CN107936086A - A kind of accurate self-assembling method of albumen of peptide nucleic acid mediation - Google Patents

A kind of accurate self-assembling method of albumen of peptide nucleic acid mediation Download PDF

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Publication number
CN107936086A
CN107936086A CN201711247179.0A CN201711247179A CN107936086A CN 107936086 A CN107936086 A CN 107936086A CN 201711247179 A CN201711247179 A CN 201711247179A CN 107936086 A CN107936086 A CN 107936086A
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China
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albumen
nucleic acid
pna
peptide nucleic
assembling method
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CN201711247179.0A
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CN107936086B (en
Inventor
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
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Changzhou University
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Changzhou University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids

Abstract

The invention belongs to biological technical field, and in particular to a kind of accurate self-assembling method of albumen of peptide nucleic acid mediation.By polypeptide sequence by one section of special PNA sequence of mark on target protein, and efficiently, accurately combined with DNA profiling by PNA, facilitate the accurate assembling of multiple target proteins.

Description

A kind of accurate self-assembling method of albumen of peptide nucleic acid mediation
Technical field
The invention belongs to biological technical field, and in particular to a kind of accurate self-assembling method of albumen of peptide nucleic acid mediation.
Background technology
Peptide nucleic acid (Peptide nucleic acid, PNA) is used as rigging.Peptide nucleic acid be by Nielsen et al. in Artificial synthesized high molecular polymer (Nielsen, the P.E. similar to DNA and RNA of invention in 1991；Egholm,M.；Berg, R.H.；Buchardt,O.Science 1991,254,1497-1500).It is primarily present following characteristics：(1) superpower combination power. Peptide nucleic acid can form good alkali with DNA (or RNA) under the driving of Watson-Crick hydrogen bonds and Hoogsteen hydrogen bonds Basigamy Thermodynamic parameters.Further, since base mispairing between PNA/DNA than more unstable between DNA/DNA, is added The characteristics of PNA electroneutral, can not produce electrical charge rejection effect between complementary DNA chain, therefore between PNA and DNA (or RNA) Binding ability be better than DNA (or RNA) itself.(2) superpower stability.Since artificial synthesized main chain backbone structure can not be by Internal various proteolytic enzymes and identified with sour hydrolase, therefore either PNA is in itself, or PNA participates in combination Any duplex structure is degraded all without by the enzyme in microbial body.
Self assembly currently for albumen is based primarily upon with lower platform：(1) skelemin.Utilize the phase between protein-protein Interaction, the method by building fusion skelemin, makes target protein be assembled in the presence of skelemin.But This method exist interact it is poor, can not preferably control ratio and too big etc. disadvantage of skelemin molecular weight between albumen End；(2) nano-particle.It is that target protein is carried out from group in nanoparticle surface using the interaction between albumen-nano-particle Dress.But this method exists and can not accurately control ratio between albumen, be unable to control albumen configuration when assembled, potential source biomolecule poison The drawbacks such as property；(3) dsDNA molecules.Using the interaction between albumen-dsDNA, target protein is set to be formed on dsDNA from group Dress.This method presence selectivity is low, is unable to control assembling ratio, can not realize the drawbacks such as accurate self assembly.
The content of the invention
The technical problem to be solved in the present invention is：In order to overcome albumen can not accurately be controlled in self assembly, poor specificity The problems such as, limit its popularization in the biological fields such as multi-enzyme system, biology sensor, protein structure regulation and control and use.To solve The above problem, the present invention provide a kind of accurate self-assembling method of albumen of peptide nucleic acid mediation：
(1) fusion protein of the label containing active peptides is prepared,
Specifically, the N-terminal or C-terminal in albumen have merged polypeptide sequence of the length for 21 natural amino acids；
(2) the non-native polypeptide probe of the PNA sequences containing targeting is prepared,
Specifically, being connected with the non-native polypeptide that length is 10 base PNA sequences in N-terminal or C-terminal, (polypeptide length is 21 Amino acid)；
(3) will be obtained in the fusion protein for the label containing active peptides that obtained in step (1) and step (2) containing targeting The non-native polypeptide probe of PNA sequences carries out Covalent bonding together,
What Covalent bonding together here specifically referred to：Non-natural X on cysteine and polypeptide probe on fusion protein Covalent bonding together between amino acid, is probably identification a certain site when covalent bond is formed between conventional polypeptide, in other words marker bit Point is random, and binding ability is not strong, and recognition site is only to be fixed in 21 amino acid on fusion protein in this patent Cysteine, has quite high reaction identification specificity；
(4) product of step (3) Covalent bonding together is subjected to controllable self assembly in ssDNA sequences,
SsDNA sequences refer to single-stranded DNA sequence of the length comprising one section of linking arm for 26 bases, it is used as and combines mould Plate.
The mode of the accurate self assembly of albumen provided by the invention synthesizes simply, and assembling is accurate, and specificity is high, bio-compatibility It is good.By polypeptide sequence by one section of special PNA sequence of mark on target protein, and by PNA and DNA profiling efficiently, precisely Combination, the accurate assembling of multiple target proteins is facilitated, so as to be carried for biological technical fields such as multi-enzyme system, biology sensors Pervasive assembly platform is supplied.
Brief description of the drawings
Fig. 1 is the HPLC phenograms of the non-native polypeptide (PNA-P1) containing PNA sequences.
Fig. 2 is the HPLC phenograms of the non-native polypeptide (PNA-P2) containing PNA sequences.
Fig. 3 is after the fusion protein (EGFP-CCE) containing active peptides label is reacted with PNA-P1 and PNA-P2 SDS-PAGE phenograms.Wherein the first swimming lane is albumen Marker, and the second swimming lane is natural EGFP-CCE, and the 3rd swimming lane is The EGFP-CCE and compound EGFP-PNA1 after PNA-P1 covalent bonds, the 4th swimming lane are covalently tied for EGFP-CCE and PNA-P2 Compound EGFP-PNA2 after conjunction.
Fig. 4 is the SDS-PAGE after fusion protein (the mCherry-CCE and PNA-P2) reaction containing active peptides label Phenogram.Wherein the first swimming lane is albumen Marker, and the second swimming lane is natural mCherry-CCE, and the 3rd swimming lane is Cherry- The CCE and compound mCherry-PNA2 after PNA-P2 covalent bonds.
Fig. 5 is self assembly design sketch of the EGFP-PNA1 from ssDNA templates under different stoichiometric numbers.With protein content/ The increase of template amount, product are converted into the compound after two EGFP-P1 assemblings.
Fig. 6 is mechanism figures of the EGPF-PNA1 from the self assembly under different stoichiometric numbers of ssDNA templates.With protein content/ The increase of template amount, predicts the existence form of different products.
Fig. 7 is that EGFP-PNA1 and mCherry-PNA2 form 1 under the action of ssDNA templates:The effect of 1 accurate self assembly Fruit is schemed.
Fig. 8 is the bonding mechanism of the accurate self assembly of albumen of peptide nucleic acid mediation, wherein (1) contains active peptides in this patent The fusion protein of label, (2) non-native polypeptide probe of PNA sequences containing targeting, the active peptides label on (3) fusion protein with The Covalent bonding together of non-native polypeptide, (4) ssDNA sequences.
Embodiment
Embodiment 1
Vector construction：
(1) by two sections of single-stranded DNA sequences
5’-CAAATCTGAAGAGTC-TTATGAATGTGCTGCCTTAGAGAAGGAAGTTGCAGCGTTAGA GAAGGAAGTTGCTGCATTAGAGAAGTAGA-3 ' and
5’-AGCTTCTACTTCTCTAATGCAGCAACTTCCTTC-TCTAACGCTGCAACTTCCTTCTCTA AGGCAGCACATTCATAAGACTCTTCAGATTTGAGCT-3 ' carries out taking off fire, and pure using PCR purification kits progress product Change；
(2) pET28m-EGFP and pET28m-mCherry plasmids are subjected to double digestion using Sac I and Hind III, and Electrophoresis product is recycled using DNA plastic recovery kits；
(3) product of above-mentioned (1) and (2) is mixed, enzyme is carried out under T4DNA connection enzyme effects and is even reacted；
(4) product of (3) is converted, colony screening and sequencing obtain plasmid pET28m-EGFP-CCE and pET28m- mCherry-CCE。
Protein expression and purifying：
(1) the plasmid pET28m-EGFP-CCE and pET28m-mCherry-CCE of above-mentioned structure are transformed into Escherichia coli In BL21 competent cells, and choose monoclonal and be incubated overnight into 5mL LB culture mediums；
(2) product in (1) is transferred in 500mL LB solution and is cultivated at 37 degree, made when OD600 reaches 0.6-0.8 Induced with IPTG, subsequent 16 degree of overnight incubations；
(3) product in (2) is collected by centrifugation, and uses ultrasonication, by albumen supernatant and Ni-NTA after centrifuging again Purification column is incubated, and carries out gradient elution using imidazole solution；
(4) product in (3) is subjected to SDS-PAGE analyses, collects target protein and carry out solution replacement, obtain product EGFP-CCE and mCherry-CCE (such as attached drawing 3,4).
Embodiment 2
Method of the present invention is using conventional solid phase Fmoc methods, i.e., by fmoc-protected monomer amino on solid-phase resin Expose amino after sour (or peptide nucleic acid) deprotection, peptide is formed by the carboxyl of amino acid (or peptide nucleic acid) in condensation reaction and solution Key, so that amino acid (or peptide nucleic acid) is connected on resin, makes peptide chain extend from C-terminal to N-terminal：
Resin and connection molecule：The resin of solid phase Fmoc methods selection is RinkResin.It is this Resin has extraordinary swellability, can make preferably to carry out condensation reaction between peptide chain, and have enough cyberspaces to expire The ever-increasing peptide chain of foot.Using HBTU and HOBt as connection molecule, peptide molecule is fixed on resin；
Monomer：Monomer used in synthesis is the a-amino acid (or peptide nucleic acid) through chemical modification；
Reactions steps：
The first step, first amino acid is covalently attached on resin
Appropriate condensing agent such as HBTU and HOBt are added, makes to form common fat with resin by protected amino acid c-terminus to complete The fixation of amino acid；
Second step, deprotection
Fmoc on amino is removed using 20% piperidines of basic solvent, exposes amino；
3rd step, activation and crosslinking
Carboxyl on next amino is activated using activator HBTU and HOBt, is crosslinked with the amino on resin, forms peptide Key；
4th step, repeats second step and the 3rd step, iterative cycles addition single amino acid (or peptide nucleic acid), until having synthesized Into；
5th step, when being related to alpha-non-natural amino acid synthesis, needs first selectivity to slough Dap amino acid side chain amino protecting groups, And it is set to be condensed under the action of EDC and HOBt with chloracetic acid；
Synthesis post processing：
(1) elute and be deprotected：Peptide chain is eluted from branch with deprotection agent trifluoroacetic acid (TFA), and is removed Protection group.
(2) HPLC analyses purifying, freezes and preserves.
Using the above method synthesize two non-native polypeptide sequences be
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)。
Embodiment 3
The fusion protein of the present invention and the covalent bond of polypeptide probe are based on the cysteine and polypeptide on fusion protein The spontaneous reaction between non-natural X amino acid on probe：
The fusion protein that embodiment 1 is obtained carries out concentration mensuration using BCA kits, and fusion protein is dissolved into 100 μM of concentration；
The polypeptide probe that embodiment 2 obtains is passed through into A260Determination of uv absorption concentration, and polypeptide probe is dissolved into 500 μM of concentration；
It is as follows to configure 200 μ L reaction systems：Above-mentioned 50 μM of fusion protein final concentration, 100 μM of aforementioned polypeptides probe final concentration, 20mM HEPES, 150mM NaCl, 0.5mM TCEP, at room temperature reaction overnight, and using SDS-PAGE detections reaction efficiency (such as Fig. 3,4)；
Post-reaction treatment：
Solution after reaction is separated by exclusion chromatography column, and target efflux is collected and concentrated, is finally stored dense Spend for 20 μM.
The covalent cross-linking product EGFP-PNA-1 of fusion protein and polypeptide probe and ssDNA templates Td (sequence 5 '- CTGAGTGTAGTACTGTGACTGAGTGTAG-3 ', the reverse complemental binding site containing two PNA-1) in different proportion Combination effect.As seen from Figure 5, when the ratio of EGFP-PNA-1 and Td is 1:When 10, main component is to combine one in solution The Td of a EGFP-PNA-1 albumen and free Td；When the ratio of EGFP-PNA-1 and Td is 1:When 1, main component is in solution Combine the Td of an EGFP-PNA-1 albumen；When the ratio of EGFP-PNA-1 and Td is 2.5:When 1, the main component in solution To combine the Td of two EGFP-PNA-1.More accurate concentration titrations and the relation that principal component in solution speculates are as shown in Figure 6.
Covalent cross-linking product EGFP-PNA-1, mCherry-PNA-2 and ssDNA template Tb of fusion protein and polypeptide probe (sequence 5 '-CTGAGTGTAGTACTGTGAAATCCGTAGT-3 ', the reverse complemental respectively containing a PNA-1 and PNA-2 Binding site) combine effect.As seen from Figure 7, when EGFP-PNA-1, mCherry-PNA-2 and Tb are incubated in the solution Afterwards, EGFP-PNA-1 and mCherry-PNA-2 is formd 1 in Tb templates:1 accurate self assembly.

Claims (5)

  1. A kind of 1. accurate self-assembling method of albumen of peptide nucleic acid mediation, it is characterised in that：The method is,
    (1) fusion protein of the label containing active peptides is prepared；
    (2) the non-native polypeptide probe of the PNA sequences containing targeting is prepared；
    (3) the PNA sequences containing targeting will be obtained in the fusion protein of the label containing active peptides obtained in step (1) and step (2) The non-native polypeptide probe of row carries out Covalent bonding together；
    (4) product of step (3) Covalent bonding together is subjected to controllable self assembly in ssDNA sequences.
  2. 2. the accurate self-assembling method of albumen of peptide nucleic acid mediation as claimed in claim 1, it is characterised in that：In step (1), The N-terminal or C-terminal of albumen have merged polypeptide sequence of the length for 21 natural amino acids.
  3. 3. the accurate self-assembling method of albumen of peptide nucleic acid mediation as claimed in claim 1, it is characterised in that：In step (2), N-terminal or C-terminal are connected with non-native polypeptide of the length for 10 base PNA sequences.
  4. 4. the accurate self-assembling method of albumen of peptide nucleic acid mediation as claimed in claim 3, it is characterised in that：The non-natural Polypeptide length is 21 amino acid.
  5. 5. the accurate self-assembling method of albumen of peptide nucleic acid mediation as claimed in claim 1, it is characterised in that：Institute in step (4) The ssDNA sequences stated refer to single-stranded DNA sequence of the length comprising one section of linking arm for 26 bases.
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Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105445350A (en) * 2015-11-13 2016-03-30 南京理工大学 Electrochemical DNA (Deoxyribose Nucleic Acid) biosensor based on peptide nucleic acid and preparation method of electrochemical DNA biosensor
CN106163570A (en) * 2014-01-21 2016-11-23 美国卫生和人力服务部 CGAP PNA multivalence peptide nucleic acid(PNA) ligand presen-tation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106163570A (en) * 2014-01-21 2016-11-23 美国卫生和人力服务部 CGAP PNA multivalence peptide nucleic acid(PNA) ligand presen-tation
CN105445350A (en) * 2015-11-13 2016-03-30 南京理工大学 Electrochemical DNA (Deoxyribose Nucleic Acid) biosensor based on peptide nucleic acid and preparation method of electrochemical DNA biosensor

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BERGER O等: "Molecular self - assembly using peptide nucleic acids", 《PEPTIDE SCIENCE》 *
FLORY J D等: "PNA-peptide assembly in a 3D DNA nanocage at room temperature", 《JOURNAL OF THE AMERICAN CHEMICAL SOCIETY》 *
曾芳等: "修饰性肽核酸的合成研究进展", 《中国药学杂志》 *

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