentration, and pH. Conditions, ELP or ELP-containing fusion proteins can be precipitated or dissolved in different states, and when the original conditions are restored, the ELP or ELP-containing fusion protein can return to its original state.

 In the present invention, specifically, the method for expressing a purified protein of interest, the unlabeled recombinant protein, and the target protein involved in the cyclic digestion purification using a protease containing ELP include a relatively broad protein. Specifically, it includes viral proteins, bacterial proteins, yeast expressed proteins, mammalian proteins, and the like.

 In the present invention, ELP - 3C and ELP - SUMO are only two specific examples, and of course, are not limited to these two types, and enzyme enzymes having enzymatic activity are included in the scope of the present invention. These enzymatically active proteases act on the ELP-containing fusion protein containing the corresponding cleavage site, thereby cleaving the substrate into the target protein and the ELP fusion partner; and these digested fusion proteins cannot act on themselves. The ELP fusion partner refers to a protein other than the target protein containing ELP, and the ELP fusion partner and the target protein have an enzyme cleavage site, which can be digested into a target protein and an ELP fusion partner.

The expression vector referred to in the present invention has a general meaning, that is, a vector containing a promoter sequence, which is capable of efficiently promoting the transcription of the inserted target gene, and then translating the protein product encoded by the inserted gene. The promoter of the expression vector is usually homologous to its receptor, such as an expression vector containing E. coli as a receptor, the promoter of which is derived from the E. coli system, and the promoter expressed in the vaccinia virus is derived from the vaccinia virus. The vector is mainly a plasmid or a virus, including plant viruses, animal viruses, and phage. Commonly used are: plasmid vectors carrying Escherichia coli, such as pQE, pMAL, pUC, etc., and lambda phage and Escherichia coli artificial chromosome (BAC), Yeast-based yeast artificial chromosome (YAC) and pRS series yeast plasmid vector, pcDNA series plasmid and pEGFP series plasmid hosted on mammalian cells (animal/E. coli shuttle plasmid).

 The prokaryotic expression vector is usually a plasmid, and the typical expression vector has the following components: a coding sequence for selection of a marker, a promoter for controllable transcription, a transcriptional regulatory sequence (transcription terminator, ribosome binding site), a multi-restriction enzyme digestion Site linker and sequences autonomously replicated in the host. The pET system is the most powerful system for expressing recombinant proteins in E. coli. The gene of interest was cloned into the pET plasmid vector and subjected to strong transcriptional and translational (selective) signals by the phage T7; expression was induced by T7 RNA polymerase provided by the host cell. Under non-inducing conditions, the system allows the gene of interest to be completely silent without transcription. Cloning of the gene of interest with a host strain that does not contain T7 RNA polymerase avoids plasmid instability due to possible toxicity of the protein of interest to the host cell. If a non-expressing host cell clone is used, expression of the protein of interest can be initiated by two methods: infecting the host cell with a lambda CE6 phage bearing T7 RNA polymerase controlled by the A pL and pi promoters, or transferring the plasmid into An expression cell with a T7 RNA polymerase gene under the control of lacUV5. In the second case, expression can be initiated by the addition of IPTG to the bacterial culture medium. The two T7 promoters, as well as a variety of host cells with different levels of inhibition of background expression, constitute an extremely flexible and efficient system for optimal expression of various proteins of interest.

 The viral vector is a viral particle that allows the foreign gene to enter the cell by interaction of the viral envelope protein with the host cell membrane. The vector used in the present invention includes commonly used viral vectors, including adenovirus, adeno-associated virus, retrovirus, semliki forest virus (sFv) vector and the like. In addition, the application of baculovirus vectors to mammalian cells has received much attention in recent years because of its unique advantages over other viral vectors, such as the large production of viral particles by insect cells; A mammalian cell, but has no replication ability in the cell, and has high biosafety; it can insert a foreign gene of up to 38 kb.

The most commonly used is the autogra-phacalifornica multi-nuclear polyhedrosis virus (MNPV), which is called AcMNPV or AcNPV. The virus It is the prototype of the baculoviridae Baculoviridae. It is a large, double-stranded DNA virus with a coat that can infect more than 30 lepidopteran insects and is widely used as a vector for gene expression systems. The AcNPV virus is used as an expression vector for a foreign gene, and a recombinant virus is usually constructed by replacing the polyhedrin gene with a foreign gene by a method of homologous recombination in vivo. Since the polyhedrin gene promoter began transcription and translation 18 to 24 hours after infection, it lasted for 70 hours. The replacement of the polyhedrin gene by the foreign gene does not affect the infection and replication of the progeny virus, which means that the recombinant virus does not require the function of the helper virus. Other baculoviruses as expression vectors are mainly NP~ (bombyx moil, BmNP~) from silkworm. Since the in vivo system of silkworm larvae is suitable for large-scale production and production of foreign proteins, and has low cost, it has a good application prospect.

 In the yeast expression system, the Pichia pastoris expression system is considered to be one of the most effective expression systems. The Pichia pastoris expression system has a variety of secretory expression plasmids, and many proteins are highly secreted and expressed in Pichia pastoris. Extracellular expression requires the addition of a signal peptide sequence at the N-terminus of the foreign protein to direct the recombinant protein into the secretory pathway.

 Compared with the prior art, the present invention has the following beneficial effects:

 The present invention utilizes the reverse temperature phase transition characteristic of ELP to provide a series of ELP fusion proteins which can be used in protein expression and purification as well as recyclable enzymes having immobilized enzyme properties. The problem of low protein expression or formation of insoluble inclusions, and the need to utilize complex purification procedures in protein purification. The ELP fusion protein has the characteristics of a solid phase enzyme, which facilitates continuous production and automation for industrial mass production. DRAWINGS

 Figure 1 shows the induced expression and purification of ELP-3C protease and its SDS-PAGE electrophoresis for purification of GST protein. M: protein molecular weight standard; 1 : total bacterial protein induced by ELP-3C protease; 2: cleavage supernatant induced by ELP-3C protease; 3: centrifugation supernatant after precipitation of ELP-3C protease by salt; 4: purification Post-ELP-3C protease; 5: purified GST-ELP; 6: ELP-3C protease digested GST-ELP; 7: pre-cleaved product; 8: purified GST.

Figure 2 is an SDS-PAGE electropherogram showing the optimal digestion concentration of the GST-ELP fusion protein by ELP-3C protease digestion. 1 : GST-ELP; 2: ELP-3C protease: GST-ELP = 1: 10 (w/w); 3: ELP-3C protease: GST-ELP = 1: 50 (w/w); 4: ELP-3C protease: GST-ELP = 1: 100 (w/w); 5: ELP-3C protease: GST-ELP = 1: 500 (w/w); 6: ELP-3C protease: GST-ELP = 1: 1000 (w/w); 7: ELP-3C protease: GST-ELP = 1: 5000 (w/w); 8: ELP-3C protease: GST-ELP = 1: 10000 (w/w).

 Figure 3 is a SDS-PAGE electropherogram of the purification of GFP protein by ELP-3C protease. M: protein molecular weight standard; 1 : total bacterial protein induced by ELP-GFP; 2: cleavage supernatant induced by ELP-GFP; 3: centrifugation supernatant after precipitation of ELP-GFP by salt; 4: ELP after purification - GFP; 5: ELP-3C protease digests ELP-GFP; 6: pre-cleaved product; 7: purified GFP.

 Figure 4 shows the purified GFP protein detected under a fluorescence microscope.

 Figure 5 is a SDS-PAGE electropherogram of the purification of MBP protein by ELP-3C protease. M: protein molecular weight standard; 1 : total bacterial protein induced by ELP-MBP; 2: cleavage supernatant induced by ELP-MBP expression; 3: centrifugation supernatant after precipitation of ELP-MBP by salt; 4: ELP after purification - MBP; 5: ELP-3C protease digestion of ELP-MBP; 6: precipitation of the product; 7: purified MBP.

 Figure 6 is a SDS-PAGE electropherogram of purification of TRX protein by ELP-3C protease. M: protein molecular weight standard; 1 : total bacterial protein induced by ELP-TRX; 2: cleavage supernatant induced by ELP-TRX expression; 3: centrifugation supernatant after precipitation of ELP-TRX by salt; 4: ELP after purification -TRX; 5: ELP-3C protease digestion ELP-TRX; 6: precipitation digestion product; 7: purified TRX.

 Figure 7 is a SDS-PAGE electropherogram of the PEG protein purified by continuous digestion. 1 : The first ELP-3C protease cleaves ELP-GFP-purified GFP; 2: The second ELP-3C protease cleaves ELP-GFP-purified GFP; 3: The third ELP-3C protease cleaves ELP -GFP purified after GFP; 4: GFP after encapsulation of ELP-GFP by the fourth ELP-3C protease; 5: GFP after encapsulation of ELP-GFP by the fifth ELP-3C protease; 6: sixth time ELP-3C protease cleaves GFP after purification of ELP-GFP. detailed description

In order to make the present invention easier to understand, the present invention will be further described below in conjunction with the specific embodiments. Should The experimental methods in the following examples, which do not specify the specific conditions, are usually in accordance with conventional conditions, for example

The conditions described in the molecular clone implementation room manual by Sambrook et al.

 Example 1

 Preparation of pET23a-ELP plasmid in kit for protein expression purification

 Preparation of ELP-3C protease in a kit for purification of protein expression

 Construction of ELP-3C protease expression vector and transformation of host bacteria

 The ELP gene was synthesized according to the method reported by Meyer and Chilkoti et al. in 1999 (Meyer, DE, and Chilkoti, A. 1999. Purification of recombinant proteins by fusion with thermally-responsive polypeptides. Nat Biotechnol 17: 1112-1115.) The nucleotide sequence 歹'J is shown in SEQ ID NO. After digestion with the Ecorl and C-terminal Hindlll of the ELP gene, the corresponding restriction sites in the commercial prokaryotic expression vector pET23a were cloned to form a pET23a-ELP vector. The pET23a-ELP plasmid was transformed into Escherichia coli DH5a strain, and the pET23a-ELP plasmid was extracted after culture expression. At the same time, the 3C protease gene was synthesized according to the gene sequence of human rhinovirus 3C protease (Accession No.: M12168) published in genbank in NCBI, and its nucleotide sequence is shown in SEQ ID N0.2, and cloned into pbluscript vector. The pbluscript-3C vector was formed. The pbluscript-3C plasmid was transformed into Escherichia coli DH5a strain, and the pbluscript-3C plasmid was extracted after culture expression. pbluscript-3C was digested with Hindl and C-terminal Notl endonucleases at the N-terminus of the 3C protease gene, and the purified 3C gene fragment was cloned into the corresponding restriction site of the pET23a-ELP vector, and the 3C protease gene was located in the ELP gene. At the C-terminus, the ELP-3C protease expression vector pET23a-ELP-3C was obtained.

The constructed expression vector pET23a-ELP-3C was transformed into E.co/.BL21 by CaCl ₂ method according to the method of Sambrook (Sambrook, et al. 1989, Molecular doing. Cold Spring Harbor Labroratory Press. USA). DE3) strain, the transformed strain was cultured in LB medium containing ampicillin (100 g/mL).

 Example 2: Expression and purification of recombinant ELP-3C protease

A single colony of the expression host strain BL21 (DE3) containing the recombinant plasmid pET23a-ELP-3C was inoculated into an Amp+ LB liquid-enriched medium, and cultured at 37 ° C for 225 rpm for 12 hours as a seed fungus. Seeding The bacterium was inoculated in fresh Amp + TB medium at a ratio of 1:100, and cultured at 37 ° C with vigorous shaking until the OD 600 was about 1.0, and the temperature was adjusted to 18 ° C for 24 hours. 100 ml of the induced bacterial liquid was centrifuged and centrifuged at 5000 rpm for 5 minutes. The cells were resuspended in 8 ml of ice-cold PBS, and then ultrasonically lysed, with a power of 200 W and sonication for 15 minutes. The lysate was centrifuged at 12000 g for 10 minutes at 4 ° C to remove insoluble bacterial proteins. Solid NaCl was added to the centrifugation supernatant and dissolved, and the solution concentration was 2 M. The cleavage supernatant was changed from clarification to turbidity at room temperature. After the turbid lysate supernatant was centrifuged at room temperature for 5 minutes at high speed, the supernatant was removed. The precipitate was resuspended by adding pre-cooled PBS, the precipitate was blown off and placed on ice until all the precipitates were dissolved. This protein solution was centrifuged at 12000 g for 5 minutes at 4 ° C to remove the insoluble precipitate. The supernatant obtained by centrifugation was repeatedly salted to agglomerate the protein, centrifuged, the protein precipitate was dissolved, and the step of insoluble precipitation was removed by centrifugation until all the hybrid proteins were removed to obtain a purer target protein (see Fig. 1).

 Example 3: Purification of GST protein by ELP-3C protease

 A single colony of the pGEX-ELP expression host strain BL21 (DE3) containing the recombinant plasmid was inoculated into an Amp+ LB liquid-enriched medium, and cultured at 37 ° C for 225 rpm for 12 hours as a seed fungus. The seed bacteria were inoculated into fresh Amp+ LB rich medium at a ratio of 1:100, and cultured at 37 °C with vigorous shaking until O.D. 600 was about 1.0, 0.5 mM IPTG was added, and expression was induced at 37 ° C for 4 hours. After the bacteria were collected, the solution was lysed by adding solid NaCl to a concentration of 2 M, triggering GST-ELP agglutination and centrifuging to precipitate, using 3C digestion buffer (50 mM Tris, 150 mM NaCl, ImM EDTA, pH 8.0, ImM DTT). Dissolve. ELP-3C protease was added at 1:100 and digested at 5 °C for 16 hours. After digestion, solid NaCl was added to make the solution concentration 2 M, and the ELP and ELP-3C proteases were triggered to agglutinate and centrifuged to remove. The supernatant was collected for purification of GST protein (see Figure 1).

 Example 4: Enzymatic digestion of ELP-3C protease The optimal digestion concentration of GST-ELP fusion protein

 Enzyme digestion of 100 g GST-ELP protein substrate, according to ELP-3C: GST-ELP mass ratio 1: 10, 1: 50, 1: 100, 1: :500, 1: 1000, 1: 5000, 1: 10000 The ELP-3C protease was mixed and cleaved at 5 ° C for 16 hours. After the end of the digestion reaction, SDS-PAGE electrophoresis was performed (see Fig. 2).

 Example 5: Purification of GFP protein by ELP-3C protease

Single colony inoculation of expression host strain BL21 (DE3) containing recombinant plasmid pET23a-ELP-GFP It was cultured in an LB liquid-enriched medium of Amp+ at 37 ° C for 225 rpm for 12 hours as a seed fungus. The seed bacteria were inoculated in fresh Amp + TB medium at a ratio of 1:100, and cultured at 37 ° C with vigorous shaking until the OD 600 was about 1.0, and the temperature was adjusted to 18 ° C for 24 hours. After the bacteria were collected, the solution was lysed, and the solution concentration was 2 M by adding solid NaCl. The ELP-GFP was triggered to agglutinate and centrifuged to precipitate, and 3C digestion buffer (50 mM Tris, 150 mM NaCl, ImM EDTA, pH 8.0, ImM DTT) was used. Dissolve. The ELP-3C protease was added at 1:100 and digested at 5 °C for 16 hours. After digestion, solid NaCl was added to a solution concentration of 2 M, and ELP and ELP-3C proteases were triggered to agglutinate and removed by centrifugation. The supernatant was collected for purification of GFP protein (see Figure 3). When the purified GFP protein was observed under a fluorescence microscope, significant fluorescence was detected (see Figure 4).

 Example 6: Purification of MBP protein by ELP-3C protease

 The single colony of the expression host strain BL21 (DE3) containing the recombinant plasmid pET23a-ELP-MBP was inoculated into Amp+ LB liquid-enriched medium, and cultured at 37 ° C for 225 rpm for 12 hours as a seed fungus. The seed bacteria were inoculated in fresh Amp + TB medium at a ratio of 1:100, and cultured vigorously at 37 ° C until O.D. 600 was about 1.0, and the temperature was adjusted to 18 ° C for 24 hours. After the bacteria were collected, the solution was lysed, and the solution concentration was 2M by adding solid NaCl. The ELP-MBP was triggered to agglutinate and centrifuged to precipitate. The 3C digestion buffer (50 mM Tr, 150 mM NaCl, ImM EDTA, pH 8.0, ImM DTT was used. ) Dissolve. ELP-3C protease was added at 1:100 and digested at 5 °C for 16 hours. After digestion, solid NaCl was added to make the solution concentration 2 M, and the ELP and ELP-3C proteases were triggered to agglutinate and removed by centrifugation. The supernatant was collected as purified MBP protein (see Figure 5).

 Example 7: Purification of TRX protein by ELP-3C protease

The single colony of the expression host strain BL21 (DE3) containing the recombinant plasmid pET23a-ELP-TRX was inoculated into an Amp+ LB liquid-enriched medium, and cultured at 37 ° C for 225 rpm for 12 hours as a seed fungus. The seed bacteria were inoculated in fresh Amp + TB medium at a ratio of 1:100, and cultured at 37 ° C with vigorous shaking until the OD 600 was about 1.0, and the temperature was adjusted to 18 ° C for 24 hours. After the bacteria were collected, the solution was lysed by adding solid NaCl to a concentration of 2 M, triggering GST-GFP to agglutinate and centrifuging to precipitate, using 3C digestion buffer (50 mM Tri, 150 mM NaCl, ImM EDTA, pH 8.0, ImM). DTT) is dissolved. The ELP-3C protease was added at 1:100 and digested at 5 °C for 16 hours. Add solid after digestion NaCl was brought to a solution concentration of 2 M, and ELP and ELP-3C proteases were triggered to agglutinate and removed by centrifugation. The supernatant was collected as purified TRX protein (see Figure 6).

 Example 8: Detection of ELP-3C protease recycling activity

 The ELP-GFP protein was digested with 1 mg of ELP-GFP protein, and ELP-3C protease was added at a mass ratio of ELP-3C:ELP-GFP of 1:100. After digesting at 20 °C for 4 hours, solid NaCl was added to digest the solution to a concentration of 2 M, and the mixture was centrifuged at room temperature for 5 minutes at high speed, and the supernatant sample was retained. The pellet was resuspended in a digestion buffer solution (50 mM Tr, 150 mM NaCl, 1 mM EDTA, pH 8.0, ImM DTT) containing 1 mg of ELP-GFP protein, and continuously soaked to dissolve the precipitate, and then continued for 20°. C was digested for 4 hours. Repeat the above steps 6 times. Each purified GFP protein was subjected to SDS-PAGE protein electrophoresis (see Figure 7). It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of the present invention. Although the present invention is described in detail with reference to the preferred embodiments, those skilled in the art The technical solutions of the present invention are modified or equivalently substituted without departing from the spirit and scope of the technical solutions of the present invention.

Claims

A method for purifying expression and purification of a tag-free recombinant protein, comprising the steps of: (1) a gene encoding a fusion protein of a target protein gene and an ELP-containing fusion partner, and expressing the fusion protein A, Wherein the fusion protein A has at least one protease cleavage site at the junction of the ELP and the ELP-containing fusion partner;  (2) mixing the fusion protein A obtained in the step (1) with a fusion protein B containing ELP, and the fusion protease B can act on the restriction site of the fusion protein A, but does not act on itself;  (3) allowing the mixture of step (2) to interact, wherein fusion protein B degrades fusion protein A into an ELP-containing fusion partner and a target protein;  (4) changing at least one of the temperature, the salt ion concentration or the pH of the mixed solution of the step (3), and after the aggregate is present, it is centrifuged, and the supernatant solution is taken as the purified target protein. The method of expressing and purifying a tag-free recombinant protein according to claim 1, wherein the nucleotide sequence of the monomer of the ELP is as shown in SEQ ID NO. The method for purifying expression and purification of a tag-free recombinant protein according to claim 1, wherein the ELP is a tetramer, a pentamer, a hexamer, a heptamer, and an eight of an ELP monomer. Polymer or 9-mer. The method for expression and purification of a tag-free recombinant protein according to claim 1, wherein the fusion protease B comprises an ELP-3C or ELP-SUMO protease. 5. A label-free recombinant protein, which is obtained by the following method: (1) a gene encoding a fusion protein of a target protein gene and an ELP-containing fusion partner, and expressing the fusion protein C, wherein The fusion protein C in the ELP and the melt containing the ELP At least one protease digestion site is present at the junction of the partner;  (2) mixing the fusion protein C obtained in the step (1) with a fusion protein D containing ELP, and the fusion protease D can act on the cleavage site of the fusion protein C, but does not act on itself;  (3) allowing the mixture of step (2) to interact, wherein fusion protein D degrades fusion protein C into an ELP-containing fusion partner and a target protein;  (4) changing at least one of the temperature, the salt ion concentration or the pH of the mixed solution of the step (3), and after the aggregate is present, it is centrifuged, and the supernatant solution is taken as the purified target protein. The method for purifying expression and purification of a tag-free recombinant protein according to claim 5, wherein the nucleotide sequence of the monomer of the ELP is as shown in SEQ ID NO. The method for purifying expression and purification of a tag-free recombinant protein according to claim 5, wherein the ELP is a tetramer, a pentamer, a hexamer, a heptamer, and an eight of an ELP monomer. Polymer or 9-mer. The method of expressing and purifying a tag-free recombinant protein according to claim 5, wherein the fusion protease D comprises an ELP-3C or ELP-SUMO protease. 9. An ELP-containing fusion protein, characterized in that the fusion protein comprises an elastin-like polypeptide (ELP) and at least one protein or polypeptide of interest, the protein or polypeptide of interest being capable of forming a fusion protein with ELP. Formally expressing, the fusion protein can be in a dissolved or precipitated state at at least one different temperature, different salt ion concentration or different pH values, and the fusion protein can be adjusted by temperature, salt ion Concentration or pH is worth purifying. The fusion protein according to claim 9, wherein the nucleotide sequence of the monomer of the elastin-like polypeptide is as shown in SEQ ID NO. The fusion protein according to claim 9, wherein the elastin-like polypeptide comprises a tetramer, a pentamer, a hexamer, a heptamer, an octamer or a 9-mer of a monomer. . 12. A method for expressing a purified fusion protein comprising ELP, comprising the steps of: (1) connecting a gene of a target protein and a fusion partner comprising an ELP to a fusion protein gene, and expressing the fusion protein E;  (2) changing at least one of the temperature, the salt ion concentration or the pH value of the fusion protein E obtained in the step (1), and after centrifugation, the obtained aggregate is an ELP-containing fusion protein. The method of expressing an ELP-containing fusion protein according to claim 12, wherein the nucleotide sequence of the monomer of the ELP is as shown in SEQ ID NO. The method for expressing a purified ELP-containing fusion protein according to claim 12, wherein the ELP is a tetramer, a pentamer, a hexamer, a heptamer, an octamer of an ELP monomer. Body or 9-mer. The method for expressing a purified ELP-containing fusion protein according to claim 12, wherein the fusion protease E comprises an ELP-3C or ELP-SUMO protease. A kit for purification of protein expression, characterized in that the kit comprises an ELP-containing plasmid for preparing a fusion protein F and a fusion protein G, and the fusion protein G contains A target protein or polypeptide, which is capable of cleaving the fusion protein G to obtain a protein or polypeptide of interest. The kit for purification of protein expression according to claim 16, wherein the fusion protein F comprises an ELP-3C or an ELP-SUMO protease. The kit for purification of protein expression according to claim 16, wherein the fusion protein F is capable of cleaving the fusion protein G into an ELP and a target protein. A recyclable enzyme having an immobilized enzyme property, characterized in that the enzyme is a fusion protease comprising ELP, and the ELP can be in a dissolved or precipitated state depending on temperature, salt ion concentration or pH. The recyclable enzyme having an immobilized enzyme property according to claim 19, wherein the nucleotide sequence of the monomer of the ELP is as shown in SEQ ID NO. The recyclable enzyme having immobilized enzyme characteristics according to claim 19, wherein the ELP is a tetramer, a pentamer, a hexamer, a heptamer of an ELP monomer, Octameric or nonamer. The recyclable enzyme having immobilized enzyme characteristics according to claim 19, wherein the fusion protease comprises ELP-3C or ELP-SUMO protease. A method for cyclic enzymatic purification using a protease comprising ELP, comprising the steps of:  (1) mixing the fusion protein with the substrate to carry out a digestion reaction;  (2) adjusting the temperature, salt ion concentration or pH value of the solution after the digestion reaction, and centrifuging after the aggregate is present;  (3) The aggregate obtained after centrifugation is added to the enzymatic hydrolysate, and after it is dissolved, the substrate is added, and the enzyme digestion reaction is carried out, and the step (2) is repeated. A method for cyclic enzymatic cleavage using a protease comprising ELP according to claim 23, wherein the substrate in the step (1) is an ELP fusion protein containing a protein of interest.