CN107936086B

CN107936086B - Accurate self-assembly method of peptide nucleic acid mediated protein

Info

Publication number: CN107936086B
Application number: CN201711247179.0A
Authority: CN
Inventors: 王建浩; 王建鹏; 邱琳; 蒋鹏举; 柳丽; 刘晓骞; 朱志兰; 马路平; 郭倩倩; 杜炫呈; 王政
Original assignee: Changzhou University
Current assignee: Changzhou University
Priority date: 2017-12-01
Filing date: 2017-12-01
Publication date: 2021-09-28
Anticipated expiration: 2037-12-01
Also published as: CN107936086A

Abstract

The invention belongs to the technical field of biology, and particularly relates to a peptide nucleic acid mediated protein accurate self-assembly method. A specific PNA sequence is marked on the target protein through a polypeptide sequence, and the PNA is efficiently and accurately combined with a DNA template, so that the accurate assembly of a plurality of target proteins is facilitated.

Description

Accurate self-assembly method of peptide nucleic acid mediated protein

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a peptide nucleic acid mediated protein accurate self-assembly method.

Background

Peptide Nucleic Acid (PNA) serves as an assembly template. Peptide nucleic acids are synthetic high molecular polymers similar to DNA and RNA invented by Nielsen et al in 1991 (Nielsen, P.E.; Egholm, M.; Berg, R.H.; Buchardt, O.science 1991,254, 1497-1500). It mainly has the following characteristics: (1) and the binding force is super strong. Peptide nucleic acids can form good base-pairing interactions with DNA (or RNA) driven by Watson-Crick hydrogen bonds as well as Hoogsteen hydrogen bonds. In addition, because the base mismatch between PNA and DNA is more unstable than that between DNA and DNA, and because the electroneutrality of PNA cannot generate charge repulsion with the complementary DNA strand, the binding capacity between PNA and DNA (or RNA) is stronger than that of DNA (or RNA) itself. (2) And (4) super stability. Since the backbone structure of synthetic backbone is not recognized by various proteolytic and hydrolytic enzymes in vivo, neither PNA itself, nor any double stranded structure of PNA involved in binding is degraded by enzymes in the body of microorganisms.

Current self-assembly for proteins is based primarily on the following platform: (1) a scaffold protein. The target protein is assembled in the presence of the skeleton protein by a method for constructing a fusion skeleton protein by utilizing protein-protein interaction. However, the method has the disadvantages of poor interaction, incapability of well controlling the ratio of proteins, too large molecular weight of skeleton protein and the like; (2) nanoparticles. The protein-nanoparticle interaction is utilized to perform self-assembly of the target protein on the surface of the nanoparticle. However, the method has the defects that the ratio between proteins cannot be accurately controlled, the configuration during protein assembly cannot be controlled, the potential biological toxicity is high and the like; (3) a dsDNA molecule. The target protein is self-assembled on the dsDNA by utilizing the interaction between the protein and the dsDNA. The method has the defects of low selectivity, incapability of controlling the assembly proportion, incapability of realizing accurate self-assembly and the like.

Disclosure of Invention

The technical problem to be solved by the invention is as follows: in order to overcome the problems that the protein cannot be accurately controlled during self-assembly, the specificity is poor and the like, the popularization and the application of the protein in the biological fields of multi-enzyme systems, biosensors, protein structure regulation and the like are limited. In order to solve the above problems, the present invention provides a peptide nucleic acid mediated protein precision self-assembly method:

(1) preparing a fusion protein containing an active polypeptide tag,

specifically, a polypeptide sequence with the length of 21 natural amino acids is fused at the N end or the C end of the protein;

(2) preparing a non-natural polypeptide probe containing a targeting PNA sequence,

specifically, a non-natural polypeptide (the length of the polypeptide is 21 amino acids) with the length of 10 basic groups of PNA sequences is connected to the N end or the C end;

(3) covalently bonding the fusion protein containing the active polypeptide tag obtained in the step (1) with the non-natural polypeptide probe containing the targeting PNA sequence obtained in the step (2),

the covalent bonding here means in particular: the cysteine on the fusion protein is combined with the covalent bond between the unnatural X amino acid on the polypeptide probe, a certain site can be recognized when the covalent bond is formed between the conventional polypeptides, in other words, the labeling site is random, the combining ability is not strong, and the recognition site in the patent is only the cysteine fixed in 21 amino acids on the fusion protein, and has quite high reaction recognition specificity;

(4) performing controllable self-assembly on the products of the covalent bond combination in the step (3) on the ssDNA sequence,

ssDNA sequence refers to a single-stranded DNA sequence of 26 bases in length comprising a linker arm, which serves as a binding template.

The protein provided by the invention is simple to synthesize in an accurate self-assembly mode, accurate in assembly, high in specificity and good in biocompatibility. A specific PNA sequence is marked on the target protein through a polypeptide sequence, and the PNA is efficiently and accurately combined with a DNA template to facilitate the accurate assembly of a plurality of target proteins, so that a universal assembly platform is provided for the biotechnology fields of multienzyme systems, biosensors and the like.

Drawings

FIG. 1 is a HPLC characterization of a non-native polypeptide containing a PNA sequence (PNA-P1).

FIG. 2 is a HPLC characterization of a non-native polypeptide containing a PNA sequence (PNA-P2).

FIG. 3 is a representation of SDS-PAGE of fusion proteins containing active polypeptide tags (EGFP-CCE) after reaction with PNA-P1 and PNA-P2. The first lane is a protein Marker, the second lane is a natural EGFP-CCE, the third lane is a compound EGFP-PNA1 formed by covalently binding the EGFP-CCE with PNA-P1, and the fourth lane is a compound EGFP-PNA2 formed by covalently binding the EGFP-CCE with PNA-P2.

FIG. 4 is a SDS-PAGE representation of the reaction of fusion proteins containing active polypeptide tags (mCherry-CCE with PNA-P2). The first lane is a protein Marker, the second lane is natural mCherry-CCE, and the third lane is a compound mCherry-PNA2 formed by covalently bonding Cherry-CCE and PNA-P2.

FIG. 5 is a graph showing the effect of EGFP-PNA1 on self-assembly with ssDNA templates at different stoichiometry. With increasing amount of protein/template, the product was converted into two complexes after assembly of EGFP-P1.

FIG. 6 is a schematic diagram of the mechanism of the self-assembly of EGPF-PNA1 with ssDNA template at different stoichiometry. As the amount of protein/template increased, the presence of different products was predicted.

FIG. 7 is a graph showing the effect of EGFP-PNA1 and mCherry-PNA2 on 1:1 precision self-assembly by ssDNA template.

FIG. 8 shows the mechanism of peptide-nucleic acid mediated protein self-assembly, wherein (1) the fusion protein containing active peptide tag, (2) the non-natural polypeptide probe containing targeting PNA sequence, (3) the covalent linkage between the active peptide tag and the non-natural polypeptide on the fusion protein, and (4) ssDNA sequence.

Detailed Description

Example 1

Vector construction:

(1) two single-stranded DNA sequences

5 '-CAAATCTGAAGAGTC-TTATGAATGTGCTGCCTTAGAGAAGGAAGTTGCAGCGTTAGA GAAGGAAGTTGCTGCATTAGAGAAGTAGA-3' and

5 '-AGCTTCTACTTCTCTAATGCAGCAACTTCCTTC-TCTAACGCTGCAACTTCCTTCTCTA AGGCAGCACATTCATAAGACTCTTCAGATTTGAGCT-3' is subjected to annealing, and a PCR purification kit is used for product purification;

(2) carrying out double enzyme digestion on pET28m-EGFP and pET28m-mCherry plasmid by using Sac I and Hind III, and recovering an electrophoresis product by using a DNA gel recovery kit;

(3) mixing the products of (1) and (2), and carrying out enzyme-linked reaction under the action of T4DNA ligase;

(4) and (4) transforming, cloning, screening and sequencing the product of the step (3) to obtain plasmids pET28m-EGFP-CCE and pET28 m-mCherry-CCE.

Protein expression and purification:

(1) the plasmids pET28m-EGFP-CCE and pET28m-mCherry-CCE which are constructed above are transformed into escherichia coli BL21 competent cells, and are selected to be monoclonal to 5mL LB culture medium for overnight culture;

(2) transferring the product in (1) to 500mL LB solution and culturing at 37 degrees, inducing with IPTG when OD600 reaches 0.6-0.8, then culturing overnight at 16 degrees;

(3) centrifuging and collecting the product in the step (2), breaking the wall by using ultrasonic waves, centrifuging again, incubating the protein supernatant with a Ni-NTA purification column, and performing gradient elution by using an imidazole solution;

(4) and (3) carrying out SDS-PAGE analysis on the product in the step (3), collecting the target protein, and carrying out solution replacement to obtain products EGFP-CCE and mCherry-CCE (shown in attached figures 3 and 4).

Example 2

The method adopts a conventional solid phase Fmoc method, namely, a solid phase resin is deprotected by Fmoc protected monomer amino acid (or peptide nucleic acid) to expose amino, and peptide bond is formed with carboxyl of the amino acid (or peptide nucleic acid) in a solution through condensation reaction, so that the amino acid (or peptide nucleic acid) is connected to the resin, and a peptide chain is extended from a C end to an N end:

resin and linker molecule: the resin selected by the solid phase Fmoc method is Rink

And (3) resin. The resin has very good swelling property, can better perform condensation reaction between peptide chains, and has enough network space to meet the growing peptide chains. HBTU and HOBt are used as connecting molecules to fix the polypeptide molecules on resin;

monomer (b): the monomer used for synthesis is chemically modified alpha-amino acid (or peptide nucleic acid);

the reaction steps are as follows:

in the first step, the first amino acid is covalently attached to the resin

Adding appropriate condensing agent such as HBTU and HOBt to make the carboxyl terminal of the protected amino acid form co-lipid with resin to complete the fixation of the amino acid;

second, deprotection

Removing Fmoc on the amino group by using an alkaline solvent of 20% piperidine to expose the amino group;

third step, activation and crosslinking

Activating carboxyl on the next amino by using activators HBTU and HOBt, and crosslinking with amino on the resin to form peptide bonds;

fourthly, repeating the second step and the third step, and repeatedly and circularly adding monomer amino acid (or peptide nucleic acid) until the synthesis is finished;

fifthly, when the unnatural amino acid is synthesized, selectively removing amino protective groups of side chains of Dap amino acids, and condensing the amino protective groups with chloroacetic acid under the action of EDC and HOBt;

and (3) post-synthesis treatment:

(1) elution and deprotection: the peptide chain was eluted from the tree branches with the deprotection agent trifluoroacetic acid (TFA) and the protecting groups were removed.

(2) HPLC analysis and purification, freeze-drying and storage.

The two non-natural polypeptide sequences synthesized by the method are

CCK-PNA-1:gactcacatc-KXALKEKVAALKEKVAALKE-NH₂(wherein X is (2S) -2-amino-3- [ (2-chloroacetyl) amino)]propanoic acid)；

CCK-PNA-2:ttaggcatca-KXALKEKVAALKEKVAALKE-NH₂(wherein X is (2S) -2-amino-3- [ (2-chloroacetyl) amino)]propanoic acid)。

Example 3

The covalent binding of the fusion protein of the invention to the polypeptide probe is based on a spontaneous reaction between cysteine on the fusion protein and the non-natural X amino acid on the polypeptide probe:

the fusion protein obtained in example 1 was subjected to concentration measurement using the BCA kit, and the fusion protein was dissolved to a concentration of 100 μ M;

the polypeptide probe obtained in example 2 was passed through A₂₆₀And dissolving the polypeptide probe to a concentration of 500. mu.M;

a200. mu.L reaction system was prepared as follows: the fusion protein final concentration of 50M, the polypeptide probe final concentration of 100M, 20mM HEPES, 150mM NaCl, 0.5mM TCEP, room temperature overnight reaction, and the use of SDS-PAGE detection of reaction efficiency (see figure 3, 4);

and (3) post-reaction treatment:

the reacted solution was separated by exclusion chromatography, and the target effluent was collected and concentrated to a final stock concentration of 20. mu.M.

The covalent cross-linking product of the fusion protein and the polypeptide probe EGFP-PNA-1 was bound to ssDNA template Td (sequence 5'-CTGAGTGTAGTACTGTGACTGAGTGTAG-3', containing two PNA-1 reverse complementary binding sites) at different ratios. As can be seen from FIG. 5, when the ratio of EGFP-PNA-1 to Td is 1:10, the solution is mainly composed of Td bound to one EGFP-PNA-1 protein and free Td; when the ratio of EGFP-PNA-1 to Td is 1:1, the solution contains Td bound to an EGFP-PNA-1 protein as a major component; when the ratio of EGFP-PNA-1 to Td is 2.5:1, the major component in the solution is Td binding two EGFP-PNA-1. The relationship between the more accurate concentration titration and the estimation of the main component in the solution is shown in FIG. 6.

Covalent cross-linking products of the fusion protein and the polypeptide probe, namely EGFP-PNA-1, mCherry-PNA-2 and ssDNA template Tb (with the sequence of 5'-CTGAGTGTAGTACTGTGAAATCCGTAGT-3', each containing a reverse complementary binding site of PNA-1 and PNA-2), are combined. As can be seen in FIG. 7, when EGFP-PNA-1, mCherry-PNA-2 and Tb were incubated in solution, precise self-assembly of EGFP-PNA-1 and mCherry-PNA-2 on Tb template 1:1 was formed.

Claims

1. A peptide nucleic acid mediated precise protein self-assembly method, which is characterized in that: the method comprises the following steps of,

(1) preparing a fusion protein containing an active polypeptide tag;

(2) preparing a non-natural polypeptide probe containing a targeting PNA sequence;

(3) covalently bonding the fusion protein containing the active polypeptide tag obtained in the step (1) with the non-natural polypeptide probe containing the targeting PNA sequence obtained in the step (2);

(4) performing controllable self-assembly on the products of the covalent bond combination in the step (3) on the ssDNA sequence;

in the step (1), a polypeptide sequence with the length of 21 natural amino acids is fused at the N end or the C end of the protein;

in the step (2), a non-natural polypeptide with the length of 10 basic groups of PNA sequences is connected to the N end or the C end;

the ssDNA sequence in the step (4) is a single-stranded DNA sequence which comprises a connecting arm and is 26 bases in length;

the non-native polypeptide sequence is:

CCK-PNA-1: gactcacatc-KXALKEVAKVAALKE-NH 2, wherein X is (2S) -2-amino-3- [ (2-chloroacetyl) amino ] propanoic acid;

CCK-PNA-2: ttaggcatca-KXALKEVAKVAALKE-NH 2, wherein X is (2S) -2-amino-3- [ (2-chloroacetyl) amino ] propanoic acid.

2. The method of claim 1, wherein the peptide nucleic acid-mediated precise self-assembly of proteins comprises: the length of the non-natural polypeptide is 21 amino acids.