CN111690039B - Self-assembly polypeptide probe for identifying 6xHis tag protein, preparation method and application - Google Patents
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Abstract
The small molecular polypeptide compound R-FF prepared by the inventionPYGK (NTA), using small molecule polypeptide FFPThe N end of YGK is connected with a functional group, the C end is connected with NTA, the small molecule polypeptide compound forms nano hydrogel (nanofiber) through alkaline phosphatase catalytic self-assembly under the condition of divalent metal ions, the nano hydrogel can enter cells in a form of self-swallowing, and the functional group can be matched to indicate the subcellular distribution of the recombinant histidine target protein in the cells and research the interaction between the cells and the recombinant histidine target protein.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a self-assembly polypeptide probe for identifying 6XHis tag protein, a preparation method and application thereof.
Background
Since the first report about 30 years ago, nitrilotriacetic acid (NTA)/Histidine Tag (HT) technology has become a powerful tool in bioscience for single-step isolation and purification of proteins and enzymes modified at the N-terminus or N-terminus. However, since the NTA group forms a metal chelate with a divalent metal ion in a PBS solution with PH 7.4 in the form of a negative ion, these complexes have relatively poor cell permeability, and it is difficult to rapidly permeate a cell membrane to enter the interior of a cell to recognize the 6xHis recombinant protein in a living cell. Such as Metal Chelate Affinity Chromatography (MCAC), although NTA attachment resins are used to immobilize nickel ions (Ni)2+) The method is mainly used for separating recombinant protein from cell lysate or bacterial liquid and cannot be applied to the inside of cells. Viable cell labeling techniques require high specificity, high label density and cell permeability of the labeling molecule to target the protein of interest. Thus, selective and rapid labeling of proteins in living cells is a significant challenge.
Disclosure of Invention
The invention aims to solve the problems in the prior art, a functional group R and NTA are connected through a short peptide FFPYGK to form a small molecule polypeptide compound of R-FFPYGK (NTA), and under the condition of divalent metal ions, the self-assembly is catalyzed by alkaline phosphatase to form a nanofiber, the nanofiber can enter cells in a self-endocytic form and is used for indicating the subcellular distribution of intracellular recombinant histidine target protein and researching the intracellular interaction with the recombinant histidine target protein.
The invention relates to a self-assembly polypeptide probe for identifying 6xHis tag protein, and the polypeptide probe R-FFPYGK (NTA) has the following structural formula:
r is a functional group;
wherein, R is one of-Bio, -ANA and-G-Nap;
the structural formulas of-Bio, -ANA and-G-Nap are respectively shown as follows in sequence:
the invention also discloses a preparation method of the self-assembly polypeptide probe for identifying the 6xHis tag protein, which mainly comprises the following steps:
(1) the synthesis process of the intermediate compound Fmoc-Lys (NTA) -COOH is as follows:
i) taking tert-butyl bromoacetate, potassium carbonate and N6-Cbz-L-lysine benzyl ester hydrochloride for reaction; adding hydrochloric acid solution after reaction, extracting and drying to obtain N6-Cbz-Lys (NTA) -OtBu;
ii) dissolving N6-Cbz-Lys (NTA) -OtBu in methanol solution, adding palladium carbon, and reacting at room temperature under the condition of introducing hydrogen to obtain NH2-Lys(NTA)-COOH；
iii) taking NH2-lys (nta) -COOH is dissolved in sodium bicarbonate solution, Fmoc-OSu is dissolved in acetonitrile, the two are mixed and react at room temperature to obtain Fmoc-lys (nta) -COOH;
(2) the synthesis process of Bio-FFpYGK (NTA), ANA-FFpYGK (NTA), Nap-GFFpYGK (NTA) is as follows:
iv) connecting Fmoc-Lys (NTA) -COOH synthesized in (1) to a solid phase carrier 2-chlorotrityl chloride resin in a solid phase synthesizer, and reacting for 1-3 hours by using dichloromethane as a solvent;
v) removing a protecting group Fmoc by using a piperidine dimethylformamide solution, putting a polypeptide condensing agent HBTU into the solid phase synthesizer in the step iv), and sequentially and circularly adding Fmoc-glycine, Fmoc-phosphotyrosine, Fmoc-phenylalanine and Fmoc-glycine, wherein the N end of the polypeptide is respectively capped by biotin, ANA and naphthylacetic acid, so that Bio-FFpYGK (NTA), ANA-FFpYGK (NTA) and Nap-GFFpYGK (NTA) can be respectively obtained;
vi) the prepared short peptide was cleaved from the resin using TFA as a cleavage solution, and the obtained Bio-FFpYGK (NTA), ANA-FFpYGK (NTA), Nap-GFFpYGK (NTA) were separated.
The invention also discloses a method for applying the self-assembly polypeptide probe for identifying the 6xHis tag protein to a small molecule hydrogel nanofiber transmission system, and R-FFPYGK (NTA) small molecule compound and divalent metal ion combine to form NTA metal complex, under the action of cell membrane surface alkaline phosphatase (ALP), self-assembly forms into nanofiber that can enter cells in endocytosis mode.
Wherein the divalent metal ion is Zn2+、Cu2+、Co2+、Ni2+One kind of (1).
It should be noted that all amino acids used in the present invention are all natural amino acids in L configuration.
The glycine linked to Nap in Nap-GFFpYK (NTA) plays a role in linking, and is used for maintaining a carbon chain with the same length as biotin and ANA, and is mainly used for strict biological control experiments.
The invention has the following beneficial effects:
(1) the small molecular polypeptide compound R-FF prepared by the inventionPYGK (NTA), using small molecule polypeptide FFPThe N end of YGK is connected with a functional group, the C end of YGK is connected with NTA, the small molecule polypeptide compound forms nano hydrogel (nanofiber) through catalysis and self-assembly of alkaline phosphatase under the condition of divalent metal ions, the nano hydrogel can enter cells in a form of self-swallowing, and the functional group can be matched to indicate the subcellular distribution of the recombinant histidine target protein in the cells and research the interaction between the cells and the recombinant histidine target protein;
(2) the small molecular polypeptide compound R-FF of the inventionPThe raw materials used in the YGK (NTA) preparation process are various amino acids required by cultured cells every day, and do not generate toxic or side effect on the cells, and the MTT results of the prepared Bio-FFpYGK (NTA), ANA-FFpYGK (NTA) and Nap-GFFpYGK (NTA) show that the cell growth is not inhibited;
(3) the nano hydrogel prepared by the invention has good biocompatibility and good three-dimensional appearance (about 10nm nano-fiber), can more easily enter tumor cells, and can be widely applied to the aspects of biomedicine and biological materials.
Drawings
FIG. 1 is a scheme showing the scheme of the chemical synthesis of Fmoc-L-Lys (NTA) -COOH;
FIG. 2 shows R-FFPChemical synthesis scheme of YGK (NTA);
FIG. 3 is a mass spectrum of Bio-FFpYGK (NTA);
FIG. 4 is a mass spectrum of ANA-FFpYGK (NTA);
FIG. 5 is a mass spectrum of Nap-GFFpYGK (NTA);
FIG. 6 shows Nap-GFFpYGK (NTA) (Ni)2+)、Bio-FFpYGK(NTA)(Ni2+)、ANA-FFpYGK(NTA)(Ni2+) Three-dimensional topography images of three kinds of small molecular hydrogel observed by a transmission electron microscope;
FIG. 7 shows ANA-FF in AD293 cellsPYGK (NTA) indicates the nuclear entry of the protein with 6xHisARNT, DIPA is the blue fluorescence of nuclear dye with wavelength of 488nm under 408nm excitation light; ANA has an excitation wavelength of 488nm and emits green fluorescence with a wavelength of 520 nm.
FIG. 8 shows Bio-FFPYGK (NTA) (Ni) in AD293 cells2+) Validation graph for ARNT protein with 6XHis interaction with HIF-1 α and HIF-2 α.
Detailed Description
Unless defined otherwise, technical terms used in the following examples have the same meanings as commonly understood by one of ordinary skill in the art to which the present invention belongs. The test reagents used in the following examples, unless otherwise specified, are all conventional biochemical reagents; the experimental methods are conventional methods unless otherwise specified. The present invention will be described in detail with reference to examples.
EXAMPLE Synthesis of Fmoc-L-Lys (NTA) -COOH
Tert-butyl bromoacetate (20mmol, 3.9 g), potassium carbonate (20mmol, 2.8 g), N6-Cbz-L-lysine benzyl ester hydrochloride (10mmol, 4.07 g) were taken in a round bottom flask (500 mL) containing 150 mL of N, N-Dimethylformamide (DMF), and reacted at 65 ℃ for 12 hours. Adding 300 ml of 1mol/L hydrochloric acid solution, extracting with 200 ml of ethyl acetate three times respectively, collecting ethyl acetate layers, and 100 g of anhydrous sulfuric acidSodium was dried for 12 hours. Purification on a silica gel column gave 4.5g of Cbz-Lys (NTA) -OtBu in 75.25% yield. 4.5g (7.53mmol) of N6-Cbz-Lys (NTA) -OtBu were dissolved in 250 ml of methanol solution, 2 g of 10% palladium on carbon was added, and the reaction was carried out at room temperature for 24 hours under the introduction of hydrogen gas to obtain 2.25 g of NH2-Lys (NTA) -COOH. 2.25 g (6mmol) of NH were taken2-Lys (NTA) -COOH was dissolved in 50 ml of saturated sodium bicarbonate solution, 2.03 g (6mmol) of Fmoc-OSu was dissolved in 50 ml of acetonitrile, and the two were mixed, reacted at room temperature for 24 hours, and purified by silica gel column chromatography to obtain 2.79 g of Fmoc-Lys (NTA) -COOH as a white solid with a yield of 77.92%.
The synthesis steps of this example can be referred to in FIG. 1.
Example two R-FFPSynthesis of YGK (NTA)
Synthesis of R-FF by solid phase synthesisPYGK (NTA), the specific synthesis steps are as follows:
(1) 0.75mmol (700 mg) of the dichloro resin was weighed into a solid phase synthesizer, 20 ml of Dichloromethane (DCM) was added to immerse the resin and swell the resin for 20 minutes, DCM solvent was removed, 1mmol (596 mg) of Fmoc-L-Lys (NTA) -COOH in DCM was added and 800. mu.l of N, N-Diisopropylethylamine (DIPEA) was added. The reaction was carried out for 2 hours. After which it was washed five times with DCM for 2 minutes each. Using volume ratio DCM: methanol: DIPEA ═ 17: 2: 1 for 30 minutes. The reaction mixture was washed 5 times with 20 ml of DCM solution each time for 1 minute. Each time with 20 ml of DMF solution, 5 washes for 2 min each time. Then 20 ml of 20% piperidine in DMF was added and reacted for 30 minutes to remove the Fmoc protecting group and expose the amino group for the next reaction.
(2) 1mmol of Fmoc-glycine (297 mg), 1ml of DIPEA, 1mmol of polypeptide condensing agent HBTU (380 mg) were added, reacted for 2 hours, washed 5 times for 2 minutes with 20 ml of DMF solution. 20 ml of 20% piperidine in DMF was added and reacted for 30 minutes to remove the Fmoc protecting group and expose the amino group for the next reaction.
(3) Repeating the experiment step (2), wherein the amino acid protected by Fmoc added each time is only required to be replaced, and the amino acids are sequentially linked on the resin; the last added end capping group is: ANA-COOH, biotin, naphthylacetic acid.
(4) After the reaction was complete, the reaction mixture was washed 5 times with 20 ml of DMF solution for 2 minutes each. The reaction mixture was washed 5 times with 20 ml of DCM solution each time for 1 minute. With 95% TFA (TFA: H)2O: TIS 95%: 2.5%: 2.5%) for 1 hour, washing twice with 20 ml of 1% TFA in dichloromethane, collecting the total cleavage solution, removing the solution by rotary evaporation, and separating with high performance liquid to obtain polypeptide compounds Bio-FFpYGK (NTA), ANA-FFpYGK (NTA), Nap-GFFpYGK (NTA).
The synthesis steps of this embodiment can be referred to fig. 2;
the mass spectra of Bio-FFpYGK (NTA), ANA-FFpYGK (NTA), Nap-GFFpYGK (NTA) are shown in FIG. 3, FIG. 4 and FIG. 5, respectively.
EXAMPLE three Small molecule hydrogels R-FFPYGK(NTA)(Ni2+) Formation of
The formation steps of the small molecule hydrogel are as follows:
(1) 2.00mg of the obtained polypeptide compound was weighed, 1ml of PBS (pH 7.4 buffer) was added to adjust the pH to 7.4 with sodium carbonate to form a probe solution, and the divalent metal ion Ni having the same concentration as NTA in the probe solution was added2+. NTA is first centrifuged with the metal to form a metal complex. Adding alkaline phosphatase solution (final enzyme concentration of 90U/ml), standing overnight, and turning the bottle upside down to obtain Nap-GFFpYGK (NTA) (Ni)2+)、Bio-FFpYGK(NTA)(Ni2+)、ANA-FFpYGK(NTA)(Ni2+)。
(2) Sucking about 15 microliters of colloid by using a pipette gun, dropwise adding the colloid on a silicon wafer with a clean surface, spin-coating to ensure that the colloid is uniformly distributed on the silicon wafer, and observing the appearance by using a transmission electron microscope (see figure 6); the appearance of the nano-fiber is about 10nm of nano-fiber which is uniformly dispersed.
Example biological applications of tetra R-FFPYGK (NTA)
(1) Construction of C6His tagged ARNT vector
Human ARNT protein coding region DNA fragments were synthesized according to GenBank public data. Connecting PTOPO vector, transforming competent cell, picking single clone and sequencing. The PTOPO-ARNT is constructed correctly. His sequence and restriction sites BamHI and HindIII were introduced by PCR. The vector of PTOPO-C6His-ARNT, PTOPO-N6His-ARNT and PTOPO-ARNT was successfully constructed. PTOPO-C6His-ARNT, PTOPO-N6His-ARNT and PTOPO-ARNT are digested, PCDNA3.0 vector connects C6His-ARNT, N6His-ARNT and ARNT to PCDNA3.0 vector, and PCDNA3.0-C6His-ARNT, PCDNA3.0-N6His-ARNT and PCDNA-ARNT plasmids are constructed.
(2) AD293 cells were transfected with ARNT plasmids with 6XHis tags at the N-and C-termini, all three plasmids being expressed in both cells. Under the condition of hypoxia, ARNT in the cell can be specifically combined with hypoxia inducible factor HIF and enter the nucleus to play the role of nuclear transcription factor. Using ANA-FFPYGK (NTA) fluorescent indicator probe, which binds to ARNT with 6XHis tag after entering cells, and ANA-FF is induced by hypoxiaPYGK (NTA) can enter the nucleus with ARNT. The fluorescence intensity inside the cell nucleus was significantly enhanced as seen by confocal microscopy. Under the condition of hypoxia, apigenin can inhibit the expression of hypoxia inducible factor, reduce the combination of ARNT with HIF-1 alpha and HIF-2 alpha, make the amount of entering cells less, and weaken the fluorescence intensity of cell nucleus. Thus ANA-FFPYGK (NTA) can specifically identify ARNT protein with 6XHis tag at N-terminal and C-terminal in AD293 cells. That is, the ARNT protein with the 6XHis tag can be tagged, as shown in FIG. 7.
(3) AD293 was also transfected with ARNT plasmid with 6XHis tag at the N-and C-termini. Under the induction of hypoxia, ARNT and hypoxia inducible factor HIF are specific to form heterodimer. The use of enrichment of Bio-FFPYGK (NTA) probe, streptavidin purification resin, successfully enriched ARNT protein, and interaction with ARNT HIF-1. alpha. and HIF-2. alpha. see FIG. 8, can be used to study the 6XHis tag protein and interaction with 6XHis tag protein relationship.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents, improvements and the like that fall within the spirit and principle of the present invention are intended to be included therein.
Claims (4)
1. A self-assembly polypeptide probe for recognizing 6xHis tag protein, characterized in that the polypeptide probe R-FFPYGK (NTA) has the following structural formula:
r is one of-Bio and-ANA;
-Bio and-ANA are respectively shown as follows:
2. the method for preparing a self-assembled polypeptide probe capable of recognizing 6 XHis-tagged protein according to claim 1, which comprises the steps of:
(1) the synthesis process of the intermediate compound Fmoc-Lys (NTA) -COOH is as follows:
i) taking tert-butyl bromoacetate, potassium carbonate and N6-Cbz-L-lysine benzyl ester hydrochloride, and putting the tert-butyl bromoacetate, the potassium carbonate and the N6-Cbz-L-lysine benzyl ester hydrochloride into N, N-dimethylformamide for reaction; after reaction, adding hydrochloric acid solution, extracting and drying to obtain N6-Cbz-Lys (NTA) -OtBu;
ii) dissolving N6-Cbz-Lys (NTA) -OtBu in methanol solution, adding palladium carbon, and reacting at room temperature under the condition of introducing hydrogen to obtain NH2-Lys(NTA)-COOH；
iii) taking NH2-lys (nta) -COOH is dissolved in saturated sodium bicarbonate solution, Fmoc-OSu is dissolved in acetonitrile, the two are mixed and react at room temperature to obtain Fmoc-lys (nta) -COOH;
(2) the synthesis of Bio-FFpYGK (NTA), ANA-FFpYGK (NTA) is as follows:
iv) connecting Fmoc-Lys (NTA) -COOH synthesized in (1) to a solid phase carrier 2-chlorotrityl chloride resin in a solid phase synthesizer, and reacting for 1-3 hours by using dichloromethane as a solvent;
v) removing a protecting group Fmoc by using a dimethyl formamide solution of piperidine, taking a polypeptide condensing agent HBTU, putting the polypeptide condensing agent HBTU into the solid phase synthesizer in the step iv), and sequentially and circularly adding Fmoc-glycine, Fmoc-phosphotyrosine and Fmoc-phenylalanine, wherein the N end of the polypeptide is respectively capped by biotin and ANA to obtain Bio-FFpYGK (NTA) and ANA-FFpYGK (NTA);
vi) the prepared short peptide was cleaved from the resin using TFA as a cleavage solution, and the obtained Bio-FFpYGK (NTA) and ANA-FFpYGK (NTA) were separated, respectively.
3. The application of the self-assembled polypeptide probe for recognizing 6XHis tag protein in the preparation of small molecule hydrogel nanofiber delivery system as claimed in claim 1, wherein R-FFPYGK (NTA) small molecular compound and divalent metal ion combine to form NTA metal complex, under the action of cell membrane surface alkaline phosphatase, self-assembly forms into nanofiber that can enter cells in endocytosis mode.
4. The application of the self-assembled polypeptide probe for recognizing the 6 XHis-tagged protein in the preparation of the small molecule hydrogel nanofiber delivery system according to claim 3, wherein the divalent metal ion is Zn2+、Cu2+、Co2+、Ni2+One kind of (1).
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Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102120756A (en) * | 2010-12-07 | 2011-07-13 | 南开大学 | Taxol-based small molecule hydrogel-nanosphere transmission system and preparation method thereof |
| WO2013091661A2 (en) * | 2011-12-23 | 2013-06-27 | Aarhus Universitet | Proteolytic resistant protein affinity tag |
| CN107929752A (en) * | 2017-11-02 | 2018-04-20 | 南方医科大学 | A kind of taxol anticancer nano medicine being formed in situ |
| CN109957000A (en) * | 2017-12-14 | 2019-07-02 | 南开大学 | A kind of polypeptide derivative and preparation method and application promoting cell Proliferation |
| CN110456053A (en) * | 2018-05-07 | 2019-11-15 | 中国科学院化学研究所 | A kind of divalent target polypeptide probe and preparation method thereof |
| CN110721314A (en) * | 2019-10-11 | 2020-01-24 | 江苏恒泰生物科技有限公司 | Anti-tumor nano-drug and preparation method thereof |
-
2020
- 2020-07-02 CN CN202010626263.9A patent/CN111690039B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102120756A (en) * | 2010-12-07 | 2011-07-13 | 南开大学 | Taxol-based small molecule hydrogel-nanosphere transmission system and preparation method thereof |
| WO2013091661A2 (en) * | 2011-12-23 | 2013-06-27 | Aarhus Universitet | Proteolytic resistant protein affinity tag |
| CN107929752A (en) * | 2017-11-02 | 2018-04-20 | 南方医科大学 | A kind of taxol anticancer nano medicine being formed in situ |
| CN109957000A (en) * | 2017-12-14 | 2019-07-02 | 南开大学 | A kind of polypeptide derivative and preparation method and application promoting cell Proliferation |
| CN110456053A (en) * | 2018-05-07 | 2019-11-15 | 中国科学院化学研究所 | A kind of divalent target polypeptide probe and preparation method thereof |
| CN110721314A (en) * | 2019-10-11 | 2020-01-24 | 江苏恒泰生物科技有限公司 | Anti-tumor nano-drug and preparation method thereof |
Non-Patent Citations (3)
| Title |
|---|
| Identification of Small Molecule Inhibitors of Human As(III) S-Adenosylmethionine Methyltransferase (AS3MT);Hui Dong等;《Chem. Res. Toxicol.》;20151117;第28卷(第12期);全文 * |
| Live-Cell Targeting of His-Tagged Proteins by Multivalent N-Nitrilotriacetic Acid Carrier Complexes;Ralph Wieneke等;《Journal of the American Chemical Society》;20140919;第136卷(第40期);全文 * |
| Opioid peptides: synthesis and biological activity of new endomorphin analogs;Biondi, Barbara等;《International Journal of Peptide Research and Therapeutics》;20060324;第12卷(第2期);全文 * |
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