KR101300672B1 - Method for producing soluble foreign protein using specific intracellular cleavage system - Google Patents

Abstract

The present invention relates to a method for producing a foreign protein, characterized in that the cleavage of the water-soluble expression promoting protein from the fusion protein in the cell using a sequence specific protein cleavage enzyme. Using a method for producing a foreign protein using intracellular cleavage of the fusion protein according to the present invention can efficiently remove the fusion tag from the fusion protein, thereby producing a water-soluble foreign protein.

Description

Method for producing soluble foreign protein using specific intracellular cleavage system

The present invention relates to a method for producing a foreign protein, characterized in that the cleavage of the water-soluble expression promoting protein from the fusion protein in the cell using a sequence specific protein cleavage enzyme.

E. coli is a host that is still the most popular host for the production of recombinant proteins because of its simple operation, fast growth rate, and high production efficiency, but many heterologous proteins expressed in E. coli are insoluble aggregates. body) has a problem that is expressed in the form. In order to overcome the problem of expression of aggregated proteins in Escherichia coli, an approach for fusing a water-soluble enhancing tag to the N-terminus of the aggregate-prone protein has been taken (Braun P et al., High throughput protein production). for functional proteomics.Trends Biotechnol. (2003), 21: 383-388). To date, various water-soluble enhancement tags such as maltose binding protein, glutathione S-transferase, thiorredoxin, SUMO, NusA and the like are well known.

This fusion approach is indispensable for rapidly producing large quantities of functionally active proteins in the post-genome era. For structure-function studies of most proteins, the fused tags should be removed using sequence-specific protein proteases such as enterokinase, Factor Xa, thrombin, and the like. However, these protein cleavage enzymes have the inherent activity of cutting out unwanted sites of the protein and are also expensive (Choi SI et al., Recombinant enterokinase light chain with affinity tag: expression from Saccharomyces cerevisiae and its utilities). in fusion protein technology.Biotechnol Bioeng. (2001), 75: 718-724). Accordingly, in order to utilize the fusion approach, a recombinant protein cleavage enzyme having low specificity and specificity capable of accurately recognizing a sequence is required. For example, enterokinase (DDDDK /) produced from Pichia Pastoris and the E. coli cytoplasm is commercially available. Enterokinase works very efficiently and can produce proteins with the original N-terminal form when cleaved. There is an advantage. However, in spite of these advantages, in some cases, the enzyme is not only non-specifically cleaved but also expensive to cut the fusion protein. In addition, tobacco etch virus (TEV) protein cleavage enzyme specifically cleaves between Q and G (or S) of the recognized sequence ENLYFQ / G (or S), and other amino acid residues are substituted in place of G or S. Cutting is also possible. This feature makes it possible to make target proteins with native N-terminus (Waugh DS et al., The P1 'specificity of tobacco etch virus protease. Biochem. Biophys. Res. Commun. (2002), 294: 949 -955). However, in order to obtain the target protein from the fusion protein in vitro, the process of removing the fusion tag by sequence specific protein cleavage enzyme and then separating and purifying the target protein separated through cleavage is time and cost. It is consumed a lot. To overcome this problem, a method of cleaving a fusion tag in a cell through co-expression of sequence-specific protein cleavage enzymes such as TEV protease, ubiquitin-specific protease, and SUMO-specific protease has been applied (Kapust RB et al. Controlled intracellular processing of fusion proteins by TEV protease.Protein Expr.Purif. (2000), 19: 312-318). However, when ubiquitin-specific protease and SUMO-specific protease are used, only their respective fusion tags can be cut. TEV protein cleavage enzymes can be applied to any fusion tag but have a low water solubility.

Therefore, the present inventors have tried to provide a way to remove the fusion tag using TEV protein cleavage enzyme having high water solubility and activity, by simultaneously expressing the soluble protein and the target protein of the water-soluble expression promoting protein and TEV protein cleavage enzyme The present invention was completed by confirming that the fusion tag was cut in the cell to efficiently produce the water-soluble target protein.

The object of the present invention is to prepare a first expression vector in which a gene encoding a water-soluble expression promoting protein and a gene encoding a foreign protein are linked to a first linker;

(b) preparing a second expression vector in which a gene encoding a water-soluble expression promoting protein and a gene encoding a protein cleavage enzyme are linked to a second linker;

(c) preparing a transformant by introducing the first and second expression vectors into a host cell; And

(d) culturing the transformant to induce the expression of a recombinant fusion protein and to provide a method for producing a foreign protein comprising the step of cleaving a water-soluble expression promoting protein.

In order to achieve the above object,

(a) preparing a first expression vector in which a gene encoding a water-soluble expression promoting protein and a gene encoding a foreign protein are linked to a first linker;

(b) preparing a second expression vector in which a gene encoding a water-soluble expression promoting protein and a gene encoding a protein cleavage enzyme are linked to a second linker;

(c) preparing a transformant by introducing the first and second expression vectors into a host cell; And

(d) culturing the transformant to induce the expression of the recombinant fusion protein and provide a method for producing a foreign protein comprising the step of cleaving a water-soluble expression promoting protein.

In the present invention, the first linker and the second linker may be characterized in that the sequence is cleaved by the protein cleavage enzyme, wherein step (d) is by self-cutting from the second recombinant fusion protein The isolated protein cleavage enzyme may cleave the first linker of the first recombinant fusion protein, thereby cleaving the water-soluble expression promoting protein from the first recombinant fusion protein. In addition, the expression of the recombinant fusion protein of step (d) may be characterized in that occurs at 20 ℃, wherein, the water-soluble expression promoting protein of the recombinant fusion protein may be characterized in that the LysRS, the protein cleavage The enzyme may be characterized as being water soluble.

In the present invention, the first linker is a sequence cleaved by the protein cleavage enzyme and the second linker may be characterized in that the sequence is not cleaved by the protein cleavage enzyme, wherein step (d) The protein cleavage enzyme of the second recombinant fusion protein cleaves the first linker of the first recombinant fusion protein, thereby cutting the water-soluble expression promoting protein from the first recombinant fusion protein. In addition, the expression of the recombinant fusion protein of step (d) may be characterized in that it occurs at 37 ℃, wherein the amino acid sequence of the second linker may be characterized in that represented by DDDDK.

In the present invention, when the recombinant fusion protein to which the water-soluble expression promoting protein and the protein cleavage enzyme is linked at 20 ° C. is expressed, the protein cleaving enzyme is expressed in water so that the protein cleaving enzyme is a water-soluble expression promoting protein and protein cleaving enzyme. The linker is being cut. On the other hand, at 37 ° C., it was confirmed that the protein cleavage enzyme was expressed insoluble, so that the linker was identified by the DDDDK sequence, thereby indicating that the protein cleavage enzyme was soluble in the form of a fusion protein without cleaving the linker by the protein cleavage enzyme. Confirmed.

In another embodiment, the present invention provides a foreign protein having a tag in which a water-soluble expression promoting protein is cleaved from a recombinant fusion protein in which a water-soluble expression promoting protein and a foreign protein are fused by a protein cleavage enzyme.

In another embodiment,

(a) preparing an expression vector linking a gene encoding a water-soluble expression promoting protein, a protein cleavage enzyme recognition site, and a gene encoding a sequence specific protein cleavage enzyme cutting the protein cleavage recognition site;

(b) preparing a transformant by introducing the expression vector into a host cell; And

(c) culturing the transformant to induce expression of and obtain a recombinant fusion protein.

In another embodiment, the present invention provides a recombinant fusion protein in which a water-soluble expression promoting protein and a protein cleavage enzyme are fused.

In the present invention, the water-soluble expression promoting protein may be characterized as LysRS or RBD, the foreign protein may be characterized as BMP-2, the protein cleavage enzyme may be characterized as TEV. . In addition, the host cell may be characterized in that E. coli.

The term "foreign protein" used in the present invention is a protein that a person skilled in the art intends to produce in large quantities, and means any protein that can be expressed in a transformant by inserting a nucleotide encoding the protein into a recombinant expression vector.

As used herein, the term "recombinant fusion protein" refers to a protein in which another protein is linked or another amino acid sequence is added to the N terminus or C terminus of the sequence of the original foreign protein.

As used herein, the term "expression vector" is a linear or circular DNA molecule consisting of fragments encoding a polypeptide of interest operably linked to additional fragments provided for transcription of the expression vector. Such additional fragments include promoter and termination coding sequences. Expression vectors also include one or more replication initiation points, one or more selection markers, and the like. Expression vectors are generally derived from plasmid or viral DNA or contain elements of both.

As used herein, the term “water soluble expression promoting protein” refers to a peptide that is reported to be able to express a fusion protein in a water-soluble form and includes GST (Glutathione S transferase), maltose binding protein, ubiquitin, thioredoxin, and the like. In the present invention, the present invention is not limited thereto, and any known water-soluble expression promoting protein is used without limitation.

The term "linker" used in the present invention functions to link two proteins. When one of the two proteins is expressed as a water-soluble protein, the linker is cleaved by a protein cleavage enzyme, and when expressed as an insoluble protein. It is characterized in that the linker is not cut.

Using a method for producing a foreign protein using intracellular cleavage of the fusion protein according to the present invention can efficiently remove the fusion tag from the fusion protein, thereby producing a water-soluble foreign protein.

Figure 1 confirms the intracellular cleavage of TEV protein cleavage enzymes.
Figure 2 confirms the expression of LysRS-e-TEV protease fusion protein (20 ℃).
Figure 3 shows the results of SDS-PAGE analysis of LysRS-TEV protease.
Figure 4 confirms the simultaneous expression of LysRS-TEV protease and RBD-BMP-2 and the cleavage of intracellular fusion proteins.
Figure 5 shows the intracellular cleavage and soluble BMP-2 of the RBD-BMP-2 fusion protein.

Protein expression Plasmid  making

In this study, pGE-LysRS expressing plasmid was used as a basic framework for the production of protein expressing plasmid (Choi SI et al., Protein solubility and folding enhancement by interaction with RNA, PLoS ONE (2008), 3: e2677). The expression cassette for fusion to LysRS consists of the T7 promoter-LysRS-TEV protease (or enterokinase) recognition site-multi cloning site (KpnI-BamHI-EcoRV-SalI-HindIII). Genes expressing TEV protein cleavage enzymes were characterized by PCR amplification using 5'-TAC CTG GGA TCC GGA GAA AGC TTG TTT AAG GG-3 ', 5'-CAG CTT GTC GAC CTA ATT CAT GAG TTG AGT CG-3' primers. Got through. PCR products were cut with BamHI / SalI restriction enzyme and cloned into BamHI / SalI of pGE-LysRS containing the TEV protein cleavage site. As a result, the result was pGE-LysRS-TEV protease. In addition, pGE-LysRS-e-TEV protease was also prepared by cloning the above PCR DNA product into BamHI / SalI of pGE-LysRS containing an enterokinase recognition site. Both plasmids contain the antibiotic Ampicillin resistance gene as a selection marker. PGE-RBD-BMP-2 plasmid was also prepared to make RBD-BMP-2 fusion protein. The linker peptide between RBD and BMP-2 contains a TEV cleavage recognition site, followed by six histidine (hexa-histine) tags that allow the production of native BMP-2 through cleavage of the TEV cleavage enzyme. pGE-RBD-BMP-2 plasmids were constructed via overlapping PCR. DNA capable of expressing RBD is 5'-AAT TTC TAG AAA TAA TTT TGT TTA ACT TTA AGA AGG AGA TAT ACA TAA T-3 'and 5'-CTC CTT CTC TCG CAC GTG GTA GTG GTA GTG GTA CTT TTG GAC- Obtained by PCR amplification using a 3 'primer. DNA fragments expressing BMP-2 include 5'-CAT CAC CAT GAA AAC CTG TAT TTT CAG GGC CAA GCC AAA CAC AAA CAG-3 'and 5'-AAT TGG TAC CTT AGC GAC AGC CAC AAC CCT C-3' primers Obtained via PCR amplification. The two previous PCR results were thawed from agarose gel. DNA fragments expressing the RBD-BMP-2 fusion protein were obtained by overlapping PCR using the dissolved DNA fragments as template, the second and third primers above. The resulting DNA fragment was cut with XbaI / KpnI restriction enzyme and cloned into the vector with the same XbaI / KpnI site to the final pGE-RBD-BMP-2 plasmid. pGE-RBD-BMP-2 has an antibiotic kanamycin resistance gene as a selection marker.

Protein expression

BL21 (DE3) containing pGE-LysRS-TEV protease or pGE-LysRS-e-TEV protease was overnight at 37 ° C in LB containing 200 μg / ml antibiotic ampicillin and 34 μg / ml antibiotic chloramphenicol. Incubated. 1 ml of cells were diluted in fresh 17 ml of LB under the same antibiotic conditions and incubated at 30 ° C. until an OD ₆₀₀ value of 0.5 was obtained, followed by incubation at 20 ° C. for 5 hours after treatment with 1 mM IPTG. 0.3 ml of PBS was added to the cell harvest corresponding to 10 ml of the culture solution, mixed evenly, and cell ultrasonic grinding was performed. Total cell lysates, solubles, and insolubles divided by centrifugation were analyzed by SDS-PAGE (Kim CW et al., N-terminal domains of native multidomain proteins have the potential to assist de novo folding of their downstream domains in vivo by acting as solubility enhancers, Protein Sci. (2007), 16: 635-643). The previous protein expression method was applied to the expression of BMP-2 and RBD-BMP-2 as it was, except that 7.5 μg / ml kanamycin was used instead of the antibiotic ampicillin. Simultaneous expression of RBD-BMP-2 and LysRS-TEV protease was performed in the same manner except for the use of the antibiotic 200 μg / ml ampicillin and 7.5 μg / ml kanamycin.

Western blot  Analysis method

The expressed proteins were separated via SDS-PAGE and then transferred to PVDF membranes (Membrane, Millipore). The membrane was blocked for 1 hour with 5% skim milk and washed three times with TBST buffer containing Tris-buffered saline (pH7.6) and 0.05% Tween 20. The following anti-BMP-2 antibody (1: 500) was treated overnight at 4 ° C. After three washes, anti-mouse antibody (1: 20,000) with horseradish peroxidase was reacted for 45 minutes at room temperature. After three washes, the membrane was treated with WEST-ZOL (Intron, Korea) and the film was developed and fixed.

Hereinafter, the present invention will be described in detail by the following examples. However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

TEV  Protein cleavage enzymes ( protease Intracellular expression)

In order to construct an intracellular cleavage system in Escherichia coli, TEV protein cleavage enzyme having precise specificity and cleavage activity was selected. However, TEV protein protease itself has a strong aggregation tendency when expressed alone in E. coli. For water-soluble expression of TEV protein cleavage enzyme, it was fused to the C-terminus of E. coli lysyl tRNA synthetase (LysRS), a powerful water solubility enhancing tag. Previously, LysRS has been reported to strongly soluble in various agglutination proteins (Choi SI et al., Protein solubility and folding enhancement by interaction with RNA, PLoS ONE (2008), 3: e2677). LysRS-TEV protease contains a standard sequence site recognized by TEV cleavage enzyme at the linking peptide site between LysRS (56 kDa) and TEV cleavage enzyme (27 kDa). TEV cleavage enzyme was separated from the LysRS tag and was found to be mostly water soluble at 20 ° C. The fusion form of LysRS-TEV protease (83 kDa) was almost quantitatively cleaved on SDS-PAGE and converted to Tev (27 kDa) and LysRS (56 kDa) (FIG. 1).

On the other hand, at a high induction temperature such as 37 ℃, a substantial portion of the TEV cleavage cleaved off was observed as insoluble. Another LysRS-TEV protease (hereinafter referred to as 'LysRS-e-TEV protease'), which was converted to the DDDDK sequence of TEV cleavage enzyme, was constructed, and the fusion protein was highly soluble and expressed in an uncut fusion protein state. (FIG. 2). These results suggest that LysRS-Tev protease is expressed in water-soluble and active form, recognizes cleavage recognition sites, and induces cleavage itself to convert Tev and LysRS.

BMP Expression of the -2 fusion protein

To enhance the water solubility of BMP-2, BMP-2 was fused to the C-terminus of the human lysyl tRNA synthetase N-terminal domain (RBD) in the form of RBD-BMP-2. While BMP-2 itself alone was almost completely insoluble in water, most of RBD-BMP-2 was expressed in water-soluble form and the expression level of RBD-BMP-2 (22.4 kDa) was also high (FIG. 3). These results indicate that RBD is a suitable water soluble enhancement tag for the expression of BMP-2 in E. coli.

Intracellular Cleavage of Fusion Proteins

RBD-BMP-2 and LysRS-TEV protease were simultaneously expressed to monitor intracellular cleavage of the fusion protein in E. coli. 4 shows that TEV cleavage enzyme produced by autocleavage from LysRS-TEV protease efficiently cleaved RBD to produce authentic BMP-2. TEV cleavage separated on SDS-PAGE overlapped with RBD-BMP-2. The results of the separated RBD on SDS-PAGE clearly showed that the fusion tag was efficiently removed in the cell by TEV cleavage enzyme (FIG. 4). The finally released BMP-2 (12.8 kDa) was almost always found to be water soluble, which was also verified by additional Western blot experiments (FIG. 5).

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

Claims (22)

(a) preparing a first expression vector in which a gene encoding a first water soluble expression promoting protein and a gene encoding a foreign protein are linked to a first linker;
(b) preparing a second expression vector in which a gene encoding a second water soluble expression promoting protein and a gene encoding a protein cleavage enzyme are linked to a second linker;
(c) preparing a transformant by introducing the first and second expression vectors into a host cell; And
(d) expressing the first recombinant fusion protein with the first expression vector and expressing the second recombinant fusion protein with the second expression vector.
(e) cleaving the first water soluble expression promoting protein from the first recombinant fusion protein by the protein cleavage isolated by autocleavage from the second recombinant fusion protein cleaving the first linker of the first recombinant fusion protein. Wherein the first linker is a sequence cleaved by the protein cleavage enzyme.


delete delete The method of claim 1,
Expression of the recombinant fusion protein of step (d) is a method for producing a foreign protein, characterized in that at 20 ℃.
5. The method of claim 4,
The method for producing a foreign protein, characterized in that the water-soluble expression promoting protein of the first recombinant fusion protein is LysRS.
The method of claim 1,
The protein cleavage enzyme is a water-soluble method for producing a foreign protein, characterized in that.
The method of claim 1,
Wherein said first linker is a sequence cleaved by said protein cleavage enzyme and said second linker is a sequence not cleaved by protein cleavage enzyme.
delete 8. The method of claim 7,
Expression of the recombinant fusion protein of step (d) is a method for producing a foreign protein, characterized in that at 37 ℃.
The method of claim 9,
A method for producing a foreign protein, characterized in that the amino acid sequence of the second linker is represented by DDDDK.
The method of claim 1,
The method of producing a foreign protein, characterized in that the water-soluble expression promoting protein of the second expression vector is LysRS or RBD.
The method of claim 1,
The foreign protein is a method for producing a foreign protein, characterized in that the BMP-2.
The method of claim 1,
The protein cleavage enzyme is a method for producing a foreign protein, characterized in that the TEV.
The method of claim 1,
The host cell is E. coli production method of the foreign protein, characterized in that.
delete delete delete delete delete delete delete delete

Images