Preparation method for constructing phage display cyclopeptide library based on phthalic dicarboxaldehyde

Abstract

The invention relates to the technical field of polypeptide cyclization methods, in particular to a preparation method for constructing a phage display cyclopeptide library based on phthalic dicarboxaldehyde, which comprises the following steps: carrying out click chemical reaction on phthalic dicarboxaldehyde (OPA) and lysine and cysteine in an unprotected peptide sequence under a neutral or alkaline condition to form cyclopeptide; constructing a phage cyclic peptide library on the basis of the reaction property that the OPA molecule is efficiently biocompatible with sulfydryl and amino groups; a polypeptide library containing cysteine and lysine is displayed on a bacteriophage, the polypeptide library is cyclized by utilizing OPA (o-phthalaldehyde), and a cyclic peptide ligand with specific binding is screened out aiming at a target protein. In the strategy of performing polypeptide cyclization by using o-phthalaldehyde, it is found that o-phthalaldehyde has good reaction activity with sequence peptide containing cysteine and lysine, reaction conditions are simple and mild, and the o-phthalaldehyde has good biocompatibility. The reaction system does not need organic solvents or metal catalysts and is suitable for biological systems.

Description

Technical Field

[0001] The present invention relates to the technical field of polypeptide cyclization methods, and specifically to a preparation method for constructing a phage display cyclic peptide library based on o-phthalaldehyde. Background Art

[0002] Peptide drugs have the advantages of both small molecule drugs and protein drugs, with advantages such as high stability, easy synthesis, strong affinity with the target, and good pharmacological properties, and are expected to be developed into therapeutic drugs. However, since linear peptides have no stable conformation in solution, are easily degraded by proteases, are difficult to cross membranes, and have limited structural diversity, the widespread application of peptides in biomedicine is limited. In response to these limiting factors, peptide cyclization is a frequently used supplementary strategy.

[0003] Compared with linear peptides, peptide conformational constraints can reduce the entropy loss of binding to the target, thereby increasing the affinity of binding to the target and enhancing the stability of enzymatic proteolysis. Secondly, cyclic peptides are between small molecules and biomacromolecules, combining the advantages of both, and can target protein surfaces with relatively large and shallow "pockets". Usually, the formation of cyclic peptides is based on cysteine ​​modification. The most common method is based on the spontaneous formation of disulfide bonds between cysteines for cyclization, but the cyclic peptides formed based on this are easily reduced and rearranged. Therefore, finding feasible biological cross-linking molecules to couple with amino acid residues in polypeptides to construct cyclic peptide skeletons is the main goal of researchers. The use of TBMB for alkylation modification or other modification methods based on CH activation between residues such as tryptophan and tyrosine requires metal catalysts or organic solvents, but it is not biocompatible and chemically selective. O-phthalaldehyde (OPA) is a versatile bioorthogonal proximal cross-linking agent. Portoghese reported that OPA and its derivatives were used to selectively cross-link proximal lysine and cysteine ​​to provide fluorescent probes for opioid receptors. In recent years, the proximal cross-linking reaction of OPA has been developed for the chemically selective coupling of natural peptides and proteins, as well as the construction of cyclic peptides under mild biocompatible aqueous conditions. The cyclic peptides formed by modification based on o-phthalaldehyde do not require metal catalysts and organic solvents, and the reaction is mild and the conditions are simple. They can selectively react with cysteine ​​and lysine / N-terminus in unprotected peptide sequences to form cyclic peptides. They have good biocompatibility and chemical selectivity, providing a new research direction for the de novo design of cyclic peptide drugs based on target structures.

[0004] Phage display is an efficient gene expression screening technology that can fuse foreign proteins or peptides with specific phage capsid proteins, display them on the phage surface and maintain relatively independent spatial conformation and biological activity, and can establish a direct connection between genotype and phenotype. In recent years, phage display technology has become an important means of drug development due to its high throughput, high efficiency and low-cost screening characteristics. It is widely used in the development of antibody drugs and peptide drugs, vaccine development, and protein drug modification. However, since the phage display reaction system requires that the cyclization method must be efficient and have good biocompatibility and chemical selectivity, the phage cyclic peptide library constructed based on o-phthalaldehyde has simple and mild reaction conditions, rapid and efficient, and strong specificity, which meets this requirement well and provides more possibilities for the development of peptide drugs and detection. Summary of the invention

[0005] In view of the shortcomings of the prior art, the present invention provides a method for preparing a phage-displayed cyclic peptide library based on o-phthalaldehyde, which has good biocompatibility and chemical selectivity, simple reaction conditions, no need for metal catalysts and organic solvents, and rapid and efficient reaction.

[0006] To achieve the above object, the present invention provides the following technical solutions:

[0007] A method for preparing a phage display cyclic peptide library based on o-phthalaldehyde, characterized in that it comprises the following steps:

[0008] Step 1: using o-phthalaldehyde (OPA) to react with lysine and cysteine ​​in the unprotected peptide sequence under neutral or alkaline conditions to form a cyclic peptide;

[0009] Step 2: Based on the above o-phthalaldehyde (OPA) molecule's highly efficient biocompatible reaction properties with thiol and amino groups, a phage cyclic peptide library was constructed;

[0010] Step 3: Display a peptide library containing cysteine ​​and lysine on phage, cyclize the peptide library using o-phthalaldehyde (OPA), and screen out cyclic peptide ligands with specific binding to the target protein.

[0011] Preferably, in step one, the specific method for synthesizing the cyclic peptide is: dissolving a polypeptide powder containing cysteine ​​and lysine in a solvent to obtain a solution A, dissolving o-phthalaldehyde powder to obtain a solution B, adding polypeptide A and a thiol reducing agent to a neutral or alkaline buffer solution, and after the polypeptide is fully reduced, adding solution B to react to obtain a cyclic peptide product; and analyzing and characterizing the product by high performance liquid chromatography and mass spectrometry.

[0012] Specifically, the sequence of the polypeptide containing cysteine ​​and lysine is KXnC, wherein K represents lysine, C represents cysteine, and Xn represents n random amino acids.

[0013] Specifically, the solvent may be one of water, methanol or dimethyl sulfoxide.

[0014] Specifically, o-phthalaldehyde powder can be dissolved in dimethyl sulfoxide to obtain a solution B with a concentration of 0.5 mol / L, which can be diluted with water to 50 mmol / L and 5 mmol / L for later use; or phosphate buffer can be heated and dissolved at 65° C. to obtain a 150 mmol / L stock solution for later use.

[0015] Specifically, the neutral or alkaline buffer solution is a phosphate or carbonate buffer solution with a pH of 7-10.

[0016] Specifically, 5 mol / L tris(2-carboxyethyl)phosphine (TCEP) can be used as a thiol reducing agent, and the ratio of the reducing agent in the thiol reducing agent to the polypeptide in the polypeptide solution A is ≥1:1.

[0017] Specifically, the ratio of the polypeptides in solution B to solution A is ≥1:1; the reaction temperature can be 20-28°C, and the reaction time can be 10 min-15 min.

[0018] Preferably, in step 2, based on the above-mentioned o-phthalaldehyde (OPA) molecule's highly efficient biocompatible reaction properties with sulfhydryl and amino groups, the specific method for constructing a phage cyclic peptide library can be:

[0019] Template: K-Xn-C

[0020] Among them, K represents lysine, C represents cysteine, X represents any amino acid, and n represents the number of amino acids;

[0021] The polypeptide sequence fused and expressed on the surface of the phage is: KXXXXXXXC,

[0022] Where X represents a random amino acid, and random amino acid mutations are introduced at the specified position by the NNK method:

[0023] The corresponding DNA sequence designed according to the above polypeptide sequence is:

[0024] 5'-CTAGGGATCCaagNNKNNKNNKNNKNNKNNKNNKtgcGGCGGCTCGGCCTCGGGGGC-3';

[0025] Specifically, the number of amino acids in the above DNA sequence can be increased or decreased as needed, and all changes in polypeptide configuration and other modification methods made in the present invention should be considered within the scope of protection of this patent;

[0026] Specifically, the cross-linker structure used to construct the phage display cyclic peptide library is as follows:

[0027]

[0028] Among them, R is not limited to its position and number on the benzene ring, including but not limited to alkyl, alkenyl, aminoalkyl, haloalkyl, cyanoalkyl, (aryl)alkyl, nitro, halogen, or alkyl, heterocycloalkyl, carboxyalkyl, alkylcarboxyalkyl ester, nitroalkyl.

[0029] Preferably, in step 3, the specific method for constructing the phage display cyclic peptide library can be:

[0030] S1. The pbcomb-3xga plasmid (transformed from the pbcomb-3xss plasmid) was directly amplified by PCR to construct a phagemid library using two primers:

[0031] Li-BF: 5'-CTAGGGATCCaagNNKNNKNNKNNKNNKNNKtgcGGCGGCTCGGCCTCGGGGG-3' and Li-BR: 5'-CTAGGGATCCggccgcctgggccacg-3'; the PCR product was digested with restriction endonuclease BamHI, and the digestion product was recovered by agarose gel;

[0032] S2. The digested library DNA fragments were ligated with T4 DNA ligase at 16°C for 12 to 18 hours or at 37°C for 30 minutes, and then the phagemid template was digested with restriction endonuclease DpnI and transformed into Escherichia coli TG1 chemically competent cells by electroporation to evaluate the ligation and transformation efficiency, and finally the M13 phage library displaying the random peptide was obtained.

[0033] Specifically, the method of displaying a polypeptide library containing cysteine ​​and lysine on a phage, cyclizing the polypeptide library using o-phthalaldehyde (OPA), and screening out a cyclic peptide ligand with specific binding may be:

[0034] (1) To a titer greater than 10 10 TCEP was added to a phage library containing at least one cysteine ​​and one lysine displayed at the N-terminus at a concentration of 1 mM, the phage library was reduced, solution B was added to a final concentration of 300 μM, PEG-NaCl solution was added after the reaction, the phage was placed on ice to precipitate the phage, the supernatant was removed after precipitation, and the precipitate was resuspended in a phosphate buffer / carbonate buffer solution to obtain a phage cyclic peptide library modified with o-phthalaldehyde;

[0035] (2) Screening the monolithic phage cyclic peptide library: After the target protein is immobilized on a 96-well plate, the cyclic peptide library is added and incubated. After 1 hour, the unbound and low-affinity phages are washed away, and the specifically bound phages are eluted and recovered and amplified for the next round of screening. After multiple rounds of in vitro screening, the phages bound to the target protein are effectively enriched. Single clones of the colonies are picked for DNA sequencing to decode the amino acid sequence bound to the target protein.

[0036] Specifically, in step (1), the amount of PEG-NaCl added is 1 / 5 of the total volume; the incubation time on ice can be 2 to 6 hours; the centrifugation condition can be 8000 rpm for 20 minutes;

[0037] Specifically, in step (2), the screening is performed using PTP-1B, NEK7 and hKeap1 as target proteins; the multiple rounds of in vitro screening may be performed for 3 to 5 rounds;

[0038] The coating solution was 1% NaHCO3 solution, and the blocking solution was 1% NaHCO3 solution (containing 1% BSA); after each solidification of the protein, the cells were washed with 0.1% PBS-Tween 20, and unbound phages were washed with 0.1% PBS-Tween 20 in one round of screening, and unbound phages were washed with 0.5% PBS-Tween20 in subsequent rounds of screening;

[0039] The peptide sequences screened for the target protein PTP-1B are as follows:

[0040] Ac-KTFLHWRYC-CONH2(peptide 1)

[0041] Ac-KLFLHWARC-CONH2(peptide 2)

[0042] Ac-KTLLSHWFC-CONH2(peptide 3)

[0043] Ac-KLRFHWAYC-CONH2(peptide 4)

[0044] Ac-KYAIHRYEC-CONH2(peptide 5)

[0045] The peptide sequences screened for the target protein NEK7 are as follows:

[0046] Ac-KTFPLSSFC-CONH2(peptide 6)

[0047] Ac-KWLIAQRMC-CONH2(peptide 7)

[0048] Ac-KLLFPLINC-CONH2(peptide 8)

[0049] The peptide sequences screened for the target protein hKeap1 are as follows:

[0050] Ac-KQTATGRGC-CONH2(peptide 9)

[0051] Ac-KLDPDTGEC-CONH2(peptide 10)

[0052] Ac-KQTATGEGC-CONH2(peptide 11)

[0053] Ac-KDPETGEYC-CONH2(peptide 12)

[0054] Ac-KLDSETGEC-CONH2(peptide 13)

[0055] Ac-KQSAETGEC-CONH2(peptide 14)

[0056] After the peptides with higher enrichment are synthesized, they are subjected to cyclization reaction. The affinity between the cyclic peptide ligand and the target protein is verified by biophysical experiments to test the feasibility and effectiveness of the cyclization and screening methods.

[0057] The present invention provides a method for constructing a phage display cyclic peptide library based on o-phthalaldehyde small molecules and screening cyclic peptide ligands that specifically bind to a target using the phage cyclic peptide library. The present invention first uses o-phthalaldehyde (OPA) to undergo a click chemistry reaction with a polypeptide containing cysteine ​​and lysine under neutral or alkaline conditions to synthesize a monocyclic peptide. In addition, it can further react with maleimide to obtain a derivatized cyclic peptide with other additional motif functions.

[0058] The beneficial effects of the present invention are:

[0059] 1. In the strategy of using o-phthalaldehyde for polypeptide cyclization, the present invention finds that it has good reactivity with sequence peptides containing cysteine ​​and lysine, the reaction conditions are simple and mild, and it has good biocompatibility. The reaction system does not need to add organic solvents or metal catalysts, and is suitable for biological systems. It can also further react with maleimide to generate a more stable cyclopeptide product. By investigating the yield and reaction rate of the cyclopeptide reaction, an ideal monocyclic peptide method with fast reaction and high conversion rate is obtained.

[0060] 2. The present invention constructs a phage cyclic peptide library with o-phthalaldehyde and screens the target to obtain a monocyclic peptide ligand with high affinity. The development of this OPA-based chemical selective bioconjugation and peptide cyclization expands the toolbox of protein chemical modification and cyclic peptide construction, providing possibilities for the development of polypeptide drugs, disease detection and treatment. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] Figure 1 This is a diagram of the reaction between o-phthalaldehyde and a polypeptide containing cysteine ​​and lysine.

[0062] Figure 2 This is a chromatographic characterization diagram of the reaction between o-phthalaldehyde and polypeptides containing cysteine ​​and lysine.

[0063] Figure 3 Construct a sequencing result map for the peptide library.

[0064] Figure 4 The graph shows the phage infection activity under different reaction conditions.

[0065] Figure 5 Schematic diagram for screening a monocyclic peptide phage library.

[0066] Figure 6 It is a peptide sequence enriched after screening for the target protein PTP-1B.

[0067] Figure 7 The HPLC characterization diagram of the linear peptide and cyclic peptide of peptide 1.

[0068] Figure 8 This is the result of the biolayer interference experiment of the cyclic peptide and linear peptide of peptide 1 against PTP-1B.

[0069] Fig. 9 This is the result of docking the cyclic peptide ligand molecule of PTP-1B and peptide 1.

[0070] Fig.10 It is the peptide sequence enriched after screening against the target protein NEK7.

[0071] Fig.11 The HPLC characterization diagram of the linear peptide and cyclic peptide of peptide 2.

[0072] Fig.12 This is the result of the biolayer interference experiment of peptide 2's cyclic peptide and linear peptide against NEK7.

[0073] Fig.13 This is the result of docking the cyclic peptide ligand molecule of NEK7 and peptide 2.

[0074] Fig.14It is the peptide sequence enriched after screening against the target protein hKeap1.

[0075] Fig.15 The HPLC characterization diagram of the linear peptide and cyclic peptide of peptide 3.

[0076] Fig.16 This is the result of the biolayer interference experiment of peptide 3's cyclic peptide and linear peptide against hKeap1.

[0077] Fig.17 This is the result of docking hKeap1 with the cyclic peptide ligand of peptide 3. DETAILED DESCRIPTION

[0078] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0079] A preparation method for constructing a phage display cyclic peptide library based on o-phthalaldehyde comprises the following steps:

[0080] 1) Modification of linear peptides on the surface of phage using OPA:

[0081] The peptide sequence (KGSSC) encoded at the N-terminus of the phage coat protein III (pIII) was directly amplified by PCR from the Pbcomb3xga plasmid (transformed from the pbcomb-3xss plasmid) using two primers:

[0082] GSS-fw:5'-CTAGGGATCCaagggttcttcttgcGGTGAAAACCTGTACTTCCAGGGCGGCTCGGCCTC-3'GSS-rv:5'-CTAGGGATCCGCAACGACCGTCGATAGAACCACCGGCCGCCTGG-3'.

[0083] In the phagemid vector pbcomb-3xga, the encoded peptide sequence was inserted using the BamHI single restriction site. The encoded peptide sequence in the DNA sequence of this fragment is represented by lowercase, and the restriction endonuclease cleavage site BamHI is represented by uppercase and bold;

[0084] The plasmid was transformed into E. coli TG1 cells by the click method, cultured in 1 ml LB medium for 1 h, and then inoculated into 50 mL LB medium containing 50 μg / mL ampicillin, and incubated at 37°C and 220 rpm for 1 h;

[0085] 0.5 Mm IPTG was added to the bacterial suspension of E. coli TG1 containing the Pbcomb3xga_GSS plasmid and incubated overnight at 37°C and 220 rpm;

[0086] Centrifuge the culture at 8000 rpm for 20 min, pour the supernatant containing phage into a new centrifuge tube, add 1 / 5 volume of PEG-NaCl solution, and incubate on ice for 4-6 h;

[0087] The above solution was centrifuged at 8000 rpm for 20 min, the supernatant was discarded, and the centrifuge tube was placed upside down on filter paper for 2 min to remove all residual liquid; then the phage particles were resuspended in 10 mL PBS 7.4 and the mixture was transferred to a 50 mL tube; the remaining phage residues were removed by centrifugation again, and the supernatant was carefully transferred to a new 50 mL centrifuge tube;

[0088] Add an appropriate amount of Xa Factor digestion enzyme to the phage solution and incubate at room temperature for 4 hours. Add TCEP (1 mM) to the purified phage and incubate at 37°C for 1 hour; incubate with 6-hydroxy-1,3-benzothiazole-2-carbonitrile (CBT, 0.5 mM) at 25°C for 2 hours, and wash three times with 3 mL PBS;

[0089] OPA was added to the phage sample and the resulting reaction mixture was incubated at 25°C for 20 min. The sample was then concentrated to 1 mL and washed three times with PBS. Then, 50 μL of TEV digestion enzyme was added to the blocked phage solution and incubated at room temperature for 8 h. The sample was concentrated to 1 mL using an Ultra-15 (30 KDa cutoff) centrifuge tube. The filtrate was collected and used for HPLC and mass spectrometry analysis.

[0090] 2) Construction of phage display cyclic peptide library:

[0091] Template: K-Xn-C

[0092] Where K represents lysine, X represents a random amino acid, C represents cysteine, and n represents the number of random amino acids;

[0093] The peptide sequence displayed on the phage surface is: KXXXXXXXC

[0094] The corresponding DNA sequence designed according to the polypeptide sequence displayed on the surface of the phage is:

[0095]

[0096] In the phagemid vector pbcomb-3xga, the library fragment was inserted using the BamHI single restriction site. The polypeptide framework in the DNA sequence of the fragment was indicated by lowercase and underline, and the restriction endonuclease cleavage site BamHI was indicated by uppercase and bold;

[0097] The phagemid library was constructed by direct PCR amplification of the pbcomb-3xga plasmid using two primers Li-BF: 5'-CTAGGGATCCaagNNKNNKNNKNNKNNKNNKtgcGGCGGCTCGGCCTCGGGGG-3'

[0098] and Li-BR: 5'-CTAGGGATCCggccgcctgggccacg-3'; digest the PCR product with restriction endonuclease BamHI, recover the digestion product with 1% agarose gel, connect the digestion product with T4 DNA ligase, and finally remove the phagemid template with DpnI digestion enzyme. The connected plasmid was transformed into competent Escherichia coli TG1 by electroporation to evaluate the connection and transformation efficiency. The transformant was incubated with 1mL LB medium at 37℃220rpm for one hour, 10μL sample was taken for dilution and titer determination, and 20 single clones were selected for DNA sequencing. The sequencing results are shown in Figure 3 ; In the 20 sequencing results, it can be seen that all sites are randomized and unbiased;

[0099] 3) Preparation of phage cyclic peptide library:

[0100] The above transformation sample was inoculated into 500 mL of LB medium containing 50 μg / mL carbenicillin, of which 400 mL was cultured at 37°C and 220 rpm until OD 600 Mix with 50% glycerol to 0.8-1.0 and place in -80℃ refrigerator for later use; the remaining 100mL is cultured until OD 600 After the pH value is 0.4-0.6, helper phage is added for infection for 2 hours. After adding 0.5mM IPTG and culturing at 37°C for 12-14 hours, centrifuge at 4°C and 8000rpm for 20 minutes to remove the bacterial precipitate and collect the supernatant. Add 1 / 5 volume of PEG-NaCl to precipitate the supernatant containing phage (it can be placed on ice for precipitation for 4-6 hours or at 4°C overnight), centrifuge at 4°C and 8000rpm for 20 minutes to collect the precipitate, add 2mL PBS buffer to resuspend, add 300μM OPA to cyclize at room temperature for 15 minutes, add 1 / 5 volume of PEG-NaCl to precipitate the phage library, centrifuge at 4°C and 10000rpm for 1 minute to collect the precipitate, and finally resuspend the precipitate with PBS buffer to obtain the phage cyclic peptide library.

[0101] 4) Determination of the titer of phage cyclic peptide library:

[0102] Take 10 μL of the test solution and add 990 μL of 2×YT liquid medium to dilute it. Take 10 μL of the dilution solution and add 990 μL of 2×YT liquid medium to dilute it. Then dilute it step by step to 10 -8 -10 -10 , take 10 μL of the dilution and add it to 150 μL of TG1 in the logarithmic growth phase. After standing at 37°C for 30 minutes, the culture is evenly spread on the surface of a solid plate containing 50 μg / mL carbenicillin, inverted in a 37°C constant temperature incubator and cultured for 10-12 hours. Take out and count to calculate its titer.

[0103] 5) Coating and sealing:

[0104] The target proteins PTP-1B, NEK7, and hKeap1 were diluted to 50 μg / mL with 1% NaHCO3 and coated on a 96-well plate at 4°C overnight. At the same time, a group without target protein was set up as a control. The supernatant was removed and 1% NaHCO3 (containing 1% BSA and 1% Tween20) blocking solution was added to block at 4°C for 2 hours, and then washed 6 times with 0.1% PBS-Tween 20.

[0105] 6) Combination;

[0106] The phage cyclic peptide library was added and bound at 25°C and 60 rpm for 1 h. After binding, the unbound phage particles were removed and washed 10 times with 0.1% PBS-Tween 20.

[0107] 7) Elution:

[0108] After adding 200 μL of Gly-HCl (containing 1% BSA) with pH = 2.2 and eluting at 25°C and 60 rpm for 10 min, 30 μL of Tris-HCl with pH = 9.1 was added for neutralization, and the eluate was collected. 10 μL of the eluate was taken and diluted according to the above step 3) and then infected with TG1 in the logarithmic growth phase and the titer was determined;

[0109] After repeating the above steps for four rounds of screening of the target protein (the elution concentration for the last few rounds of screening was 0.5% PBS-Tween 20), phage monoclones were randomly selected for sequencing. The sequencing results are shown in Figure 6 , 10 、14.

[0110] The peptide sequence obtained by sequencing is subjected to solid phase synthesis. The specific synthesis steps are as follows:

[0111] (1) Swelling resin:

[0112] Weigh 0.5 g of RinkAmide Resin with a degree of substitution of 0.93, pour it into a reaction bottle, and add 30 mL of DMF to swell for 1 h.

[0113] (2) Deprotection

[0114] The DMF in the reaction bottle was drained, and a 20% piperidine / DMF solution was added to remove Fmoc. The reaction was continued for 15 min. The reaction was repeated once, and the mixture was drained. The mixture was washed with DMF for 5 times and drained.

[0115] (3) Condensation reaction

[0116] The condensation was carried out using a combination of HOBT / HBTU / DIEA activators. Three equivalents of HOBT / HBTU and Fmoc-amino acid were added, dissolved in DMF and added to the reaction bottle. DIEA was added, nitrogen was agitated, the reaction was carried out for 2 h, and the mixture was dried.

[0117] (4) Detection and washing

[0118] Take a little resin from the reaction bottle, put it into a small test tube, wash it once with DMF, add ninhydrin solution and heat it at 100℃ for 3min, take it out and observe the color of the resin. If the resin does not turn purple or blue, it indicates that the condensation reaction is complete. Stop the reaction, drain, and wash it 3 times with DMF. If the color of the resin is blue, purple or other colors, it means that the reaction is not complete. Drain and wash it 3 times, weigh the same amount of the current reaction and put it into the reaction column to react again until the detection reaction is complete.

[0119] (5) Repeated condensation

[0120] Repeat steps 2-5 for the subsequent amino acid connections, and use the same algorithm to weigh different amino acids, while keeping the amounts of HOBT / HBTU / DIEA constant.

[0121] (6) Contraction

[0122] After the last amino acid is connected, repeat step 2), then wash with DCM 3 times, methanol 3 times, drain, and place the resin in a fume hood to dry.

[0123] (7) Acetylation

[0124] Add 20 mL of a mixed solution of pyridine: anhydrous dichloroethylene: acetic anhydride = 1:2:1, react at room temperature for 1 h, and drain.

[0125] (8) Inspection

[0126] Take a small amount of resin from the reaction bottle, put it into a small test tube, add ninhydrin solution and heat at 100℃ for 3min, take out and observe the color of the resin. If the resin does not turn purple or blue, it indicates that the acetylation reaction is complete. Stop the reaction, wash with DMF 3 times, DCM 3 times, and methanol 3 times, and dry. If the color of the resin is detected to be blue or purple or other colors, it indicates that the reaction is not complete. Continue to add acetylation reagent to react until the reaction is complete.

[0127] (9) Cut off

[0128] Add 20 mL of cutting agent (TFA / Tris / H2O=19 / 0.5 / 0.5) to the reaction bottle, stir and react for 2 h, collect the filtrate, remove the liquid inside by rotary evaporation, add ether for precipitation, at this time the polypeptide is precipitated to form a polypeptide ether suspension, transfer the above liquid to a 50 mL centrifuge tube, centrifuge at 7000 rpm for 5 min, pour off the supernatant, add ether for washing, repeat 2 times, vacuum dry, remove the ether residue, and the obtained solid is the crude target polypeptide.

[0129] (10) Purification

[0130] The crude product is dissolved in water or methanol, filtered, and separated and purified by high performance liquid chromatography to finally obtain a pure target polypeptide with a purity of more than 95%.

[0131] Based on the enriched peptide sequences obtained from the sequencing results, we synthesized the following peptides:

[0132] Synthetic peptides include but are not limited to: Ac-KTFLHWRYC-CONH2 (peptide 1)

[0133] Ac-KTFPLSSFC-CONH2(peptide 2)

[0134] Ac-KQTATGRGC-CONH2(peptide 3)

[0135] Ac-KDPETGEYC-CONH2(peptide 4)

[0136] Ac-KLFLHWARC-CONH2(peptide 5)

[0137] Ac-KLDSETGEC-CONH2(peptide 6).

[0138] The synthesized peptides were analyzed and characterized by HPLC.

[0139] Taking the cyclization of peptide 4 as an example, the specific cyclization method is:

[0140] The peptide was dissolved in PBS at pH 7.4 to prepare a 1 mM solution (methanol:PBS = 1:1 ratio can also be used), and o-phthalaldehyde was added to make the final concentration 300 μM, and the reaction was carried out at room temperature for 15 minutes. The chromatogram of the cyclization product is shown in Figure 2 As shown, the expected product was obtained.

[0141] The affinity of linear peptides and cyclic peptides to PTP-1B was determined by biolayer interferometry. First, the linear peptides and cyclic peptides of pepetide 1 were purified by chromatography and quantified, and diluted to 50nM, 100nM, 200nM, 400nM, 800nM, and 1.6μM with 0.05% PBS-Tween 20. The target protein PTP-1B was diluted to 20μg / mL with 0.05% PBS-Tween20. At the same time, no target protein and no peptide ligand were set as backgrounds. After deducting the background, the affinity of the target protein and the peptide ligand under different concentration gradient conditions was globally fitted. The affinity of the cyclic peptide ligand to PTP-1B was calculated to be 1.6μM, and the affinity of the linear ligand to PTP-1B was 8.3μM.

[0142] exist Fig. 9 In the study, the interaction between the target protein PTP-1B and the peptide1 cyclic peptide was simulated by molecular docking, and the results showed that the ligand produced salt bridges, hydrogen bonds and conjugated interactions with specific residues Asp 29, Arg 24, HiS 25, Asp48 and Tyr46.

[0143] The affinity of linear peptides and cyclic peptides to NEK7 was determined by biolayer interferometry. First, the linear peptides and cyclic peptides of pepetide 2 were purified by chromatography and quantified, and diluted to 50nM, 100nM, 200nM, 400nM, 800nM, and 1.6μM with 0.05% PBS-Tween 20. The target protein NEK7 was diluted to 20μg / mL with 0.05% PBS-Tween 20. At the same time, no target protein and no peptide ligand were set as the background. After deducting the background, the affinity of the target protein and the peptide ligand under different concentration gradient conditions was globally fitted. The affinity of the cyclic peptide ligand to NEK7 was calculated to be 1.9μM, and the linear ligand had almost no interaction with NEK7.

[0144] exist Fig.13 In the study, the interaction between the target protein NEK7 and the peptide2 cyclic peptide was simulated by molecular docking, and the results showed that the ligand produced salt bridges, hydrogen bonds and conjugated interactions with specific residues Lys 96, Lys 17 and Thr 172.

[0145] The affinity of linear peptides and cyclic peptides to hKeap1 was determined by biolayer interferometry. First, the linear peptide and cyclic peptide of pepetide 3 were purified by chromatography and quantified, and diluted to 0.25μM, 0.5μM, 1.0μM, 2.0μM, 4.0μM, and 8.0μM with 0.05% PBS-Tween 20. The target protein hKeap1 was diluted to 20μg / mL with 0.05% PBS-Tween 20. At the same time, no target protein and no peptide ligand were set as backgrounds. After deducting the background, the affinity of the target protein with the peptide ligand under different concentration gradient conditions was globally fitted. The affinity of the cyclic peptide ligand to hKeap1 was calculated to be 3.8μM, and the linear ligand had almost no interaction with hKeap1.

[0146] exist Fig.17 In the study, molecular docking was used to simulate the interaction between the target protein hKeap1 and the peptide3 cyclic peptide, and the results showed that the ligand formed salt bridges and hydrogen bonds with specific residues Asn 382, ​​Asn 387, Arg 415, Gln 530, and Tyr 572 in the Nrf2 binding site.

[0147] The embodiments of the present application are described above in conjunction with the accompanying drawings, but the present application is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present application, ordinary technicians in this field can also make many forms without departing from the purpose of the present application and the scope of protection of the claims, all of which are within the protection of the present application.

Claims

1. A method for preparing a phage display cyclic peptide library based on o-phthalaldehyde, characterized in that: The steps include: Step 1: using o-phthalaldehyde (OPA) to react with lysine and cysteine ​​in the unprotected peptide sequence under neutral or alkaline conditions to form a cyclic peptide; Step 2: Based on the above o-phthalaldehyde (OPA) molecule's highly efficient biocompatible reaction properties with thiol and amino groups, a phage cyclic peptide library was constructed; Step 3: Display a peptide library containing cysteine ​​and lysine on phage, cyclize the peptide library using o-phthalaldehyde (OPA), and screen out cyclic peptide ligands with specific binding to the target protein.

2. The method for preparing a phage display cyclic peptide library based on o-phthalaldehyde according to claim 1, characterized in that: In step 1, the specific method for synthesizing the cyclic peptide is as follows: dissolving a polypeptide powder containing cysteine ​​and lysine in a solvent to obtain a solution A, dissolving o-phthalaldehyde powder to obtain a solution B, adding polypeptide A and a thiol reducing agent to a neutral or alkaline buffer solution, and after the polypeptide is fully reduced, adding solution B to react to obtain a cyclic peptide product.

3. The method for preparing a phage display cyclic peptide library based on o-phthalaldehyde according to claim 1, characterized in that: The sequence of the polypeptide containing cysteine ​​and lysine is KXnC, wherein K represents lysine, C represents cysteine, and Xn represents n random amino acids; the solvent can be one of water, methanol or dimethyl sulfoxide; o-phthalaldehyde powder can be dissolved in dimethyl sulfoxide to obtain a solution B with a concentration of 0.5 mol / L, and diluted with water to 50 mmol / L and 5 mmol / L for standby use; phosphate buffer can also be heated and dissolved at 65°C to obtain a 150 mmol / L stock solution for standby use; the neutral or alkaline buffer solution is a phosphate or carbonate buffer solution with a pH of 7-10; 5 mol / L tris(2-carboxyethyl)phosphine (TCEP) can be used as a thiol reducing agent, and the ratio of the reducing agent in the thiol reducing agent to the polypeptide in the polypeptide solution A is ≥1:1; the ratio of the polypeptide in the solution B to the solution A is ≥1:1; the reaction temperature can be 20-28°C, and the reaction time can be 10 min-15 min.

4. The method for preparing a phage display cyclic peptide library based on o-phthalaldehyde according to claim 3, characterized in that: In step 2, based on the above-mentioned o-phthalaldehyde (OPA) molecule's highly efficient biocompatible reaction properties with sulfhydryl and amino groups, the specific method for constructing a phage cyclic peptide library can be as follows: Template: K-Xn-C Among them, K represents lysine, C represents cysteine, X represents any amino acid, and n represents the number of amino acids; The polypeptide sequence fused and expressed on the surface of the phage is: KXXXXXXXC, Where X represents a random amino acid, and random amino acid mutations are introduced at the specified position by the NNK method: The corresponding DNA sequence designed according to the above polypeptide sequence is: 5'-CTAGGGATCCaagNNKNNKNNKNNKNNKNNKNNKtgcGGCGGCTCGGCCTCGGGGGC-3'; The number of amino acids in the above DNA sequence can be increased or decreased as needed, and all changes in polypeptide configuration and other modification methods made in the present invention should be considered within the scope of protection of this patent; The general formula of the cross-linker structure used to construct the phage display cyclic peptide library is: Among them, R is not limited to its position and number on the benzene ring, including but not limited to alkyl, alkenyl, aminoalkyl, haloalkyl, cyanoalkyl, (aryl)alkyl, nitro, halogen, or alkyl, heterocycloalkyl, carboxyalkyl, alkylcarboxyalkyl ester, nitroalkyl.

5. The method for preparing a phage display cyclic peptide library based on o-phthalaldehyde according to claim 4, characterized in that: In step 3, the specific method for constructing the phage display cyclic peptide library can be: S1. The phagemid library was constructed by direct PCR amplification of the pbcomb-3xga plasmid (transformed from the pbcomb-3xss plasmid) using two primers: Li-BF: 5'-CTAGGGATCCaagNNKNNKNNKNNKNNKNNKtgcGGCGGCTCGGCCTCGGGGG-3' and Li-BR: 5'-CTAGGGATCCggccgcctgggccacg-3'; the PCR product was digested with restriction endonuclease BamHI, and the digestion product was recovered by agarose gel; S2. The digested library DNA fragments were ligated with T4 DNA ligase at 16°C for 12 to 18 hours or at 37°C for 30 minutes, and then the phagemid template was digested with restriction endonuclease DpnI and transformed into Escherichia coli TG1 chemically competent cells by electroporation to evaluate the ligation and transformation efficiency, and finally the M13 phage library displaying the random peptide was obtained.

6. The method for preparing a phage display cyclic peptide library based on o-phthalaldehyde according to claim 5, characterized in that: The specific method of displaying a polypeptide library containing cysteine ​​and lysine on a phage, cyclizing the polypeptide library using o-phthalaldehyde (OPA), and screening out a cyclic peptide ligand with specific binding can be: (1) To a titer greater than 10 10 TCEP was added to a phage library containing at least one cysteine ​​and one lysine displayed at the N-terminus at a concentration of 1 mM, the phage library was reduced, solution B was added to a final concentration of 300 μM, PEG-NaCl solution was added after the reaction, the phage was placed on ice to precipitate the phage, the supernatant was removed after precipitation, and the precipitate was resuspended in a phosphate buffer / carbonate buffer solution to obtain a phage cyclic peptide library modified with o-phthalaldehyde; (2) Screening the monolithic phage cyclic peptide library: After the target protein is immobilized on a 96-well plate, the cyclic peptide library is added and incubated. After 1 hour, the unbound and low-affinity phages are washed away, and the specifically bound phages are eluted and recovered and amplified for the next round of screening. After multiple rounds of in vitro screening, the phages bound to the target protein are effectively enriched. Single clones of the colonies are picked for DNA sequencing to decode the amino acid sequence bound to the target protein.

7. The method for preparing a phage display cyclic peptide library based on o-phthalaldehyde according to claim 6, characterized in that: In step (1), the amount of PEG-NaCl added is 1 / 5 of the total volume; the incubation time on ice can be 2 to 6 hours; and the centrifugation condition can be centrifugation at 8000 rpm for 20 minutes.

8. The method for preparing a phage display cyclic peptide library based on o-phthalaldehyde according to claim 6, characterized in that: In step (2), the screening is performed using PTP-1B, NEK7 and hKeap1 as target proteins; the multiple rounds of in vitro screening can be performed for 3 to 5 rounds.

9. The method for preparing a phage display cyclic peptide library based on o-phthalaldehyde according to claim 8, characterized in that: The coating solution is 1% NaHCO3 solution, and the blocking solution is 1% NaHCO3 solution (containing 1% BSA); After each protein fixation, the cells were washed with 0.1% PBS-Tween 20. Unbound phages were washed with 0.1% PBS-Tween 20 in one round of screening, and unbound phages were washed with 0.5% PBS-Tween20 in subsequent rounds of screening.

10. The method for preparing a phage display cyclic peptide library based on o-phthalaldehyde according to claim 9, characterized in that: The peptide sequences screened for the target protein PTP-1B are as follows: Ac-KTFLHWRYC-CONH2(peptide 1) Ac-KLFLHWARC-CONH2(peptide 2) Ac-KTLLSHWFC-CONH2(peptide 3) Ac-KLRFHWAYC-CONH2(peptide 4) Ac-KYAIHRYEC-CONH2(peptide 5) The peptide sequences screened for the target protein NEK7 are as follows: Ac-KTFPLSSFC-CONH2(peptide 6) Ac-KWLIAQRMC-CONH2(peptide 7) Ac-KLLFPLINC-CONH2(peptide 8) The peptide sequences screened for the target protein hKeap1 are as follows: Ac-KQTATGRGC-CONH2(peptide 9) Ac-KLDPDTGEC-CONH2(peptide 10) Ac-KQTATGEGC-CONH2(peptide 11) Ac-KDPETGEYC-CONH2(peptide 12) Ac-KLDSETGEC-CONH2(peptide 13) Ac-KQSAETGEC-CONH2(peptide 14) After the peptides with higher enrichment are synthesized, they are subjected to cyclization reaction. The affinity between the cyclic peptide ligand and the target protein is verified by biophysical experiments to test the feasibility and effectiveness of the cyclization and screening methods.

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