Patent Application
20240132545
The present disclosure relates the technical field of protein self-assembly, in particular to, a combination of peptide linkers for protein covalent self-assembly using a spontaneous isopeptide bond.
Cell function depends upon the interaction of a large number of non-covalent proteins-proteins; and the precise arrangement of proteins in a complex influences and determines their functions. Non-covalent interaction is always relatively weak, and even will be usually interrupted under the conditions such as, long period or some mechanical force. Therefore, the ability to design the interaction of covalent proteins-proteins can bring a series of new opportunities for Basic Research, Synthetic Biology, and Biotechnology.
The interaction of covalent proteins is usually mediated by a disulfide bond; but disulfide bond is reversible and not suitable for the reduction of cellular space, and even may disturb protein folding. Due to small molecular weight, peptide tag minimizes its interference on protein functions and thus, it is a common tool to analyze and modify a protein. Gene coding is conducted very easily via a peptide tag; due to its small size, biosynthesis costs and immunogenic interference introduced will be reduced. However, there is a little of higher affinity in the interaction between a peptide tag and a protein, which thus limits its effect in the formation of a stable complex.
Currently, the protein capable of spontaneously forming an isopeptide bond has been always applied to develop a peptide tag, and is covalently bound to the corresponding binding polypeptide thereof (i.e., a linker of the two parts) mutually, thus providing irreversible interaction. The protein capable of spontaneously forming an isopeptide bond can be expressed as two fragments: a peptide tag and a polypeptide binding partner. These two fragments can achieve covalent recombination via the formation of an isopeptide bond, such that molecules or components connected to the pepetide tag and the polypeptide binding partner, respectively are fused.
Isopeptide bond is an amido bond formed between side-chain carboxyl groups or side-chain amino groups. In the case of typical biological conditions, such a bond is chemically nonreversible and is resistant to most of proteases. Isopeptide bond is covalent chemically and thus able to produce ultra-strong protein binding effects. On the condition that noncovalent interaction will be dissociated rapidly, e.g., high temperature, high pressure, rigorous chemical treatment or long period, the isopeptide bond formed between a peptide tag and its polypeptide binding partner can still achieve stable binding.
There exist some proteins having very strong interaction in some Gram-positive bacteria. For example, the fibronectin-binding protein (FbaB) of Streptococcus pyogenes applied to the fields of therapeutics, biological materials and vaccines contains an adhesin domain (CnaB2); the 31st lysine (K) and the 117th aspartic acid (D) in the domain can spontaneously form an isopeptide bond under the catalysis of the 77th glutamic acid (E). In the previous scientific researches, the CnaB2 of Streptococcus pyogenes FbaB protein have been divided into a SpyTag peptide having 13 residues and a SpyCatcher peptide having 116 residues to establish a Spy Tag/SpyCatcher system, thus achieving gene coding and covalent interaction between peptides and proteins. The SpyTag/SpyCatcher system provides a simple, specific and genetic-coding method to create various biological materials, for use in multiple aspects, including biological materials, next generation sequencing (NGS), enzyme stabilization, vaccine development, etc.
Even though the SpyTag/SpyCatcher technology has been applied to multiple aspects, the reaction speed and efficiency are still lower. To improve such a situation, on the one hand, an existing SpyTag/SpyCatcher sequence, e.g., a SpyTag003/SpyCatcher003 system is modified and optimized. On the other hand, a protein capable of spontaneously forming an isopeptide bond in other bacteria can be sought, so as to establish a more excellent linking system.
The objective of the present disclosure is to overcome the above shortcomings in the prior art, and to provide a combination of peptide linkers for protein covalent self-assembly using a spontaneous isopeptide bond.
The first objective of the present disclosure is to provide a combination of peptide linkers for protein covalent self-assembly using a spontaneous isopeptide bond.
A second objective of the present disclosure is to provide a recombinant and synthetic peptide chain.
A third objective of the present disclosure is to provide a nucleic acid molecule.
A fourth objective of the present disclosure is to provide a vector.
A fifth objective of the present disclosure is to provide a cell.
A sixth objective of the present disclosure is to provide use of any of the combination in self-assembling two molecules or components using an isopeptide bond.
A seventh objective of the present disclosure is to provide a method for self-assembling two molecules or components using an isopeptide bond.
An eighth objective of the present disclosure is to provide a kit for self-assembling two molecules or components using an isopeptide bond.
A ninth objective of the present disclosure is to provide use of one or more of the combination, the nucleic acid molecule, the vector, or the cell in preparing a kit for self-assembling two molecules or components, or in self-assembling two molecules or components using an isopeptide bond.
To achieve the above objectives, the present disclosure is achieved through the following technical solution:
In the present disclosure, GvTag/SdCatcher (SEQ ID NO: 11 and SEQ ID NO: 5), PsTag/SdCatcher (SEQ ID NO: 13 and SEQ ID NO: 5), and SaTag/SdCatcher (SEQ ID NO: 14 and SEQ ID NO: 5) are applied to establish a combined polypeptide-peptide tag linking system capable of spontaneously forming an isopeptide bond. Further, corresponding amino acids are modified and optimized at the N terminal of the peptide tag GvTag such that the binding efficiency is stronger than that of the SpyTag003/SpyCatcher003 system.
Specifically, the other 10 bacteria having a CnaB2 adhesin domain sequence are found via alignment with the CnaB2 sequence of Streptococcus pyogenes. These bacteria may include Gardnerella vaginalis (Gv), Granulicatella balaenopterae (Gb), Peptostreptococcus sp (Ps), Streptococcus anginosus subsp (Sa), Streptococcus dysgalactiae (Sd), Anaerobutyricum hallii (Ah), Clostridium perfringens (Cp), Ruminococcus sp (Rs), Streptococcus constellatus (Sco), and Streptococcus pneumoniae (Spn).
In the present disclosure, to obtain an isopeptide bond system with higher binding efficiency, the corresponding CnaB2 is decomposed into a Tag peptide and a Catcher protein, to establish 10 different Tag-GFP clones and 10 different Catcher-HPF clones, and then these clones are expressed and purified in an E. coli system. Based on the yield of the expression proteins, Catcher proteins of Peptostreptococcus sp, Streptococcus anginosus subsp, and Streptococcus dysgalactiae (PsCatcher, SaCatcher, and SdCatcher) are picked out and incubated with the Tag-GFP of the 10 bacteria above in a way of permutation and combination. Protein electrophoresis and Coomassie brilliant blue are applied to screen out three combinations with more efficient binding capacity, e.g., the combination of SdCatcher and GvTag-GFP, the combination of SdCatcher and PsTag-GFP, and the combination of SdCatcher and SaTag-GFP.
These three forms of combinations are then applied to a Receptor Binding Domain (RBD) subunit nanovaccine system of the S protein of SARS-CoV-2 novel coronavirus. Results show that GvTag-RBD and SdCatcher-HP are self-assembled to a spherical tetracosemer nanoparticle. The use of GvTag/SdCatcher, PsTag/SdCatcher, and SaTag/SdCatcher has effectively improved binding efficiency compared to the existing Spy Tag/SpyCatcher system. Moreover, the protein binding efficiency is optimal when using the GvTag/SdCatcher system. The GvTag sequence is further optimized to obtain a GvTagOpti/SdCatcher system which may further improve the binding efficiency.
Mice are immunized with the RBD-ferritin subunit tetracosemer antigen nanoparticle obtained from the linking systems GvTagOpti/SdCatcher, PsTag/SdCatcher, and SaTag/SdCatcher, which may overcome the shortcoming of insufficient immunogenicity of the RBD monomer, effectively trigger stronger immunoreaction, and improve antibody titer. The linking systems GvTagOpti/SdCatcher, PsTag/SdCatcher, and SaTag/SdCatcher of the present disclosure can significantly improve the reaction efficiency of molecules or components fused to a peptide tag and polypeptide thereof, and has a simple preparation method and a high protein expression level.
Therefore, the present disclosure sets forth the following features:
A combination of peptide linkers for protein covalent self-assembly using a spontaneous isopeptide bond, including a peptide linker 1 (a polypeptide) and a peptide linker 2 (a peptide tag); the peptide linker 1 contains a peptide chain having an amino acid sequence as shown in SEQ ID NO: 5, and the peptide linker 2 contains any one or more of peptide chains having amino acid sequences as shown in SEQ ID NO: 11, SEQ ID NO: 13, and SEQ ID NO: 14.
Preferably, the peptide linker 2 contains a peptide chain having an amino acid sequence as shown in SEQ ID NO: 11.
More preferably, the peptide linker 2 contains a peptide chain having an amino acid sequence as shown in SEQ ID NO: 46 (the peptide chain as shown in SEQ ID NO: 11 further has three KVG amino acids at the N terminal).
The present disclosure further sets forth a recombinant and synthetic peptide chain, including one or more of the peptide linker 1 and/or the peptide linker 2 in the combination according to any one of claim 1 or claim 2.
The present disclosure further sets forth a nucleic acid molecule, including one or more nucleotide sequences encoding the peptide linker 1 and/or the peptide linker 2 in any one of the combinations.
The present disclosure further sets forth a vector, including the nucleic acid molecule.
The present disclosure further sets forth a cell, including the nucleic acid molecule or the vector.
The present disclosure further sets forth use of any of the combination in self-assembling two molecules or components using an isopeptide bond; the two molecules or components are connected with the peptide linker 1 (polypeptide) and the peptide linker 2 (peptide tag) in the combination, respectively.
Further, the present disclosure sets forth a method for self-assembling two molecules or components using an isopeptide bond, including:
Furthermore, the present disclosure sets forth a kit for self-assembling two molecules or components using an isopeptide bond, including: one of the peptide linker 1 (polypeptide) and the peptide linker 2 (peptide tag) in the combination, the nucleic acid molecule encoding the peptide linker 1 and the peptide linker 2 in the combination, and a cell containing a vector encoding the peptide linker 1 and the peptide linker 2 in the combination.
The present disclosure further sets forth use of one or more of the combination, the nucleic acid molecule, the vector, and the cell in preparing a kit for self-assembling two molecules or components, or in self-assembling two molecules or components using an isopeptide bond, falling within the protection scope of the present disclosure.
Compared with the prior art, the present disclosure has the following beneficial effects:
The present disclosure constructs novel protein self-assembly systems GvTag/SdCatcher, PsTag/SdCatcher, and SaTag/SdCatcher based on an isopeptide bond, which effectively solves the problem of low binding efficiency of an existing SpyTag/SpyCatcher system, can significantly improve the reaction efficiency of molecules or components fused to a peptide tag and polypeptide thereof, and has a binding efficiency stronger than that of a Spy Tag003/SpyCatcher003 system. Moreover, the GvTagOpti/SdCatcher system, the PsTag/SdCatcher system, and the SaTag/SdCatcher system of the present disclosure have a simple preparation method and a high protein expression level.
with SpyCatcher;
The present disclosure is described in detail below with reference to the accompanying drawings and specific examples. The examples are merely used to explain the present disclosure, but not construed as limiting the scope of the present disclosure. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are commercially available reagents and materials unless otherwise specified.
Bacteria having a sequence of a CnaB2 adhesin domain were found via alignment
with the CnaB2 sequence of Streptococcus pyogenes.
The sequence alignment of the Catcher polypeptides from different bacteria with SpyCatcher, the sequence alignment of the Tag peptide with Spytag, as well as structure diagrams of the fusion proteins Tag-GFP and Catcher-HPF are shown in
1. Separation of the sequence of a CnaB2 adhesin domain
10 Catcher polypeptide sequences (amino acid sequences are shown in SEQ ID NOs: 1-10, respectively) and 10 Tag peptides (amino acid sequences are shown in SEQ ID NOs: 11-20, respectively) from 10 bacteria, e.g., Gv, Gb, Ps, Sa, Sd, Ah, Cp, Rs, Sco, and Spn.
2. Construction of a Catcher-HPF fusion protein clone, a Tag-GFP fusion protein clone, and a recombinant vector thereof
The above 10 different Catcher proteins and Helicobacter pylori _Ferritin (HPF) were used to construct 10 types of Catcher-HPF fusion proteins (amino acid sequences are shown in SEQ ID NOs: 21-30) according to the schematic diagram of
The 10 different Tag proteins and green fluorescent protein (GFP) were used to construct 10 types of Tag-GFP fusion protein clones (amino acid sequences are shown in
SEQ ID NOs: 31-40) according to the schematic diagram of
The specific sequence information is specifically shown in Table 1.
Gardnerella
vaginalis (Gv)
Granulicatella
balaenopterae (Gb)
Peptostreptococcus
Streptococcus
anginosus subsp (Sa)
Streptococcus
dysgalactiae (Sd)
Anaerobutyricum
hallii (Ah)
Clostridium
perfringens (Cp)
Ruminococcus sp
Streptococcus
constellatus (Sco)
Streptococcus
pneumoniae (Spn)
3. Expression of the fusion protein Catcher-HPF and the fusion protein Tag-GFP
The prepared 20 recombinant plasmids were transformed into DH5a competent cells, and cultured overnight at 37° C.; positive clones were screened out and PCR-identified. The positive clones were sent for sequencing, and the plasmids was extracted after the sequencing result was correct.
The plasmids with correct sequencing of the 20 recombinant plasmids were transfected into BL21(DE3) to obtain recombinant strains, respectively for the expression of a prokaryotic protein; the recombinant strains were subjected to enlarged culture with kanamycin-resistant LB media at 37° C. and 220 rpm/min until 0D600 was about 0.6 (about 4 h), and then IPTG with a final concentration of 1 mM was added to induce protein expression at 16° C. and 220 rpm/min. 18 h later after the induction, bacterial cells were dissolved and disrupted ultrasonically; then the solution was centrifuged and precipitate was discarded.
Supernatant was incubated with Ni-NTA agarose (GE Healthcare) to enrich His-labeled Catcher-HPF and Tag-GFP, and then the proteins were eluted by a Tris buffer solution containing imidazole. The purified proteins were concentrated and replaced by 20 mM Tri-HCl+50 mM NaCl buffer solution (pH=7.5). The proteins were subjected to concentration determination by BCA assay and stained by Coomassie blue, to detect the expression of each protein. Anaerobutyricum hallii (Ah), Clostridium perfringens (Cp), Ruminococcus sp (Rs), Streptococcus constellatus (Sco) and Streptococcus pneumoniae (Spn).
4. Incubation of the Tag-GFP and the Catcher-HPF
Each purified Tag-GFP and each purified Catcher-HPF were incubated, respectively. Incubation of the Tag-GFP and the Catcher-HPF in a 20 mM Tri-HCl+50 mM NaCl Tris buffer solution (pH=7.5) according to a molar ratio of 1:1, i.e., 5 nmol of the Tag-GFP and 5 nmol of the Catcher-HPF were put to 1 ml of the buffer solution and incubated for 1 h at room temperature.
Coomassie blue staining results are shown in
Incubation results are shown in
PsTag/SdCatcher (SEQ ID NO: 13 and SEQ ID NO: 5) and SaTag/SdCatcher (SEQ ID NO: 14 and SEQ ID NO: 5) were screened out.
These three combined binding systems of GvTag/SdCatcher, PsTag/SdCatcher, and SaTag/SdCatcher screened in Example 2 were then applied to an RBD subunit nanovaccine system of the S protein of SARS-CoV-2 novel coronavirus.
1. Construction of recombinant plasmid
Three Tag-RBD fusion protein clones were constructed by GvTag, PsTag, and SaTag with RBD, respectively according to the schematic diagram as shown in
2. Expression and purification of the recombinant protein
The prepared recombinant plasmids were transformed into DH5a competent cells, and cultured overnight at 37° C.; positive clones were screened and PCR-identified. Deendotoxin-plasmids were extracted, and transfected into HEK293F cells in a way of lipofection transfection, 5 days alter after the transfection, the cellular supernatant was harvested after centrifuge, and then the interest protein was purified.
The supernatant was incubated with Ni-NTA agarose (GE Healthcare); His-labeled Tag-RBD was enriched and then protein elution was performed by a Tris buffer solution containing imidazole. The purified proteins were concentrated and replaced by 20 mM Tri-HCl+50 mM NaCl buffer solution (pH=7.5). The proteins were subjected to concentration determination by BCA and stained by Coomassie blue.
3. Self-assembly of Ferritin of a tetracosemer nanoparticle
5 nmol of the purified Tag-RBD protein was incubated with 5 nmol of the SdCatcher-HPF protein prokaryotically expressed and purified in Example 3 in 1 ml of buffer system (20 mM Tri-HCl+50 mM NaCl, pH=7.5) for 1 h at room temperature. After the Tag-RBD and the SdCatcher-HPF were bound together, the monomeric protein had a size of 70 KDa.
Results are as shown in
Three amino acids RVG were added at N terminal of the GvTag sequence to construct a peptide tag GvTag001 with 18 amino acids (amino acid sequence is shown in SEQ ID NO: 44: RVGNTIVMVDKLKEVPTP); three amino acids KKVG were added at N terminal of the GvTag sequence to construct a peptide tag GvTag002 with 19 amino acids (amino acid sequence is shown in SEQ ID NO: 45: KKVGNTIVMVDKLKEVPTP); three amino acids KVG were added at N terminal of the GvTag sequence to construct a peptide tag GvTagOpti with 18 amino acids (amino acid sequence is shown in SEQ ID NO: 46: KVGNTIVMVDKLKEVPTP); namely, 3 artificially modified GvTags (amino acid sequences are shown in SEQ ID NOs: 44-46, respectively) were obtained.
The 3 artificially modified GvTags (amino acid sequences are shown in SEQ ID NOs: 44-46, respectively) and RBD served to construct fusion protein clones GvTag001-RBD, GvTag002-RBD and GvTagOpti-RBD, respectively according to the schematic diagram as shown in
The GvTag001-RBD, GvTag002-RBD, and GvTagOpti-RBD proteins were purified by the method of Example 3, respectively. 5 nmol of the purified GvTag001-RBD, GvTag002-RBD and GvTagOpti-RBD were incubated with 5 nmol of the SdCatcher-HPF protein prokaryotically expressed in Example 3 in 1 ml of buffer system (20 mM Tri-HC1 +50 mM NaCl, pH=7.5) for lh at room temperature.
The results are shown in
5 nmol of the GvTagOpti-RBD protein obtained in Example 4 was bound to 5 nmol of the SdCatcher-HPF obtained in Example 3, and then diluted by a 20 mM Tri-HCl+50 mM NaCl buffer solution (pH=7.5) at the dilution ratio of 1:2, 1:5 and 1:10. The specific operating steps were as follows: 5 nmol of the GvTagOpti-RBD protein obtained in Example 4 was incubated with 5 nmol of the SdCatcher-HPF obtained in Example 3 in 1 ml of a buffer system (20 mM Tri-HC1 +50 mM NaCl) at room temperature for lh to obtain a solution A; 50 μl of the solution A was then added to 50 μl of 20 mM Tri-HC1 +50 mM NaCl buffer solution (diluted at 1:2) to obtain a solution B. 40 μl of the solution B was added to 60 μl of 20 mM Tri-HCl+50 mM NaCl buffer solution (diluted at 1:5) to obtain a solution C. 50 μl of the solution C was then added to 50 μl of 20 mM Tri-HCl+50 mM NaCl buffer solution (diluted at 1:10) to obtain a solution D. 20 μl of the solution B, 20 μl of the solution C, and 20 μl of the solution D were then pipetted into three 1.5 ml EP tubes, respectively, and 5 μl of 5×loading was added respectively to each tube for protein boiling at 100° C. After being boiled for 5 min, 20 μl of each sample was subjected to protein electrophoresis and Coomassie brilliant blue staining.
Similarly, 5 nmol of the SpyTag003-RBD protein was bound to 5 nmol of the SdCatcher003-HPF, and then diluted by a 20 mM Tri-HCl+50 mM NaCl buffer solution (pH=7.5) at the dilution ratio of 1:2, 1:5 and 1:10. The specific operating steps are described above. The diluted binding proteins were then compared in sequence.
The results are shown in
The purified GvTagOpti-RBD-SdCatcher-HPF polymeric antigen obtained from Example 4, the purified PsTag-RBD-SdCatcher-HPF polymeric antigen and SaTag-RBD-SdCatcher-HPF obtained from Example 3 were purified by molecular sieve chromatography with a Siperose6 Increase10/300 GL column (GE), to obtain the tetracosemer RBD-HPF proteins (as shown in
Balb/C mice aged at 6-8 weeks were then grouped and immunized. Each mouse was inoculated in a volume of 200 ial via subcutaneous injection. The mice were subjected to blood sampling from eye sockets on the 14th day. After standing for a period of time, the mice serum was separated out, centrifuged at 4° C. and 2800 rpm for 15 min, and then immediately used for Anti-RBD IgG ELISA test.
The results are shown in
Finally, it should be indicated that the above examples are merely used to specify the technical solutions of the present disclosure, but not construed as limiting the protection scope of the present disclosure. A person skilled in the art can further make other different forms of alterations or changes on the basis of the above description. Moreover, there is no need and no way to enumerate all the embodiments one by one. Any modification, equivalent substitution and improvement made within the spirit and principle of the present disclosure should fall within the protection scope of the claims of the present disclosure.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202110444350.7 | Apr 2021 | CN | national |
| 202110876301.0 | Jul 2021 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/083430 | 3/27/2022 | WO |
