CN115491191B - Fluorescent gold nanocluster based on chaperonin GroEL protection, preparation method and application thereof - Google Patents

Fluorescent gold nanocluster based on chaperonin GroEL protection, preparation method and application thereof Download PDF

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CN115491191B
CN115491191B CN202211299401.2A CN202211299401A CN115491191B CN 115491191 B CN115491191 B CN 115491191B CN 202211299401 A CN202211299401 A CN 202211299401A CN 115491191 B CN115491191 B CN 115491191B
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吴玉清
李洪伟
张岩
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Jilin University
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Abstract

Fluorescent gold nanocluster based on chaperonin GroEL protection, preparation method and application thereof in GroEL inhibitor screening, and belongs to the technical field of fluorescent probes. The fluorescent probe is prepared by using chloroauric acid trihydrate (HAuCl) 4 ·3H 2 O) as Au source, groEL protein as reducer and final ligand stabilizer, and the preparation of fluorescent gold nanoclusters by GroEL protein has never been reported before, and experiments prove that the fluorescent gold nanoclusters have good fluorescence stability; furthermore, groEL proteins based on chaperonin GroEL protected fluorogold nanoclusters substantially retain the original secondary structure and exhibit no loss of original activity in ATPase-like activity assays. Thus, the good luminescent properties of auncs@groel can be exploited for screening of GroEL inhibitors based on the preservation of intact protein structure and function.

Description

Fluorescent gold nanocluster based on chaperonin GroEL protection, preparation method and application thereof
Technical Field
The invention belongs to the technical field of fluorescent probes, and particularly relates to a fluorescent gold nanocluster based on chaperonin GroEL protection, a preparation method and application thereof in GroEL inhibitor screening.
Background
GroEL is a protein derived from Escherichia coli, and is composed of two stacked heptameric ring subunits, each of 57kDa in size, which has important auxiliary effects on folding and assembling of newly synthesized peptide chains [1] . It is well known that naturally correctly folded proteins play an important role in the normal physiological functions of an organism; however, misfolded proteins (i.e., non-native polypeptides) are prone to disabling by aggregation of the protein, and even causing damage. GroEL can act synergistically with the chaperone GroES (70 kDa homoheptamer) to transfer in an ATP-dependent manner an unnatural polypeptide exposed in a hydrophobic region into a GroEL cavity with hydrophobic residues; subsequently, ATP hydrolysis causes the substrate to release energy, assisting in the correct folding of the nascent peptide chain [2] . Thus, groEL and GroES play an important role in correctly folding proteins in bacteria and ultimately ensuring their proper functioning. In addition, groEL is also critical to bacterial growth, since all bacteria contain at least one GroEL homolog (essential under all conditions). Therefore, the development of GroEL targeted inhibitors can be used as an important antibacterial means with broad spectrum applicability, and can effectively avoid the generation of drug-resistant bacteria caused by antibiotics. However, the related research on GroEL inhibitors is still in the primary stage, and screening GroEL effective inhibitors has important research significance [3] . Moreover, groEL can be used as a transport machine for hydrophobic drugs due to its special cage structure and ATP utilization mechanism, and GroEL has been reported to be a natural targeting vector for tumor drugs [4] . Therefore, developing GroEL with fluorescent tag has important scientific significance for researching its action mechanism, playing its function and further expanding its application.
Metal Nanoclusters (NCs) having a smaller size (2.2 nanometers) can fluoresce when protected by an appropriate ligand. Compared with the traditional fluorescent dye, partial amino acid (amino acid, cysteine, etc.) residues in the protein can be used as a reducing agent of metal ions, so that stable and protein-protected metal nanoclusters can be formed; moreover, due to the smaller size of NCs, it is generally not apparentThe original secondary structure and biological function of the protein are obviously changed [5] . As an important protein for bacterial growth, the amino acid residue and cysteine (Cys) residue contained in GroEL protein sequence can be used as a reducing agent to reduce chloroauric acid into gold atoms and stabilize the gold atoms in the form of protective ligands, so that gold nanoclusters with fluorescence effect can be prepared. Fluorescent NCs synthesized by utilizing biological macromolecular proteins have the characteristics of high safety, good biocompatibility, high stability and the like.
In addition, the GroEL and the viral capsid protein are co-expressed in the escherichia coli, so that the soluble expression of the target protein can be improved, and the target protein can be distributed in a large amount in a supernatant solution after centrifugal purification, and the expression and purification efficiency of the target protein are effectively improved. However, groEL proteins separated from the target proteins are often discarded as waste after completion of the mission, resulting in waste. The invention utilizes the abandoned GroEL proteins to prepare fluorescent metal nanoclusters, provides a novel fluorescent material AuNCs@GroEL based on waste utilization, can especially use the novel fluorescent material AuNCs@GroEL as a fluorescent probe to reveal the interaction of GroEL and other proteins, and can screen GroEL inhibitors based on the fluorescence change of the contained AuNCs, thereby having very important scientific significance for deeply revealing the functions and the action mechanism of GroEL.
Disclosure of Invention
The invention aims to provide a fluorescent gold nanocluster (AuNCs@GroEL) based on chaperonin GroEL protection and application thereof in GroEL inhibitor screening. The preparation of the fluorescent gold nanocluster serving as a fluorescent probe material by using GroEL protein has never been reported before, and experiments prove that the fluorescent gold nanocluster has good fluorescence stability; furthermore, groEL proteins based on chaperonin GroEL protected fluorogold nanoclusters substantially retain the original secondary structure and exhibit no loss of original activity in ATPase-like activity assays. Therefore, based on the preservation of intact protein structure and function, the use of good luminescence properties of auncs@groel can be used for screening of GroEL inhibitors.
The invention first prepares a purified GroEL solution. First, 1-3 mL of Escherichia coli containing GroEL and RDPV plasmids was added to 100mL of LB medium containing 50. Mu.g/mL of kanamycin and 20. Mu.g/mL of chloramphenicol double antibody, at 37℃for 220r/min overnight, and then added at 1:200 volume ratio is inoculated into 1L LB culture medium containing the double antibody, 2g/L L-arabinose induction bacteria are added to express RDPV and GroEL at 37 ℃ and 220r/min until the OD600 value reaches 0.6-0.8, and then 100 mu L IPTG and 1mol/L IPTG are added to induce expression at 37 ℃ and 220r/min for 14-16 h; the strain after induced expression is collected by centrifugation at 3000-5000 r/min for 25-30 min, and then 8-12 mL of PBS with pH of 7.4 is added to be resuspended in a 50mL centrifuge tube; the obtained bacterial liquid is prepared according to the following ratio of 1: adding the schizolysis liquid into the mixed solution according to the volume ratio of 10 for ultrasonic crushing for 5s and 5s intermittently, wherein the effective ultrasonic time is 25-35 min, centrifuging the mixed solution for 20-30 min at 16000r/min after ultrasonic crushing, taking the supernatant, and adding a saturated ammonium sulfate solution to ensure that the final mass concentration of ammonium sulfate in the supernatant is 30%; stirring at 4 ℃ for 1h, centrifuging at 10000r/min for 25-35 min, separating supernatant and precipitate, and re-suspending the precipitate with PBS (phosphate buffer solution) in equal volume to obtain a 30% ammonium sulfate precipitate re-dissolved sample; sequentially and slowly adding sucrose solutions with mass fractions of 60%, 50%, 40% and 30% into a sucrose density gradient centrifuge tube, wherein each gradient is 2mL; taking a 30% ammonium sulfate precipitation re-dissolved sample, filling the sample into a centrifuge tube, balancing, placing the sample into an overspeed centrifuge, and centrifuging for 4 hours at 4 ℃ at 35000-40000 r/min; sucking out GroEL solution on the upper layer of the centrifuge tube, dialyzing with PBS to remove sucrose, and purifying protein with AKTA to obtain purified GroEL solution.
The invention relates to a fluorescent gold nano-cluster (AuNCs@GroEL) based on chaperonin GroEL protection, which is prepared by adding HAuCl into 100 mu L GroEL solution 4 Mixing the aqueous solution, groEL and HAuCl 4 The molar ratio of the dosage is 10-15: 6, preparing a base material; then adding 20 μl of 1M NaOH aqueous solution, mixing thoroughly, depolymerizing GroEL protein to generate amino acid and adding HAuCl 4 The gold ions in (a) are reduced to Jin Yuanzi; and then reacting for 1.5-3.0 h at 50-70 ℃, dialyzing for 24h in 1 XPBS (pH=7.4) by using a 10kDa dialysis bag after the reaction is finished, and taking out and storing at-20 ℃ for standby, wherein GroEL has the dual functions of a reducing agent and a protective ligand, wherein the solution in the bag is the fluorescent gold nanocluster (AuNCs@GroEL) based on chaperonin GroEL protection.
Optimized to obtain when GroEL and HAuCl 4 The molar ratio of (2) is 11: at 6, the fluorescence emission peak of the prepared AuNCs@GroEL is strongest. After dialysis renaturation, the fluorescent dye emits strong red light with an emission center at 675nm under the irradiation of 380nm excitation wavelength. Further, fluorescence stability monitoring was performed on auncs@groel, and the results showed (fig. 4): auNCs@GroEL has good fluorescence stability (4 ℃) within a period of one week, and can be fully used as a stable fluorescent probe for further research. Finally, by comparing the infrared spectra of GroEL and AuNCs@GroEL, it was confirmed that the secondary structure of GroEL was not destroyed after synthesis of AuNCs@GroEL. ATPase Activity of AuNCs@GroEL was measured by malachite green method, and the GroEL solution and the AuNCs@GroEL solution were first treated with folding buffer (100 mL of folding buffer aqueous solution containing 50mM Tris pH7.4, 50mM KCl, 10mM MgCl was prepared) 2 1mM DTT) to 40% and 20% of the original volume. mu.L of diluted GroEL solution and AuNCs@GroEL solution, 20. Mu.L×2mM ATP solution, 120. Mu.L of malachite green reporter solution (0.034% w/v malachite green, 1.04% w/v ammonium molybdate, 1% Tween-20 and H-soluble) were added to 96-well plates 2 1M HCl of O), absorbance at 595nm was detected [7] . The results show that: auNCs@GroEL still retains the original ATPase activity compared to GroEL protein.
Evans Blue (Evans Blue) is a small molecule inhibitor of GroEL reported in the literature [8] Evans Blue solution with final concentration of 10uM is added into AuNCs@GroEL, and the mixture is incubated in a water bath kettle at 37 ℃ for 30min, and fluorescence monitoring is carried out on the mixed solution, so that the fluorescence of the AuNCs@GroEL is quenched, and the AuNCs@GroEL can be used as a fluorescent probe for the GroEL and an inhibitor thereof, and therefore, the method has application in GroEL inhibitor screening.
Drawings
Fig. 1: SDS-PAGE electrophoresis of the protein extracted from the crushed cells after inducing the co-expression of GroEL and RDPV of Escherichia coli, wherein the first lane is a marker, the second lane is a supernatant centrifuged after ultrasonic crushing, and the third and fourth lanes are supernatant and precipitate centrifuged after precipitating the supernatant by adopting ammonium sulfate.
Fig. 2: protein distribution of GroEL protein is separated by sucrose gradient centrifugation and SDS-PAGE electrophoresis image, (a) is a layering photograph after sucrose gradient centrifugation, and (b) is a SDS-PAGE gel electrophoresis image of corresponding layering, groEL is 56kDa, and GroEL is mainly distributed in layer 1.
Fig. 3: SDS gel electrophoresis of GroEL after AKTA purification. The first lane is the eluent after the purification of the uppermost solution in the centrifuge tube of FIG. 2, and the second and third lanes are the flow-through liquid after the purification.
Fig. 4: to 400. Mu.L GroEL solution (10.4 mg/mL) was added HAuCl at various concentrations 4 The fluorescence spectrum of AuNCs@GroEL prepared later has an excitation wavelength of 380nm.
Fig. 5: (a) Transmission electron microscopy (HR-TEM) image of AuNCs@GroEL, (b) grain size distribution profile of AuNCs@GroEL. Morphology characterization was performed on auncs@groel prepared under optimized conditions (fig. 5 a). From the figure, it can be seen that the nanoparticle has a high dispersibility and a uniform particle size (fig. 5 b). The average grain size was found to be-2.94 nm by systematic analysis of about 200 grains.
Fig. 6: fluorescence change curves of AuNCs@GroEL before and after dialysis and after one month of standing; the excitation wavelength was 380nm.
Fig. 7: infrared spectra of GroEL and auncs@groel at solid absorbance.
Fig. 8: groEL and AuNCs@GroEL were diluted with buffer and ATP was added, incubated at 37℃for 45min and after addition of malachite green reporter at 595 nm.
FIG. 9a is a photograph of 40% diluted GroEL, auNCs@GroEL and protein-free ATP folding buffer after addition of ATP solution, respectively after reaction at 37deg.C for 0min, 5min, 10min, 15min, 20min (top-down) and after addition of malachite green reporter solution; FIG. 9b is a graph of absorbance versus 595nm for the three substances of FIG. 9a at different reaction times, plotted using a GraphPad for non-linear regression analysis.
Fig. 10: fluorescence spectrum after AuNCs@GroEL and Evans Blue reacted, excitation wavelength was 380nm.
The AuNCs@GroEL fluorescent probe is prepared by using chloroauric acid trihydrate (HAuCl) 4 ·3H 2 O) asAs Au source, groEL protein as reducing agent and final ligand stabilizer; the preparation method comprises the following steps:
coli containing RDPV and GroEL plasmids (strain preparation method is referred to the university of Changchun Industrial university Lei Huan's Shuoshi treatise) [8] ) Culturing overnight in 100mL LB culture medium containing 50 mug/mL kanamycin and 20 mug/mL chloramphenicol (double antibody), transferring 2mL of the overnight cultured bacterial liquid into 1L LB culture medium containing the double antibody for continuous culture, adding 100 mug and 1M IPTG and L-arabinose-induced bacterial body with the final concentration of 2g/L to express RDPV and chaperone protein (GroEL) after culturing the bacterial strain to a certain concentration (OD value of 0.6-0.8 and about 4 h), collecting bacterial liquid after protein expression, and carrying out crushing centrifugation, wherein GroEL increases the solubility of RDPV protein, so that GroEL protein is mostly present in the crushed supernatant. The supernatant was further salted out with a 30% saturated ammonium sulfate solution for 1h, centrifuged at 10000r/min for 30min, and GroEL (56 kDa) was found to be present in the precipitate after salting out by SDS-PAGE. As shown in fig. 1.
And the protein is purified by adopting an ammonium sulfate precipitation and sucrose gradient ultracentrifugation method, so that the operation is simple and the purification efficiency is high. 60%, 50%, 40% and 30% sucrose were respectively prepared, and GroEL proteins after sucrose gradient centrifugation were mainly distributed in the upper layer of the centrifuge tube (see reference point in FIG. 2 a). The proteins extracted from the tubes in layers from top to bottom were further confirmed by SDS-polyacrylamide gel electrophoresis (see FIG. 2 b), and the electrophoresis results showed that GroEL (56 kDa) was distributed mainly in the first layer of the tube. Purifying the separated GroEL protein by AKTA to obtain a purified GroEL solution; DS-Polyacrylamide gel electrophoresis (see FIG. 3 for indication) shows that the purer GroEL protein was isolated around 56 kDa.
Investigation of GroEL and HAuCl 4 As shown in FIG. 4, 5, 8, 10, 12. Mu.L of HAuCl were added to 100. Mu.L of the purified GroEL solution (10.4 mg/mL), respectively 4 (10 mM) solution and 20. Mu.L NaOH (1 mol/L). After a reaction time of 2 hours at 60℃the reaction was stopped and the resulting crude product was subjected to fluorescence spectroscopy (. Lamda.) ex =380 nm). The results show that: adding 10. Mu.L of HAuCl 4 The obtained AuNCs@GroEL fluorescent intensityThe degree is strongest. Thereby obtaining GroEL and HAuCl 4 The molar ratio of (2) is 11: the fluorescence intensity of AuNCs@GroEL prepared at 6 was the strongest.
The morphology of AuNCs@GroEL prepared under optimized conditions was characterized by HR-TEM (FIG. 5 a). From the figure, it can be seen that the nanoparticle has a high dispersibility and a uniform particle size (fig. 5 b). The average grain size was found to be-2.94 nm by systematic analysis of about 200 grains.
FIG. 6 shows fluorescence spectra of AuNCs@GroEL system before and after dialysis and one month after dialysis, and shows that AuNCs@GroEL has better fluorescence stability and stronger fluorescence intensity after being placed for 1 month.
FIG. 7 is an infrared spectrum of GroEL and AuNCs@GroEL under solid state light absorption, and an infrared absorption test is performed on GroEL and AuNCs@GroEL by using a calcium fluoride sheet.
The enzyme activity was measured by the malachite green method using ATP as a substrate and GroEL and AuNCs@GroEL respectively. If AuNCs@GroEL still has GroEL activity, it can decompose the ATP substrate into phosphate groups (Pi); phosphate radical and molybdate radical can form phosphomolybdic acid association under acidic condition, the complex and malachite green form weak bond complex as chromogenic complex, malachite green not forming complex can fade under higher acidity, chromogenic complex is stable, and chromogenic complex has obvious light absorption under 595nm [9] 。
The effect of the dilution of AuNCs@GroEL on the sensitivity of the assay was tested, and FIG. 8 is a bar graph of absorbance at 595nm after incubation at 37℃for 45min for 40%, for 20% AuNCs@GroEL and GroEL, showing a dilution of 20% AuNCs@GroEL and GroEL significantly below the dilution of 40%. Thus, a 40% dilution was selected for GroEL and AuNCs@GroEL ATPase-like assay.
The time profile of absorbance at 595nm from the sample shows that AuNCs@GroEL still retains the ATPase activity of GroEL (FIG. 9).
Evans Blue solution with final concentration of 10uM is added into AuNCs@GroEL, and the mixture is incubated in a water bath kettle at 37 ℃ for 30min, and fluorescence monitoring is carried out on the mixture, so that obvious quenching of the fluorescence of the AuNCs@GroEL is found (figure 10), which shows that interaction of the GroEL and an inhibitor can cause quenching of the fluorescence of the AuNCs@GroEL, and therefore the AuNCs@GroEL can be used as an effective fluorescent probe for screening GroEL inhibitors. Fig. 9 and 10 correspond to example 7.
Detailed description of the preferred embodiments
The following examples further illustrate the content of the present invention, but the present invention is not limited to these examples.
Sucrose, tris, naCl, KCl, naHPO used in the present invention 4 ·12H 2 O、KH 2 PO 4 Ammonium sulfate, acetic acid, methanol were purchased from Shanghai a Ding Shiji company; deionized ultrapure water was used throughout the experiment.
Example 1:
expression of GroEL in E.Coli: coli containing GroEL and RDPV plasmids (prepared according to the method in the study of raccoon parvovirus-like particles of the university of Changchun Industrial science Lei Huan) [6] ) 2mL, added to 100mL LB medium containing 50. Mu.g/mL kanamycin and 20. Mu.g/mL chloramphenicol (double antibody), at 37℃for 220r/min overnight, and then taken at 1:200 volume ratio is inoculated into 1L LB culture medium containing the double antibody, 2g/L L-arabinose induction bacteria are added to express RDPV and GroEL at 37 ℃ and 220r/min until the OD600 value reaches 0.6-0.8, and 100 mu L and 1mol/L IPTG are added to induce expression at 30 ℃ and 220r/min for 16h.
The strain after induced expression is collected by centrifugation at 4000r/min for 30min, then 10mL of PBS with pH of 7.4 is added to be resuspended in a 50mL centrifuge tube, and the quality of sediment is marked.
Example 2:
isolation and purification of GroEL: the bacterial liquid obtained in example 1 was prepared by the following steps: 10 volume ratio of the schizolysis solution (50 mM Tris, 150mM NaCl in 1L H) 2 In O, the pH value is adjusted to 8), ultrasonic crushing is carried out for 5s, the intermittent time is 5s, the effective ultrasonic time is 30min, centrifugal is carried out for 25min at 16000r/min after ultrasonic crushing, the supernatant fluid is taken, and saturated ammonium sulfate solution is added, so that the final mass concentration of ammonium sulfate in the supernatant fluid is 30%. Stirring at 4deg.C for 1 hr, centrifuging at 10000r/min for 30min,the supernatant and pellet were separated and the pellet was resuspended in Phosphate Buffered Saline (PBS) in equal volumes to give a 30% ammonium sulfate pellet reconstituted sample, and the results of SDS-PAGE examination disruption and crude purity are shown in FIG. 1.
Sequentially and slowly adding sucrose solutions with mass fractions of 60%, 50%, 40% and 30% into a sucrose density gradient centrifuge tube (5 test tubes are the same and are used for centrifugal trimming and GroEL recovery improvement), taking a 30% ammonium sulfate precipitation redissolved sample per gradient of 2mL, filling the centrifuge tube (about 5-6 mL, adding the sample to a pipe orifice but not too full), placing the trimmed sample into an overspeed centrifuge, centrifuging at 4 ℃ at 35000r/min for 4h, taking 2mL of the sample as a grading sample after centrifuging, and detecting a result of SDS-PAGE.
Gradient centrifugation and electrophoresis results (FIGS. 2a and b) it can be seen that GroEL is distributed predominantly in the upper layer of the centrifuge tube. The upper GroEL solution was aspirated, and after removal of sucrose by PBS dialysis, the protein was purified using AKTA to obtain a purified GroEL solution. SDS-PAGE showed the purified protein (FIG. 3), groEL monomer was 56KD, and the electrophoretogram showed good separation of GroEL.
Example 3:
synthesis and optimization of auncs@groel: confirmation of GroEL and HAuCl 4 Is defined in terms of the optimum molar ratio:
1. configuration of 10mM HAuCl 4 An aqueous solution was taken, and 100. Mu.L of the GroEL solution purified in example 2 (GroEL concentration 10.4mg/mL as determined by Nanodrop) was taken.
2. To 100. Mu.L of the purified GroEL solution were added 5. Mu.L, 8. Mu.L, 10. Mu.L, 12. Mu.L of HAuCl, respectively 4 The aqueous solution is fully and evenly mixed by shaking, 20 mu L of 1M NaOH aqueous solution is added for evenly mixing by shaking, and the mixture is reacted for 2 hours at 60 ℃ to prepare the AuNCs@GroEL solution.
3. The AuNCs@GroEL solution was dialyzed with a 10kDa dialysis bag against 1 XPBS (pH=7.4) for 24h for renaturation, and the solution in the bag was removed and stored at-20℃for further use.
4. Fluorescence spectroscopy (lambda) was performed on the prepared AuNCs@GroEL ex =380 nm) (fig. 4), after testing it was found that 10 μl HAuCl was added 4 The obtained AuNCs@GroEL has the strongest fluorescence intensity, and GroEL and HAuCl are calculated 4 The optimum molar ratio for the preparation of AuNCs@GroEL is 11:6.
5. the system is scaled up in equal proportion and is characterized by GroEL and HAuCl 4 Is added with HAuCl according to the optimal molar ratio 4 That is, 400. Mu.L GroEL (10.4 mg/mL) was added to 40.5. Mu.L HAuCl 4 (10 mM), the prepared AuNCs@GroEL was dialyzed with a 10kDa dialysis bag in 1 XPBS (pH=7.4) for 24 hours to recover, and the bag was taken out and stored at-20℃for use.
The morphology of AuNCs@GroEL prepared under optimized conditions was characterized by HR-TEM (FIG. 5 a). From the figure, it can be seen that the nanoparticle has a high dispersibility and a uniform particle size (fig. 5 b). The average grain size was found to be-2.94 nm by systematic analysis of about 200 grains.
Example 5:
fluorescence stability monitoring of AuNCs@GroEL: auNCs@GroEL was irradiated with 365nm ultraviolet light for Jiang Gongguang, and 1mL of AuNCs@GroEL before and after dialysis was added to a 1mL quartz cell, respectively, for fluorescence spectrum measurement (lambda) ex =380 nm), test range 395-745nm. After the test is completed, the sample is stored in a refrigerator at the temperature of minus 20 ℃, and is taken out again after 1 month for the test of fluorescence spectrum, and the result shows (figure 6): the sample after dialysis treatment has perfect protein ligand renaturation and stable protected AuNCs luminescence, which proves that the obtained AuNCs@GroEL has better fluorescence stability.
Example 6:
protein structural stability measurement in AuNCs@GroEL: 200. Mu.L of GroEL solution and AuNCs@GroEL solution were respectively dropped onto a calcium fluoride sheet, spread uniformly, left to stand at room temperature for 30 hours to evaporate water, put into an instrument for infrared test and drawn an FT-IR spectrum (FIG. 7). From the FT-IR spectrum, it can be seen that GroEL is consistent with the infrared absorption spectrum of AuNCs@GroEL, indicating that the secondary structure of AuNCs@GroEL is not significantly changed.
Example 7:
ATPase Activity measurement of GroEL and AuNCs@GroEL: inorganic phosphate released during the hydrolysis of ATP by GroEL was determined using malachite green. Malachite green report solution (containing 0.034% w/v malachite green, 1.04% w/v ammonium molybdate, 1M HCl solution and 1% by volume of tween 20) was prepared. Before analysisTween20 was added to the malachite green report solution to give a final concentration of 0.004% by mass. ATP utilization folding buffer (50 mM Tris pH7.4, 50mM KCl, 10mM MgCl) 2 1mM DTT in distilled water) to a final dilution concentration of 2mM, groEL to folding buffer in a volume ratio of 4:6 mix configuration protein samples, 20 μl protein samples were taken and GroEL mediated ATPase activity was initiated by adding 20 μl ATP solution. GroEL and AuNCs@GroEL were diluted to 40% and 20% of the original (i.e., groEL and AuNCs@GroEL were mixed with folding buffer at volume ratios of 4:6 and 2:8 to prepare protein samples), respectively, incubated at 37℃for 45min, and malachite green reporter solution was added to determine absorbance at 595nm, which indicated (FIG. 8) that the 20% diluted AuNCs@GroEL and GroEL were significantly less than 40% dilution factor for the test sensitivity. Since the GroEL hydrolysis ATP reaction was relatively rapid, the OD at 595nm was continuously monitored at room temperature and the results were analyzed by nonlinear regression using GraphPadprism (FIG. 9).
It should also be noted that the specific embodiments of the present invention are provided for illustration only and not to limit the scope of the present invention in any way, and that modifications or variations can be made by persons skilled in the art in light of the above description, and all such modifications or variations are intended to fall within the scope of the appended claims.
Reference is made to:
[1]ReddiK，MeghjiS，NairSP，etal.(1998)13(8):1260-1266.
[2]AokiK，MotojimaF，TaguchiH，etal.(2000)275(18):13755-13758.
[3]StevensM，HoweC，RayAM，etal.(2020)28(22):115710.
[4]Yuan，Yi，etal.18.2(2018):921-928.
[5]FuDY，XueYR，GuoY，etal.(2018)23141-23148.
[6] lei Huan (2020) DOI:10.27805/d.cnki.gccgy.2020.000261.
[7]SivinskiJ，NgoD，ZerioCJ，etal.(2022)36(3):e22198.
[8]Stevens M,Abdeen S,Salim N,et al.(2019),29(9):1106-1112.
[9] Xu Xingfu, zheng Riru, wang Caiyun (2013) 529-533.

Claims (4)

1. A fluorescent gold nanocluster based on chaperonin GroEL protection, characterized in that: is prepared by adding HAuCl into 100 mu LGroEL solution 4 Mixing the aqueous solution, groEL and HAuCl 4 The molar ratio of the dosage is 10-15: 6, preparing a base material; then adding 20 μl of 1M NaOH aqueous solution, mixing thoroughly, depolymerizing GroEL protein to generate amino acid and adding HAuCl 4 The gold ions in (a) are reduced to Jin Yuanzi; and then reacting for 1.5-3.0 h at 50-70 ℃, dialyzing for 24h in 1 XPBS with pH=7.4 by using a 10kDa dialysis bag after the reaction is finished, and obtaining the solution in the bag, namely the fluorescent gold nanocluster solution based on chaperonin GroEL protection.
2. A chaperonin GroEL protection based fluorescent gold nanocluster according to claim 1, characterized in that: first, 1-3 mL of Escherichia coli containing GroEL and RDPV plasmids was added to 100mL of LB medium containing 50. Mu.g/mL of kanamycin and 20. Mu.g/mL of chloramphenicol double antibody, at 37℃for 220r/min overnight, and then added at 1:200 volume ratio is inoculated into 1L LB culture medium containing the double antibody, 2 g/LL-arabinose induction bacteria are added to express RDPV and GroEL at 37 ℃ and 220r/min until the OD600 value reaches 0.6-0.8, and then 100 mu L and 1mol/L IPTG are added to induce expression at 37 ℃ and 220r/min for 14-16 h; the strain after induced expression is collected by centrifugation at 3000-5000 r/min for 25-30 min, and then 8-12 mL of PBS with pH of 7.4 is added to be resuspended in a 50mL centrifuge tube; the obtained bacterial liquid is prepared according to the following ratio of 1: adding the schizolysis liquid into the mixed solution according to the volume ratio of 10 for ultrasonic crushing for 5s and 5s intermittently, wherein the effective ultrasonic time is 25-35 min, centrifuging the mixed solution for 20-30 min at 16000r/min after ultrasonic crushing, taking the supernatant, and adding a saturated ammonium sulfate solution to ensure that the final mass concentration of ammonium sulfate in the supernatant is 30%; stirring at 4 ℃ for 1h, centrifuging at 10000r/min for 25-35 min, separating supernatant and precipitate, and re-suspending the precipitate with PBS (phosphate buffer solution) in equal volume to obtain a 30% ammonium sulfate precipitate re-dissolved sample; sequentially and slowly adding sucrose solutions with mass fractions of 60%, 50%, 40% and 30% into a sucrose density gradient centrifuge tube, wherein each gradient is 2mL; taking a 30% ammonium sulfate precipitation re-dissolved sample, filling the sample into a centrifuge tube, balancing, placing the sample into an overspeed centrifuge, and centrifuging for 4 hours at 4 ℃ at 35000-40000 r/min; sucking out GroEL solution on the upper layer of the centrifuge tube, dialyzing with PBS to remove sucrose, and purifying protein with AKTA to obtain purified GroEL solution.
3. A chaperonin GroEL protection based fluorescent gold nanocluster according to claim 1, characterized in that: groEL and HAuCl 4 The molar ratio of the dosage is 11:6.
4. use of a fluorescent gold nanocluster based on chaperonin GroEL protection according to any one of claims 1 to 3 for the screening of GroEL inhibitors.
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090298115A1 (en) * 2008-05-29 2009-12-03 Chung Yuan Christian University Fluorescent Gold Nanocluster and Method for Forming the Same
CN109773203A (en) * 2019-01-28 2019-05-21 江南大学 It is a kind of with trypsin inhibitor be synthesize template gold nanoclusters and application
CN114381257A (en) * 2022-01-21 2022-04-22 吉林大学 Ratio-type fluorescent probe of near-infrared luminescent gold nanocluster based on thiolactic acid protection and application of ratio-type fluorescent probe in silver ion detection

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20090298115A1 (en) * 2008-05-29 2009-12-03 Chung Yuan Christian University Fluorescent Gold Nanocluster and Method for Forming the Same
CN109773203A (en) * 2019-01-28 2019-05-21 江南大学 It is a kind of with trypsin inhibitor be synthesize template gold nanoclusters and application
CN114381257A (en) * 2022-01-21 2022-04-22 吉林大学 Ratio-type fluorescent probe of near-infrared luminescent gold nanocluster based on thiolactic acid protection and application of ratio-type fluorescent probe in silver ion detection

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