Method for producing TLR5 agonist protein

Abstract

The present disclosure relates to a method for producing a TLR5 agonist protein. According to the present disclosure, the TLR5 agonist protein can be easily separated and purified after biotechnological production. In particular, the fusion partner used for separation and purification is effectively removed so as to minimize the possibility of inhibiting binding to TLR5 and inducing an immune response by the fusion partner.

Claims

1. A method of producing a Toll-like receptor-5 (TLR5) agonist protein, comprising the steps of: (a) producing a vector that comprises (i) a fusion nucleic acid that encodes the TLR5 agonist protein that comprises the amino acid sequence of SEQ ID NO: 2 and the amino acid sequence of SEQ ID NO: 4; (ii) a nucleic acid that encodes ubiquitin, and (iii) a nucleic acid that encodes a tag for purification; (b) producing a fusion protein by transforming a host with the vector in step (a) and then expressing the fusion nucleic acid; (c) recovering the fusion protein by binding the fusion protein of step (b) to a column that the tag for purification binds; and (d) recovering the TLR5 agonist protein by treating the recovered fusion protein of step (c) with a ubiquitin cleavage enzyme to cleave the site to which the ubiquitin binds, wherein the ubiquitin cleavage enzyme is ubiquitin-specific protease (USP) or ubiquitin C-terminal hydrolase (UCH), wherein the fusion nucleic acid has a nucleotide sequence as set forth in SEQ ID NO: 7.

2. The method of claim 1, wherein the TLR5 agonist protein comprises a polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2 and the polypeptide having the amino acid sequence as set forth in SEQ ID NO: 4 that are bound via a linker as set forth in SEQ ID NO: 6.

3. The method of claim 1, wherein the nucleic acid encoding the ubiquitin is linked to the 5 terminal of the nucleic acid encoding the TLR5 agonist protein and the nucleic acid encoding the tag for purification is linked to the 5 terminal of the nucleic acid encoding the ubiquitin.

4. The method of claim 1, wherein the host is Escherichia coli.

5. The method of claim 1, wherein the treatment of the ubiquitin cleavage enzyme of step (d) is performed in the state that the fusion protein binds to the column.

6. The method of claim 1, wherein the treatment of the ubiquitin cleavage enzyme of step (d) is performed after the fusion protein is eluted from the column.

7. The method of claim 1, wherein the ubiquitin comprises an amino acid sequence cleaved by the ubiquitin cleavage enzyme.

8. The method of claim 1, wherein the tag for purification is a histidine tag (His-tag) or a lysine tag formed by six consecutive bindings of lysines.

9. The method of claim 1, wherein in the step (c), the fusion protein is recovered by unfolding the produced fusion protein, then binding the fusion protein to the column, and then refolding the fusion protein.

10. The method of claim 1, wherein in the step (c), the fusion protein is recovered by unfolding the produced fusion protein, then refolding the fusion protein, and then binding the fusion protein to the column.

Description

DESCRIPTION OF DRAWINGS

(1) FIG. 1 is a schematic diagram illustrating a gene sequence of K6UbKMRC011.

(2) FIG. 2 is a base sequence of the K6UbKMRC011 gene.

(3) FIG. 3 is an amino acid sequence of K6UbKMRC011.

(4) FIG. 4 is a schematic diagram illustrating pAP-K6UbKMRC011 plasmid.

(5) FIG. 5 shows analysis results of the expression levels and solubility of K6UbKMRC011 expressed in E. coli.

(6) FIG. 6 shows analysis results of SDS-PAGE of solid phase refolded K6UbKMRC011.

(7) FIG. 7 shows analysis results of cleavage by USP1 of solid phase refolded K6UbKMRC011.

(8) FIG. 8 shows results of the isolation and purification of the TLR5 agonist protein through on-column cleavage.

(9) FIG. 9 shows analysis results of cleavage of K6UbKMRC011 according to application of various ubiquitin cleavage enzymes.

BEST MODE

(10) Hereinafter, the present disclosure will be described in more detail with reference to the following Examples and Experimental examples. However, the scope of the present disclosure is not limited to the following Examples, and includes modifications of equivalent technical ideas.

Example 1: Preparation of K6UbKMRC011-Expressing E. coli

(11) (1) Gene Synthesis of K6UbKMRC011

(12) The polypeptide having the amino acid sequence as set forth in SEQ ID NO: 2 (encoded by the nucleic acid sequence as set forth in SEQ ID NO: 1) and the polypeptide having the amino acid sequence as set forth in SEQ ID NO: 4 (encoded by the nucleic acid sequence as set forth in SEQ ID NO: 3) are bound via the polypeptide (linker) having the amino acid sequence as set forth in SEQ ID NO: 6 (encoded by SEQ ID NO: 5), and then are folded to form the D0 domain and D1 domain capable of binding to TLR5.

(13) In the present Example, the fusion gene, K6UbKMRC011, is synthesized by binding the gene encoding the lysine tag (K6) formed by six consecutive bonds of lysines and the gene encoding the ubiquitin (Ub) to so-called TLR5 agonist protein (KMRC011) encoding gene in which the nucleic acid sequence as set forth in SEQ ID NO: 1 and the nucleic acid sequence as set forth in SEQ ID NO: 3 are combined with mediation by a nucleic acid sequence (linker) as set forth in SEQ ID NO: 5. K6Ub portion and KMRC011 portion were separately amplified using a primary polymerase chain reaction, and the K6Ub portion and the KMRC011 portion were connected through a secondary polymerase chain reaction to synthesize the entire gene of K6UbKMRC011 (See FIG. 1).

(14) The K6Ub portion was amplified using the primers of K6Ub-NdeI-F and KMRC011-R2 and plasmid pUC18-K6Ub containing K6Ub gene as a template. The KMRC011 portion was amplified using primers KMRC011-F2 and KMRC011-BamHI-R2 and the plasmid pUC57-KMRC011 containing the genes of SEQ ID NO: 1, SEQ ID NO: 3, and SEQ ID NO: 5 as a template (See Table 1 below).

(15) TABLE-US-00001 TABLE1 Basesequenceofprimerusedinpolymerasechain reactionforK6UbKMRC011genesynthesis No. Primer'sname Primersequence 1 K6Ub-NdeI-F Aatcatatgaagaaaaaaaagaaaaagca gattttcgtcaagact (SEQIDNO:10) 2 KMRC011-R2 Tgttgataacctgcgccatgccacctctt agccttagc (SEQIDNO:11) 3 KMRC011-F2 Gctaaggctaagaggtggcgcgcaggtta tcaaca (SEQIDNO:12) 4 KMRC011-BamHI- Attggatccttagcgcagcaggctcag R2 (SEQIDNO:13)

(16) The entire gene of K6UbKMRC011 was synthesized by overlap-extension PCR using the primers K6Ub-NdeI-F and KMRC011-BamHI-R and K6Ub and KMRC011, products of the first polymerase chain reaction, as a template. The nucleotide sequence of the synthesized K6UbKMRC011 gene and the amino acid sequence deduced from the nucleotide sequence are shown in FIG. 2 (SEQ ID NO: 7) and FIG. 3 (SEQ ID NO: 8), respectively.

(17) (2) Production of K6UbKMRC011 Expression Plasmid

(18) The amplified K6UbKMRC011 gene whose expression was regulated by IPTG was inserted into the NdeI and BamHI restriction enzyme sites of the pAP (owned by AP Technology) expression plasmid containing the tac promoter, thereby constructing the pAP-K6UbKMRC011 plasmid (See FIG. 4).

(19) (3) Production of Escherichia coli Expressing K6UbKMRC011

(20) Escherichia coli TG1/pAP-K6UbKMRC011 expressing K6UbKMRC011 was finally produced by transforming the pAP-K6UbKMRC011 plasmid into Escherichia coli TG1.

Example 2: Production of K6UbKMRC011 Using the Recombinant Escherichia coli Produced in Example 1

(21) The production ability of K6UbKMRC011 was confirmed using Escherichia coli TG1/pAP-K6UbKMRC011 prepared in the above Example. For the cultivation, medium of yeast extract 5 g/L, tryptone 10 g/L, and sodium chloride 10 g/L was used. The cells were cultured in a 500 mL baffled flask at 37 C. and 200 rpm until the absorbance at 600 nm reached 0.5-1.0, then IPTG was added to 1 mM so that the expression of K6UbKMRC011 was induced, and then cultured for 4 hours.

(22) The K6UbKMRC011-expressing Escherichia coli, which had been calibrated in 15 at an absorbance of 600 nm, was disrupted using an ultrasonicator, followed by dividing into soluble and non-soluble fractions under conditions of centrifugation at 12,000 rpm for 20 minutes, and followed by confirming the expression level and the availability of K6UbKMRC011 through SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) analysis.

(23) The intracellular expression level of the expressed K6UbKMRC011 was confirmed to be about 21% through a densitometer analysis (See FIG. 5). K6UbKMRC011 was observed in mostly the insoluble fraction, and thus K6UbKMRC011 expressed in Escherichia coli was confirmed to be an insoluble inclusion body.

Example 3: Method of Refolding Solid Phase of K6UbKMRC011 Produced in Example 2

(24) Since K6UbKMRC011 expressed in Escherichia coli was expressed as an insoluble inclusion body, solid-phase refolding was performed to recover biological activity. Escherichia coli, which was disrupted with an ultrasonicator, was centrifuged (12,000 rpm, 20 minutes), and the inclusion body of the resulting K6UbKMRC011 were solubilized with buffer A (pH 7.0, 50 mM sodium phosphate, 8 M urea). Then it was injected to the column filled with a cation exchange resin (SP Sepharose FF, GE Healthcare) equilibrated with Buffer A, and thus the solubilized K6UbKMRC011 bind to the cation exchange resin by electrostatic attraction. Buffer B (pH 7.0, 50 mM sodium phosphate) was injected into the above-mentioned column to remove the urea, and thus K6UbKMRC011 binding to the cation exchange resin was induced to be re-folded. Buffer C (pH 7.0, 50 mM sodium phosphate, 1 M NaCl) was injected into the above-mentioned column to allow the refolded K6UbKMRC011 to be eluted from the cation exchange resin, and each fraction was analyzed by SDS-PAGE (See FIG. 6).

(25) The eluted refolded K6UbKMRC011 was confirmed to have a soluble form. In order to confirm whether it is cleaved by USP1 (ubiquitin-specific protease 1), USP1 was added to the eluted refolded K6UbKMRC011 to a concentration of 10 mg/L and then was reacted at 37 C. for 12 hours.

(26) As a result of SDS-PAGE analysis, it was confirmed that most of the refolded K6UbKMRC011 was cleaved by USP1 and separated into K6Ub and KMRC011 (See FIG. 7). At this time, it was also confirmed that USP1 treatment did not decompose the internal structure of the KMRC011 protein.

(27) In addition, the N-terminal sequence of the separated KMRC011 protein was confirmed through N-terminal sequencing. As a result, it was confirmed that the K6Ub was separated to be departed while the N-terminal amino acid sequence A-Q-VI-N of the KMRC011 protein was maintained intact.

(28) From the above results, it was confirmed that the KMRC011 protein of the present disclosure was not degraded by treatment with USP1, a ubiquitin cleavage enzyme, and that K6Ub was separated while maintaining the N-terminal thereof intact.

(29) When the fusion partner is removed from the target protein, the enzyme used for the removal should not affect the structure of the target protein. The ubiquitin cleavage enzyme of the present disclosure has no influence on the structure of the target protein, KMRC011.

Example 4: Method for Separating and Purifying KMRC011 from Solid-Phase Refolded K6UbKMRC011 in Example 3on Column Cleavage

(30) The USP1 solution (10 mg/L) was purposely circulated to separate and purify only KMRC011 while the solid-phase refolded K6UbKMRC011 electrostatically bound to the cation exchange resin without elution thereof (See FIG. 8).

(31) The separated and purified KMRC011 was identified as AQVINTNSLS in the amino terminal sequence analysis. Thus, it was confirmed that the USPI clearly cleaved the immediate side of K6Ub of K6UbKMRC011 so that only KMRC011 was separated and purified.

Example 5: Analysis Results of Cleavage of K6UbKMRC011 by Application of Various Ubiquitin Cleavage Enzymes

(32) In the present embodiment, various kinds of ubiquitin cleavage enzymes other than USP1 described in Example 3 were applied to K6UbKMRC011 to confirm whether the target protein KMRC011 was completely separated from K6Ub.

(33) The various ubiquitin cleavage enzymes shown in Table 2 below were added to the refolded K6UbKMRC011 in soluble form obtained in Example 3, so as to have a concentration of 10 mg/L and reacted at 37 C. for 12 hours.

(34) TABLE-US-00002 TABLE 2 Generic Catalog name Name of Product Manufacturer No. Lot No. of Enzyme UBP AP Technology P3S021-04 USP1 Corp. Recombinant Boston Biochem, E-504 09341414A USP2 Human USP2 Inc. Catalytic Domain Recombinant Boston Biochem, E-592 28570115A USP10 Human His6 Inc. USP10 UCH-L3, human Boston Biochem, E-325 34709013 UCH-L3 recombinant Inc. Recombinant Boston Biochem, E-327 25967314A UCH-L5/ Human UCH-L5/ Inc. UCH37 UCH37

(35) The experimental results are illustrated in FIG. 9. FIG. 9 shows analysis results of cleavage of K6UbKMRC011 by application of various ubiquitin cleavage enzymes. In FIG. 9, lane 1 indicates Protein Mw size marker, lane 2 indicates K6UbKMRC011, lane 3 indicates K6UbKMRC011+USP1, lane 4 indicates K6UbKMRC011+USP2, lane 5 indicates K6UbKMRC011+USP10, lane 6 indicates K6UbKMRC011+UCH-L3, and lane 7 indicates K6UbKMRC011+UCH-L5/UCH37.

(36) As shown in FIG. 9, which is the result of SDS-PAGE analysis, it was confirmed that the refolded K6UbKMRC011 was cleaved by the ubiquitin cleavage enzymes shown in Table 2 and correctly separated into K6Ub and KMRC011. At this time, it was also confirmed that the internal structure of the KMRC011 protein was not degraded by the treatment of these enzymes.

(37) Further, as a result of confirming the N-terminal sequence of the separated KMRC011 protein through N-terminal sequencing, K6Ub was separated while the N-terminal amino acid sequence A-Q-VI-N of the KMRC011 protein was maintained intact as in Example 3.

(38) As described above, it was confirmed that all of the ubiquitin cleavage enzymes used in the present experiments correctly cleaved the immediate side of K6Ub of K6UbKMRC011. Therefore, it was confirmed that USP1-based and UCH-based ubiquitin cleavage enzymes as well as USP1 of Example 3 can cleave the immediate side of K6Ub of K6UbKMRC011 so as to separate KMRC011 without damage of KMRC011.

(39) Further, from the above-described various experimental results, it can be concluded that all types of the ubiquitin cleavage enzymes can precisely cleave the immediate side of K6Ub of K6UbKMRC011 so as to separate KMRC011 without damage of KMRC011.