CN110669109A - Enzyme-linked tag short peptide and application thereof - Google Patents

Abstract

The invention discloses an enzyme-linked tag short peptide which is characterized in that the sequence of the short peptide is shown in SEQ ID NO. 1-2. The short peptide can be rapidly chemically synthesized and is catalytically marked on a lysine site of a neighboring protein by a ligase PafA, so that in vitro marking of the protein is realized, and the protein with different functions and characteristics is obtained.

Description

Enzyme-linked tag short peptide and application thereof

Technical Field

The invention belongs to the technical field of protein surface labeling, and particularly relates to a novel enzyme-linked tag short peptide and application thereof.

Background

In vitro labeling of proteins is a widely used biological technique, and modification of proteins using biological enzymes is more specific than chemical modification. Enzymatic modification of proteins can be used to create antibody-drug conjugates, introduce fluorophores for imaging purposes, examine protein folding and interactions, and study natural post-translational modifications.

The commonly used protein modifying enzyme is BirA and the like, but most modifying enzymes need to fuse corresponding sequences capable of being modified on marked proteins, which brings difficulty to certain applications. For example, antibody proteins used in research procedures are often produced by immunized animals, and fusion of labeled sequences cannot be easily performed without sequence identification and derivation of mature in vitro expression systems.

PUP-IT (copy-based interaction tagging) utilizes the PUP ligase PafA in bacteria to capture protein interactions by labeling adjacent proteins with the small molecule protein PUP (a small protein comprising 64 amino acids). The PUP-IT system can not only mark adjacent proteins, but also capture weak interaction between proteins.

It has previously been reported that the C-terminal 28 amino acids of Pup are sufficient for PafA-mediated labeling, but when Pup is further truncated to 22 amino acids, no reaction can be triggered anymore even at very high concentrations (Liu, Q.; Zheng, J.; Sun, W.; Huo, Y.; Zhang, L.; Hao, P.; Wang, H.; Zhuang, M., A promoting-tagging system binding protein-protein). The PUP-IT system does not depend on the modification of the marked protein, but the marked fragment is far larger than the existing protein modification enzyme system, and the function of the marked protein can be changed.

Disclosure of Invention

The technical problem to be solved by the invention is to provide an enzyme-linked tag short peptide and application thereof, wherein the short peptide can be rapidly chemically synthesized and is catalytically marked on a lysine site of a neighboring protein by a ligase PafA, so that in-vitro marking of the protein is realized, and the protein with different functions and characteristics is obtained.

In order to solve the technical problems, the invention provides an enzyme-linked tag short peptide which is characterized in that the sequence of the short peptide is shown in SEQ ID NO. 1-2.

Preferably, the short peptide is modified with biotin or a fluorophore at the N-terminus.

The invention also provides application of the enzyme-linked tag short peptide.

Preferably, the application is the use of PafA mediated enzyme-linked tag short peptides as described above for labeling antibodies.

More preferably, the application comprises the steps of:

step 1: expressing a target Protein (PG) and a ligase PafA fusion, purifying in vitro or transfecting into cells for expression;

step 2: adding in-vitro synthesized biotin-labeled enzyme-linked tag short peptide, and reacting at room temperature;

and step 3: immunoblot analysis detected protein labeling results.

In the novel enzyme-linked tag short peptide (peptide 4.1), E52 and R56 are mutated to proline, which may destroy the helical structure, thereby allowing the peptide to be in a more expanded conformation. Conversely, E53, which originally faced the solvent side but mutated to isoleucine, can be released from the alpha helix structure, thereby facilitating hydrophobic interactions between the enzyme and the peptide. Peptide 4.1 is a newly identified shorter peptide that can be used for lysine binding by the PafA enzyme. Unlike the wild-type peptide, peptide 4.1 does not contain any lysine residues and therefore does not form unwanted branched modifications.

Compared with the prior art, the invention has the beneficial effects that:

the invention carries out engineering transformation on the substrate of PUP-IT, so that the substrate is shorter and shorter, and the original activity is kept, thereby leading the in vitro marking of the protein to be easier and more effective.

Drawings

FIG. 1 is a screen of peptide fragments that can be used by PafA; wherein, a is a schematic diagram of detecting PafA activity by a yeast surface display method, b is a screening strategy, and c is library activity after each round of screening;

FIG. 2 is a design of a library of peptide fragments; wherein a is the sequence of wild-type P22 and P22 libraries, b is the mutation region on the structure of the PafA-Pup complex (51A, 52E, 53E, 54F, 55V, 56R, 57S, 58Y, 59V, 60Q, 61K);

FIG. 3 is a DNA alignment of sequenced 13 randomly selected clones from the original mutation library;

FIG. 4 is a schematic representation of the identification of peptide mutants as substrates for PafA; wherein, a is sequence alignment of a plurality of clones after three rounds of sorting, and b is the labeling efficiency detected by a western blotting method when wild type P22, biotin-peptide fragment 4.1 and C-terminal 14 amino acid peptide fragment 4.1(4.1-14) are used as substrates for in vitro labeling GST-PafA;

FIG. 5 shows that peptide fragment 4.1 has activity comparable to wild-type Pup; wherein, a is enzyme kinetics research carried out by using DE28 (28 amino acids at the C end of PupE), b is enzyme kinetics research carried out by using a peptide segment 4.1, C is inhibition research carried out by using wild-type Pup to truncate P22, and d is inhibition research carried out by using the peptide segment 4.1;

FIG. 6 shows the labeling of the peptides of the present invention for antibodies (0.5. mu.M PG-PafA and 10. mu.M peptides); where a is the strategy of labeling the antibody with PUP-IT proximity labeling, b is the heavy chain of the anti-beta actin antibody can be labeled with biotin-4.1 or biotin-4.1 (14) (band of about 55 kDa), "" indicates from labeled GST-PafA or protein G-PafA, and c shows that the labeled antibody can be normally used in western blot experiments;

FIG. 7 shows the labeling of the peptide fragments of the present invention for antibodies (0.1. mu.M PG-PafA and 10. mu.M peptide); where a shows that both the heavy and light chains of the antibody can be modified by PG-PafA, and b shows that all three peptides are labeled in the same manner.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

Example 1: screening of enzyme-linked tag short peptides

(1) As shown in fig. 1a, an in vitro yeast surface marker system was first established to monitor the enzymatic activity of PafA:

recombinant PafA expressed a BCCP (biotin carboxyl carrier protein) domain at the N-terminus, Pup fused to the C-terminus of yeast protein Aga2, which was bound to Aga1 by disulfide bonds on the yeast surface; BCCP-PafA was added to suspended yeast and PafA reacted with surface Pup to form a covalent bond between the C-terminus of Pup and lysine on the BCCP domain to attach the PafA enzyme to the yeast surface. streptavidin-Alexa Fluor 647 recognizes BCCP-PafA on the surface of yeast and streptavidin-Alexa Fluor 647 recognizes biotin on the BCCP domain.

(2) A peptide library was designed based on a non-reactive 22 amino acid peptide stretch:

the Pup protein is mostly unstructured in solution, but after binding to PafA it adapts to two alpha-helical structures (fig. 2), which have an important role in the affinity between the enzyme and the substrate, for the non-reactive 22 amino acid peptide stretch (residues 43-64, P22), most of the first helix is deleted; thus, the interaction between enzyme and peptide is significantly reduced, and in order to enhance the binding between enzyme and peptide, the N-terminal 8 residues (residues 43-50) and the C-terminal 3 residues are retained, which are critical for binding and enzymatic activity, but allow the use of mild mutations at residues 51-61 (table 1). The oligonucleotides were generated by PCR to 10⁸The original mutation library, and through Sanger sequencing further confirmed the library diversity. As shown in FIG. 3, 13 clones were randomly selected from the original mutation library and sequenced, and the sequencing result showed that the library contained peptide fragments with different mutations, thereby confirming the diversity of the library.

TABLE 1 primers for P22 library construction

Oligo2 when synthesizing bases, the underlined base synthesis pattern is: 70% of the original base and 10% of each of the other three nucleotides;

(3) screening enzyme-linked tag short peptides:

as shown in FIG. 1b, the short peptides in the original mutation library obtained in step (2) were combined with ATP and Mg²⁺The reactions were allowed to react at 37 ℃ and then the modified yeast clones were sorted by flow cytometry for the next round of reaction. As the number of selection rounds increases, the enzyme concentration and reaction time decreases.

As shown in FIG. 1c, the V5 tag was fused between Aga-2 and the displayed peptides, the anti-V5 (FITC) signal indicated the surface expression level of each peptide, and the surface captured BCCP-PafA was labeled with streptavidin (Alexa Fluor 647). After three rounds of selection, we observed a highly enriched subpopulation of yeast showing enhanced modification of BCCP-PafA. We selected 20 yeast clones for Sanger sequencing and identified 17 clones encoding conserved peptide sequences (table 2).

TABLE 2 amino acid alignment of the Single clones from the last round of screening compared to wild-type P22 for the P22 mutant sequence

As shown in fig. 4a, 6 amino acid positions (51/52/53/55/56/61) of the 11 variation positions were mutated compared to the wild-type peptide sequence, and more hydrophobic residues were introduced into the peptide, resulting in potentially higher affinity between the peptide and the enzyme.

As shown in FIG. 4b, the labelling efficiency was determined by Western blotting using the wild-type P22 peptide (wild-type Pup truncated P22), biotin-peptide fragment 4.1 and C-terminal 14 amino acid peptide fragment 4.1(4.1-14) as substrates for in vitro labelling of GST-PafA. The self-labelling of GST-PafA labelled with peptide 4.1 was significantly more pronounced compared to the wild-type P22 peptide. Furthermore, after removal of residues 43-50, even shorter peptides (4.1-14) comprising only 14 amino acids from the 4.1C-terminus of the peptide can also be coupled under the same experimental conditions.

Example 2: peptide fragment 4.1 enzyme kinetics assay

As shown in FIG. 5, (a) and (b) are Km value determination experiments for FITC-DE28 or FITC-4.1 and PafA.

The procedure was carried out using peptide fragments (FITC-DE28:0.7812,0.156,0.312,0.625,1.25,5,10,15, or 20. mu.M) (FITC-4.1:0.156,0.312,0.625,1,1.25,2.5,5, and 10. mu.M) and GST-PafA at various concentrations, taking out certain reaction products at 20s, 40s, 60s 3 time points, respectively, and terminating the reaction, running the gel and developing with Typhoon.

(c) And (d) is a competition experiment with wild-type P22 and peptide 4.1 with DE 28.

The method is that 1 mu M FITC-DE28 and 0.1 mu M GST-PafA are mixed and then peptide segments with different concentrations are added, and the concentration of peptide 4.1 is as follows: 200,150,100,75,50,25,20,12.5,6.25,3.125, 1.560. mu.M. Peptide P22 concentrations were: 700,500,350,175,100,50,25,12.5,6.25,3.125, 1.56. mu.M, reaction buffer was added and the reaction was carried out for 10 min. And (5) after finishing, performing gum running and carrying out tryphon development.

The N-terminus of each peptide fragment was fused to FITC and self-labeled GST-PafA was measured. As shown in fig. 5a, 5b, the Km value for peptide 4.1 is similar to that of peptide DE28, indicating that the affinity between PafA and the substrate peptide is fully reconstituted in peptide 4.1, i.e. the structure after binding of PafA to peptide 4.1 is not the same as the structure after binding of PafA to peptide DE 28.

As shown in fig. 5c, 5d, in the self-labeling reaction with FITC-DE28, different amounts of unlabeled P22 and peptide 4.1 were added to inhibit the reaction, and free peptide 4.1 significantly inhibited DE28 modification compared to inhibition of wild-type Pup peptide P22.

Example 3: use of peptide 4.1 for PafA-mediated protein labelling

As shown in FIG. 6a, this example provides the use of peptide 4.1 for the labeling of antibodies to a PafA-mediated protein, as follows:

step 1: fusing Protein G (PG) Protein with the N end of PafA, constructing on a pGEX6p-1 vector, transforming the plasmid into a BL21 expression strain, inducing expression at 16 ℃, and purifying the Protein by using GST beads;

step 2: b, adding HRV 3C protease into the fusion protein purified in the step a to cut off the GST protein to obtain PG-PafA;

and step 3: after incubating PG-PafA and IgG obtained in step 2 for 10 minutes, N-terminal biotin-modified peptide fragment (10. mu.M) and 10 Xreaction buffer (200mM Tris8.0,100mM ATP, and 150mM Mg2+) were added and reacted at room temperature for 50 minutes;

and 4, step 4: after the reaction, 5ml of 2.5% BSA blocking solution was added;

and 5: SDS-PAGE and immunoblotting detect the labeled antibody.

As shown in fig. 6b, Protein G (PG) was fused to PafA in the presence of ATP and biotin 4.1 (or shorter peptide biotin 4.1-14) using 0.5 μ M PG-PafA and 10 μ M peptide fragments, and the heavy chain of the anti- β actin antibody was labeled with-4.1 or biotin-4.1 (14) (band of about 55 kDa), "" indicated self-labeled GST-PafA or protein G-PafA, indicating that peptides 4.1 or 4.1-14 could be used for the proximity tag.

FIG. 7a, both 4.1 and 4.1-14, using 0.1. mu.M PG-PafA and 10. mu.M peptide fragments, can modify the heavy and light chains of IGG.

FIG. 7b, increasing the reactive concentration of PG-PafA, 4.1 and DE28 modified IGG heavy and light chains to a comparable extent.

Further validation of peptide-labeled antibodies:

as shown in fig. 6c, anti-beta actin antibody was first labeled, and then whole cell lysates were blotted in an immunoblot experiment using the labeled antibody; streptavidin-HRP was used as a secondary antibody instead of anti-IgG antibody-conjugated HRP. Only if the anti-beta actin antibody is labeled with biotin-4.1, the streptavidin-HRP can be recognized and used for signal detection in Western blot experiments.

Strong chemiluminescence bands were detected in western blot experiments (fig. 6c), indicating that labeling did not disrupt antibody-antigen recognition. Addition of free protein g (pg) to the blot mixture after the labeling reaction further reduced the background signal.

SEQUENCE LISTING

<110> Shanghai science and technology university

<120> enzyme-linked tag short peptide and application thereof

<130>PCN1192735

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<170>PatentIn version 3.5

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Ile Asp Gly Leu Leu Glu Asn Asn Gly Pro Ile Phe Ala Pro Ser Tyr

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Val Gln Tyr Gly Gly Glu

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Gly Pro Ile Phe Ala Pro Ser Tyr Val Gln Tyr Gly Gly Glu

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ggccgcctat tcaccacctt tctgaacgta agaacgaacg aattcctccg cgttgttttc 60

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ctaaccctct tctcggtctc gattctacgg agaacctgta cttccagagc ggcggatcca 60

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Claims (5)

1. An enzyme-linked tag short peptide is characterized in that the sequence of the short peptide is shown in SEQ ID NO. 1-2.

2. The enzyme-linked tag short peptide according to claim 1, wherein the N-terminus of the short peptide is modified with biotin or a fluorophore.

3. Use of the enzyme-linked tag short peptide according to claim 1 or 2.

4. The use of the enzyme-linked tag short peptide according to claim 3, wherein the use is for labeling an antibody by using the enzyme-linked tag short peptide according to claim 1 or 2 mediated by PafA.

5. The use of the enzyme-linked tag short peptide according to claim 4, wherein the use comprises the steps of:

step 1: the target protein and the ligase PafA are subjected to fusion expression, and are purified in vitro or transfected into cells for expression;

step 2: adding in-vitro synthesized biotin-labeled enzyme-linked tag short peptide, and reacting at room temperature;

and step 3: immunoblot analysis detected protein labeling results.

Images