ZIPPER STRUCTURE THAT HELPS THE FORMATION OF PROTEIN DIMER AND APPLICATION THEREOF

Abstract

The present invention relates to the field of genetic engineering, and provides a zipper fastener structure of promoting formation of a protein dimer and application thereof. The zipper fastener can be applied to dimerization of proteins of the same type and dimerization of proteins of different types, and can also be applied to polypeptide cycle formation, polypeptide dimerization, and polypeptide extension. A ESAT6-CFP 10 dimer having an approximately native conformation can be obtained, and the dimer has better solubility, and has a better stimulating effect on memory T cells than a ESAT6-CFP10 fusion protein capable of linear fusion expression. A dimer zipper fastener can assist the formation of a more stable cyclic polypeptide, and a CCP polypeptide added with a dimer fastener can improve the detection rate for citrullinated autoantibodies in serum of a rheumatoid arthritis patient.

Claims

1. A method for promoting formation of a protein dimer or a cyclic peptide, comprising introducing a dimer zipper fastener part into the terminal part of a peptide chain, wherein: 1) the dimer zipper fastener part comprises at least 2 charged amino acid residues; 2) the dimer zipper fastener part comprises uncharged spacers, wherein the spacer is 1 to 5 amino acids in length; 3) at least one of the spacers comprises at least one cysteine residue; and 4) two dimer zipper fastener parts are bound by electrostatic interaction of charged amino acids and disulfide bonds are formed via the cysteine residues in the spacers.

2. The method according to claim 1, wherein the charged amino acids are symmetrically or asymmetrically located at both sides of the spacer.

3. The method according to claim 2, wherein the charged amino acids are positive-charged amino acids or negative-charged amino acids, wherein the positive-charged amino acids are selected from the group consisting of lysine, arginine, and histidine, and the negative-charged amino acids are selected from the group consisting of aspartic acid and glutamic acid.

4. The method according to claim 1, wherein N-terminus and C-terminus of at least one of the peptide chains are linked to the dimer zipper fastener part, respectively.

5. The method according to claim 1, wherein at least one of the peptide chains comprises a tag sequence; preferably, each of two peptide chains of comprises one tag sequence.

6. The method according to claim 1, wherein one peptide chain of the protein dimer is ESAT6, the other peptide chain is CFP10; the cyclic peptide comprises a CCP linear amino acid sequence, and the dimer zipper fastener parts are located at N-terminus and C terminus of the CCP linear amino acid sequence, respectively.

7. A protein or polypeptide comprising a dimer zipper fastener, wherein at least one of the terminal parts of the protein or polypeptide is linked to the dimer zipper fastener part, wherein the dimer zipper fastener part is characterized in that: 1) the dimer zip per fastener part comprises at least 2 charged amino acid residues; 2) the dimer zipper fastener part comprises uncharged spacers, wherein the spacer is 1 to 5 amino acids in length; 3) at least one of the spacers comprises at least one cysteine residue, preferably; and 4) two dimer zipper fastener parts are bound by electrostatic interaction of charged amino acids and disulfide bonds are formed via the cysteine residues in the spacers.

8. The protein or polypeptide according to claim 7, wherein the charged amino acids are symmetrically or asymmetrically located at both sides of the spacer.

9. The protein or polypeptide according to claim 7, wherein the protein is constructed of ESAT6 and CFP10; the dimer zipper fastener parts are located at C-terminus of ESAT6 and C-terminus of CFP10.

10. An expression vector, comprising a nucleotide sequence, wherein the nucleotide sequence is able to express the peptide chain comprising the dimer zipper fastener part according to claim 8.

11. A method for preparing a zipper fastener-type protein dimer or a cyclic peptide, comprising, constructing an expression vector, wherein the expression vector is the expression vector according to claim 10; and, expressing by transforming into an expression strain or cell with the expression vector, then isolating, and purifying the zipper fastener-type protein dimer or the cyclic peptide.

12. The method of claim 11, wherein the peptides in the zipper fastener-type protein dimers are ESAT6 and CFP10, respectively.

13. The method of claim 12, wherein at least one of the ESAT6 and the CFP10 comprises a tag sequence.

14. (canceled)

15. A kit, wherein the kit comprises the protein or polypeptide according to claim 9.

16. (canceled)

17. A kit for detecting an anti-citrullinated protein autoantibody, comprising the protein or polypeptide according to claim 19.

18. The method according to claim 1, wherein an integral structure of the cyclic peptide is KKCK-CCP linear amino acid sequence-DCDD.

19. The protein or polypeptide according to claim 7, wherein the protein is CCP linear amino acid, and the dimer zipper fastener parts are located at N-terminus and C-terminus of the CCP linear amino acid sequence, respectively.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0102] FIG. 1 is a schematic diagram of protein dimers with dimer zipper fasteners, wherein: 1) charged amino acids are symmetrically located at both sides of the spacer containing cysteines, and a number of the charged amino acids is 2 to 9; 2) charged amino acids are asymmetrically located at both sides of the spacer containing cysteines, and are all located at N-terminus of a target protein; 3) charged amino acids are symmetrically located, and the dimer zipper fastener parts are located at C-terminus of a target protein; 4) charged amino acids are asymmetrically located, and the dimer zipper fastener parts are located at C-terminus of a target protein; 5) charged amino acids are crosswise located, and the dimer zipper fastener parts are located at C-terminus of a target protein; and 6) a dimer of a native protein has an end-to-end structure, and a dimer zipper fastener part including a linker peptide can be located at N-terminus and C-terminus of the protein, respectively, thus a right three-dimensional structure conformation can also be formed.

[0103] FIG. 2 is a schematic diagram of cyclic peptides with dimer zipper fastener parts. The dimer zipper fastener parts for forming the cyclic peptide can be symmetrically located or asymmetrically located, or can be crosswise located.

[0104] FIG. 3 is a schematic diagram of a peptide chain dimer with a dimer zipper fastener and an extended peptide including a dimer zipper fastener.

[0105] FIG. 4 is an SDS-PAGE pattern of zipper fastener-type protein dimers expressed by different IPTG-induced strains.

[0106] FIG. 5 is a Coomassie brilliant blue-stained SDS-PAGE gel pattern of a purified zipper fastener-type protein dimer. SDS and reducing agent B-mecaptoethanol broke the disulfide bond, resulting in the decomposition of a native protein dimer into two monomers, while the protein before the reduction was clearly in a dimeric state.

[0107] FIG. 6 shows the effect of zipper fastener-type protein dimer antigens at various concentrations on IFN-γ stimulation levels. The zipper fastener-type protein dimer had relatively strong stimulation activity, and can reach saturation at a relatively lower concentration.

[0108] FIG. 7 shows the effect of stimulating temperature on T cell secreting IFN-γ.

[0109] FIG. 8 shows the effect of stimulating time on T cell secreting IFN-γ.

[0110] FIG. 9 shows the effect of proteins having different structures on T cell secreting IFN-γ, a zipper fastener-type protein dimer has a stronger activity. The zipper fastener-type protein dimer had a stronger effect on stimulating T cells to secrete IFN-γ at the same concentration as the protein dimer without a zipper fastener structure, and thus the T cells secreted more IFN-γ.

[0111] FIG. 10 shows the comparison of sensitivity and specificity of zipper fastener-type CCP cyclic peptide with a conventional disulfide bond CCP cyclic peptide in diagnosing rheumatoid arthritis.

[0112] FIG. 11 shows the performance of the zipper fastener-type CCP cyclic peptide and the conventional CCP cyclic peptide in testing 26 different rheumatoid arthritis samples. S/CO values were higher in 12 samples tested by zipper fastener-type CCP cyclic peptide, which turned a negative result (undetectable) to a positive result (detectable).

DETAILED DESCRIPTION

[0113] The technical solutions of the present disclosure will be further explained below in conjunction with the examples. The specific examples described herein are only for the purpose of interpreting the present disclosure, but are not to limit the present disclosure. In addition, it should be noted that, for ease of description, the drawings only show a part of the structure related to the present disclosure, instead of all the structures.

[0114] The specific techniques or conditions not specified in the embodiments are carried out in accordance with the techniques or conditions described in the literature in the field, or in accordance with the product instructions. All the reagents or instruments not indicated with the specific manufacturer are conventional products that are commercially available.

EXAMPLE 1

Construction of Zipper Fastener-Type ESAT6-CFP10 Protein Dimer Expression Vector

[0115] An amino acid set with negative charges was added to C-terminus of ESAT6 gene, and an amino acid set with positive charges was added to C-terminus of CFP10 gene. A His-tag for purification was added at the front of the CFP10 gene.

[0116] An expressed amino acid sequence of the ESAT6 was DYKDDDDKGG-MAEMKTDAATLAQEAGNFERISGDLKTQIDQVESTAGSLQGQWRGAAGTAAQAAV VRFQEAANKQKQELDEISTNIRQAGVQYSRADEEQQQALSSQMGF-GGDDKDD.

[0117] An expressed amino acid sequence of the CFP10 was: HHHHHHGG-MTEQQWNFAGIEAAASAIQGNVTSIHSLLDEGKQSLTKLAAAWGGSGSEAYQGVQQ KWDATATELNNALQNLARTISEAGQAMASTEGNVTGMFA-GGKKCKK.

[0118] ESAT6 and CFP10, to which the above-mentioned sequences had been added, were inserted to a pET expression vector at both ends of IRES sequence, respectively, thus obtaining an expression plasmid. After purification, the expression plasmid was transformed into BL21(DE3) competent cells.

Expression and Purification of the Recombinant Protein

[0119] Successfully constructed single colonies were selected and cultured at 37° C. in 2 liters culture. After IPTG induction for 4 h, the cells were centrifuged, collected, and disrupted by ultrasonic. After Ni-column affinity purification, obtain the interest protein, i.e. zipper fastener-type protein dimer ESAT6-CFP10 (named E6C10 for short).

[0120] The protein was measured for concentration by SDS-PAGE. The result is shown in FIG. 4. It can be seen from FIG. 4 that a soluble dimer could be obtained 4 hours after induction at 37° C. It can be seen from FIG. 5 that the soluble dimer could be further purified by Ni affinity column to achieve a purity above 90%. The purified protein was subjected to endotoxin-removing treatment before a memory T cell stimulation test.

Memory T Cell Stimulation Test for the Zipper Fastener-Type Protein Dimer

[0121] In this experiment, fresh peripheral whole blood from tuberculosis patients was used to compare the effect of the zipper fastener dimer antigens in different concentrations (2 μg/ml, 4 μg/ml and 6 μg/ml) on stimulation levels (i.e., IFN-γ levels).

[0122] 1) Nine fresh peripheral whole blood samples were collected from two healthy people and seven tuberculosis patients, and each of the whole blood samples was divided into 5 aliquots (1 mL for each). Stimulation was performed using the zipper fastener dimer protein in three different concentrations, a negative control substance, and a positive control substance.

[0123] 2) 45 samples were incubated dormant at 37° C. for 20 hours.

[0124] 3) Samples were centrifuged to collect plasma supernatants.

[0125] 4) IFN-γ levels were measured by Human IFN-γ Elisa kit (a standard double antibody sandwich ELISA kit with lowest detectable concentration of IFN-γ at 5 pg/mL).

[0126] The test results were shown in Table 1 and FIG. 6 (the results of the negative control and the positive control were not shown).

TABLE-US-00003 TABLE 1 Effect of the zipper fastener-type protein dimer antigen in different concentrations on IFN-γ stimulation level. Protein Protein Protein dimer dimer dimer Sample No. (2 μg/ml) (4 μg/ml) (6 μg/ml) Healthy person 81 0.023 0.017 0.017 Healthy person 82 0.021 0.026 0.019 TB344tubercu1osis 1.162 1.182 1.168 TB348tuberculosis 0.405 0.463 0.442 TB349tuberculosis 0.143 0.14 0.135 TB351tuberculosis 0.657 0.665 0.688 TB354 tuberculosis 0.38 0.386 0.395 TB355tuberculosis 0.206 0.209 0.207 TB357tuberculosis 0.219 0.233 0.237

[0127] It can be seen from FIG. 6 that, in the test to the seven tuberculosis patients and two healthy people, IFN-γ level of the samples from the tuberculosis patients were approximately plateaued upon stimulating the cells with the zipper fastener type protein dimer antigen obtained in Example 1 in 2 μg/mL, and cannot be further increased (except for two samples in which IFN-γ level was slightly increased) even if concentrations of the zipper fastener type protein dimers were increased to 4 μg/mL and 6 μg/mL. In the following tests of the present disclosure, stimulation was performed using the antigens in a concentration of 2 μg/ml.

Selecting Culturing Temperature for Zipper Fastener-Type Protein Dimer Stimulated Cells

[0128] In this experiment, effects of culturing temperatures (25° C., 30° C., 37° C., 38° C., and 39° C.) were tested in fresh peripheral whole blood of tuberculosis patients according to a detailed process as follows:

[0129] 1) Fresh peripheral whole blood samples were collected from four tuberculosis patients, and each of samples was divided into 10 aliquots. For 5 aliquots, stimulation was performed using the zipper fastener dimer (in a final concentration of 2 μg/mL). As for the other 5 aliquots, a negative control substance was used.

[0130] 2) The samples from the 4 patients were incubated at 25° C., 30° C., 37° C., 38° C., and 39° C. for 22 hours.

[0131] 3) The samples as well as the negative control were centrifuged to collect plasma supernatants.

[0132] 4) The IFN-γ level in the plasma supernatants were measured by Human IFN-γ Elisa kit (a standard double antibody sandwich ELISA kit with a lowest detectable concentration of IFN-γ at 5 pg/mL).

[0133] The test results were shown in Table 2 and FIG. 7.

TABLE-US-00004 TABLE 2 Effect of culturing temperature on IFN-γ expression 25° C. 30° C. 37° C. 38° C. 39° C. Negative Negative Negative Negative Negative Tuberculosis 0.052 0.026 0.025 0.121 0.038 0.126 0.050 0.127 0.142 0.092 TB405 Tuberculosis 0.044 0.041 0.136 0.417 0.069 1.423 0.053 0.864 0.04 0.904 TB407 Tuberculosis 0.024 0.029 0.039 0.03 0.017 0.032 0.026 0.036 0.024 0.044 TB410 Tuberculosis 0.038 0.034 0.039 0.118 0.047 0.284 0.057 0.182 0.152 0.168 TB408

[0134] It can be seen from FIG. 7 that the IFN-γ level upon stimulation varies at the culturing temperature between 30° C. and 38° C. The INF-γ level upon stimulation was the highest at the culturing temperature of 37° C., revealing the best stimulation effect. No stimulation effect was shown at the culturing temperature of 25° C. Nonspecific response was observed in some of the negative control samples at the culturing temperature of 39° C. Therefore, in the present disclosure, the culturing temperature may range from 30° C. to 38° C., preferably 37° C.

Selecting Culturing Time for Zipper Fastener Type Protein Dimer Stimulated Cells

[0135] In this experiment, effects of culturing times (14 h, 18 h, 20 h, 22 h, 24 h, 26 h) on stimulation levels (i.e., IFN-γ stimulation levels) were assessed in fresh peripheral whole blood of tuberculosis patients.

[0136] 1) Fresh peripheral whole blood was collected from tuberculosis patients. Stimulation was performed using the zipper fastener protein dimer (in a final concentration of 2 μg/mL) in the whole blood sample.

[0137] 2) The whole blood sample upon stimulation was incubated at 37° C. for 12 h, 16 h, 18 h, 20 h, 22 h, 24 h, 26 h, and 28 h. The whole blood sample without stimulation was incubated under the same conditions and serves as a negative control.

[0138] 3) The samples as well as the negative control were centrifuged to collect plasma supernatants.

[0139] 4) The samples were measured for IFN-γ level using Human IFN-γ Elisa kit (a standard double antibody sandwich ELISA kit with the lowest detectable concentration of IFN-γ at 5 pg/mL).

[0140] The test results are shown in FIG. 8. It could be seen from FIG. 8 that the stimulation level (i.e., IFN-γ level) approximately plateaued after incubation for 20 h. Therefore, in the present disclosure, a preferably culturing time was 20 h. The experiment result was not affected when the culturing time was 20 h plus or minus 2 to 4 h.

Effect of Zipper Fastener Type Protein Dimer and Linear Fusion Protein on Stimulation Level

[0141] In this experiment, fresh peripheral whole blood from tuberculosis patients was used to compare the effect of the zipper fastener type protein dimer and linear fusion protein on stimulation level (i.e., IFN-γ stimulation levels).

[0142] 1) Fresh peripheral blood samples were collected from seven tuberculosis patients, and each of the whole blood samples was stimulated with the zipper fastener type protein dimer and a linear fusion protein (in a final concentration of 2 μg/mL), respectively.

[0143] 2) The stimulated whole blood samples were incubated at 37° C. for 20 h. The whole blood samples upon no stimulation by a stimulus were incubated under the same conditions and serves as a negative control.

[0144] 3) The samples as well as the negative control were centrifuged to collect plasma supernatants.

[0145] 4) The samples were measured for IFN-γ level using Human IFN-γ Elisa kit (a standard double antibody sandwich ELISA kit with the lowest detectable concentration of IFN-γ at 5 pg/mL).

[0146] The test results are shown in FIG. 9. It could be seen from FIG. 9 that compared with the linear fusion protein antigen, the zipper fastener-type protein dimer antigen had a significantly better stimulation effect, and more IFN-γ was produced in the whole blood sample stimulated with the zipper fastener-type protein dimer.

[0147] In conclusion, in the present disclosure, it can be concluded by investigating stimulation conditions that there is the highest cytokine level, the best stimulation effect and thus the best detection effect, when a concentration of the zipper fastener-type protein dimer was 2 μg/mL and cells are stimulated at a stimulating temperature of 37° C. for 20 h to 22 h.

EXAMPLE 2: USE OF CYCLIC PEPTIDE IN CCP DETECTION

[0148] Based on conventional CCP, positions of the disulfide bond were changed, and dimer zipper fasteners on both sides of the polypeptide were added, then a new structure of the polypeptide was generated as follows: KKCK-CCP-DCDD. With the zipper fastener cyclic peptide, the positive rate of detecting autoimmune antibody in rheumatoid arthritis serum was increased by 10%, indicating that stability of cyclization is very important to detection sensitivity of CCP, and improvement of stability of the cyclic peptide can further increase the detection sensitivity of CCP in detecting an anti-citrullinated protein autoantibody.

[0149] A plate was coated with a streptavidin-dimer zipper fastener cyclic peptide CCP (in a concentration of 5 μg/ml), and blocked with skimmed milk. HRP-goat anti-human antibody as a secondary antibody was used for testing a sample. CCP ELISA assay kit (Euro Diagnostica) was used as a control.

[0150] Referring to FIG. 10, in 95 RA patients, the positive rate for CCP without the dimer zipper fastener was 78%, and a positive rate for CCP with the dimer zipper fastener was 89%. In 71 healthy people, specificity of CCP with the dimer zipper fastener was 91%, which is slightly lower than that of CCP (98%).

[0151] FIG. 11 shows the performance of the zipper fastener-type CCP cyclic peptide and the conventional CCP cyclic peptide in testing 26 different rheumatoid arthritis samples. S/CO values were higher in 12 samples tested by zipper fastener type CCP cyclic peptide, which turns a negative result (undetectable) to a positive result (detectable).

[0152] The above description is only the preferred embodiments of the present disclosure and the applied technical principles. One of ordinary skill in the art will understand that the present disclosure is not limited to the specific embodiments described herein. For one of ordinary skill in the art, various obvious changes, adjustments, and substitutions can be made without departing from the protection scope of the present disclosure. Therefore, although the present disclosure has been described in more detail through the above embodiments, the present disclosure is not limited to the above embodiments. Without departing from the concept of the present disclosure, more other equivalent embodiments may be included. The scope of the present disclosure is defined by the scope of the appended claims.