CN116298316A - Method for inducing collagen hybridization peptide to maintain single-chain collagen hybridization capability and application - Google Patents
Method for inducing collagen hybridization peptide to maintain single-chain collagen hybridization capability and application Download PDFInfo
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Classifications
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/5306—Improving reaction conditions, e.g. reduction of non-specific binding, promotion of specific binding
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/78—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
Abstract
The invention relates to the technical field of protein engineering, and discloses a method for inducing collagen hybridization peptide to keep single-chain conformation and collagen hybridization capability and application thereof. The method comprises the following steps: (1) Dissolving a triple-helical polypeptide (CHP) in a denaturant solution to produce a polypeptide solution; (2) And adding the polypeptide solution into an aqueous phase buffer solution and/or pure water for dilution and activation. According to the invention, the three-helix tendency of the CHP is inhibited by using a denaturing agent to replace heating or chemically modifying the CHP precursor, and the hydrogen bond structure between three helices of the CHP is destroyed by using the denaturing agent, so that the CHP is denatured and unwound, and a single-chain conformation is maintained, and the three-helix process of the CHP is simple and controllable; and a large amount of aqueous buffer solution and/or pure water are added to dilute the denaturant, so that little denaturant remains in the diluted CHP solution, and the CHP is recovered to the original folding capability and collagen hybridization capability.
Description
Technical Field
The invention relates to the technical field of protein engineering, in particular to a method for inducing collagen hybridization peptide to keep single-chain collagen hybridization capability and application thereof.
Background
Collagen is the most abundant protein in mammals, provides mechanical support for connective tissues such as tendons, skin, bones and cartilage, and plays roles of cell adhesion, migration, tissue repair and the like in life organs. Collagen synthesis and degradation are subtly coordinated during tissue development and homeostasis, but collagen molecular denaturation by excessive collagen remodeling is also associated with many pathological states, such as cancer, osteoporosis, arthritis, and fibrosis. The structural feature of collagen molecules is the general Gly-Xaa-Yaa triple helix repeat motif in the amino acid sequence, where proline (Pro, P) and hydroxyproline (Hyp, O) are often present at Xaa and Yaa positions, respectively. The periodic repetition of glycine at each third residue causes the triple collagen chains to form a stable triple helix tightly around a common helical axis. Triple helix serves as a marker structure for collagen. Triple helix polypeptides with GPO (G: glycine, P: proline, O: hydroxyproline) as the repeat unit are standard polypeptide molecules that mimic the triple helix structure of natural collagen. At the same time, however, polypeptides of the single-stranded GPO repeat sequence are capable of specifically hybridizing to molecules that degrade or denature collagen, and are therefore referred to as triple-helical collagen hybridizing peptides (Collagen hybridizing peptide, CHP). Thus, the imaging technique of fluorescent-labeled CHP on degraded or denatured collagen can be used to identify collagen that is degraded or denatured.
However, for CHP-collagen hybridization, the strong triple helix propensity of the CHP chain is both driving force and barrier. CHP has a strong ability to self-form homologous triple helices under physiological conditions, and the binding of homologous triple helices to collagen has little driving force. Therefore, prior to hybridization applications (e.g., tissue staining), it is often necessary to heat the CHP solution to generate monomeric CHP. Although the pre-heating protocol may rapidly cool the polypeptide solution to room temperature to avoid thermal tissue damageBut the preheating process results in complex histological and in vivo applications and it is difficult to completely quantify the imaging results due to the uncertain monomer concentration. Another approach involves CHP precursors with engineered chemical modifications. This method forms caged CHPs by attaching a photolyzable Nitrobenzyl (NB) group to a central glycine, which cannot undergo triple helix folding, but which can restore the collagen hybridization ability of the caged CHPs by uv treatment. Although the above method is performed by a pair (GPO) n The modification of the sequence promotes the peptide probe to maintain the monomeric conformation, but this approach greatly increases the complexity of maintaining the CHP single strand and does not avoid the potential damage caused by uv pretreatment.
Therefore, there is an urgent need to develop a simple strategy to maintain the single-stranded state of CHP while maintaining its collagen hybridization capability.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides a method for inducing collagen hybridization peptide to maintain single-chain collagen hybridization capability and application thereof. The method not only denatures and unwinds triple helix Collagen Hybridization Peptide (CHP) to maintain single chain conformation, but also maintains collagen hybridization capability of CHP.
The invention is characterized in that: according to the invention, CHP freeze-dried powder (original CHP is in a triple helix state) is dissolved in a denaturing agent to prepare a polypeptide solution, so that a hydrogen bond structure among three helix chains of the CHP is destroyed, the characteristic of self-aggregation is lost by denaturation and unwinding (CHP treated by the denaturing agent is in a single-chain conformation), and then a large amount of aqueous solution is added to dilute the denaturing agent, so that the single-chain CHP is restored to the original folding conformation and collagen hybridization capability.
In a first aspect, the invention provides a method of inducing a collagen hybridizing peptide to retain single-chain collagen hybridizing ability.
Specifically, the method comprises the following steps:
(1) Dissolving triple-helical Collagen Hybrid Peptide (CHP) in a denaturant solution to prepare a polypeptide solution;
(2) And adding the polypeptide solution into an aqueous phase buffer solution and/or pure water for dilution and activation.
Preferably, in step (1), the CHP is a lyophilized powder.
Preferably, in step (1), the CHP is (GPO) n Is a positive integer.
Further preferably, n is 1 to 20.
Still more preferably, said n is 6-10.
Preferably, in step (1), the concentration of the polypeptide solution is 50 to 600. Mu.M.
Further preferably, in step (1), the concentration of the polypeptide solution is 70 to 500. Mu.M.
Still more preferably, in step (1), the concentration of the polypeptide solution is 75 to 400. Mu.M.
Preferably, in the step (1), the dissolution is performed by stirring, and the temperature is room temperature.
Preferably, in step (1), the denaturant solution includes at least one of urea, guanidine hydrochloride, and guanidine isothiocyanate.
Preferably, the urea concentration is 4-10M; for example 4M, 5M, 6M, 7M, 8M, 9M, 10M.
Preferably, the concentration of guanidine hydrochloride is 2-10M; for example 2M, 3M, 4M, 5M, 6M, 7M, 8M, 9M, 10M.
Preferably, the concentration of the guanidine isothiocyanate is 1-8M; for example 1M, 2M, 3M, 4M, 5M, 6M, 7M, 8M.
Preferably, in step (2), the dilution factor is 30-50 times; for example 30 times, 35 times, 40 times, 45 times, 50 times, 60 times.
Preferably, in step (2), the aqueous buffer solution comprises a phosphate buffer salt solution.
Further preferably, the phosphate buffered saline solution is a 1 XPBS solution.
The second aspect of the invention provides an application of a method for inducing collagen hybridization peptide to maintain single-chain collagen hybridization capability in preparation of a targeted denatured collagen biosensor, pathological tissue staining and in vivo imaging.
Specifically, the CHP single chain prepared by the method is used for constructing a targeted denatured collagen biosensor, a detection kit, pathological tissue staining and living body imaging.
Compared with the prior art, the invention has the following beneficial effects:
according to the invention, the three-helix tendency of CHP is inhibited by treating the CHP precursor with a denaturing agent, and the hydrogen bond structure of the CHP three-helix is destroyed by the denaturing agent, so that the CHP is denatured, unwound, loses the self-aggregation property and keeps a single-chain conformation, and the three-helix process of the CHP is simple and controllable; then, a large amount of aqueous buffer solution and/or pure water is added to dilute the denaturant, so that the denatured and unwound CHP is restored to the original conformation, and the CHP is restored to the original folding capacity and collagen hybridization capacity.
Drawings
FIG. 1 is a graph showing the CD spectra and thermal denaturation of 2M guanidine hydrochloride, 6M urea after treatment of CHP6 compared to pure water (negative control);
FIG. 2 is a graph showing the gelatin binding results after CHP9 treatment with three denaturants;
FIG. 3 is a graph showing the effects of two denaturants on the staining of despin collagen and type I collagen by CHP9 and type I collagen antibody (Col I) in heart tissue of mice with myocardial infarction;
FIG. 4 is a graph showing the effect of 6M guanidine hydrochloride treatment on the staining of the unwinding collagen and type I collagen of CHP9 and type I collagen antibodies (Col I) in different tumor tissues;
FIG. 5 is a fluorescence imaging of the whole body skeletal system of mice after treatment with 8M urea with CHP 7;
FIG. 6 is a fluorescence imaging of the systemic skeletal system of mice after 8M urea treatment of CHP7 with skin removed;
FIG. 7 is a fluorescence imaging of isolated mouse bone tissue after treatment of CHP7 with 8M urea;
FIG. 8 is a fluorescence imaging of isolated mouse organ tissue after treatment of CHP7 with 8M urea;
FIG. 9 is a graph showing the results of gelatin binding assays after CHP9 treatment in different ways.
Detailed Description
In order to make the technical solutions of the present invention more apparent to those skilled in the art, the following examples will be presented. It should be noted that the following examples do not limit the scope of the invention.
The starting materials, reagents or apparatus used in the following examples are all available from conventional commercial sources or may be obtained by methods known in the art unless otherwise specified.
Different denaturant solutions induce structural changes in CHP 6.
CHP6 (GPO) 6 The freeze-dried powder is respectively dissolved into a solution of 6M urea or 2M guanidine hydrochloride to prepare a CHP6 polypeptide solution with the volume of 320 mu L and the concentration of 75 mu M, so that the polypeptide structure reaches the final stable single-chain state.
CD value detection:
CD spectra were collected on a JASCO J-1500CD spectrophotometer and CD measurements of the polypeptide solution were recorded in a quartz dish with a path length of 0.1 mm. Wherein, the water treatment group is CHP6 freeze-dried powder and pure water 1:1 mixing parameters (scan speed: 20nm/min; data pitch: 0.1nm; bandwidth: 5nm; digital integration time: 16 s) were used to scan the CD spectra. The hot melt profile was obtained by measuring ovality at 225nm at 4℃to 55℃at a heating rate of 0.5℃per minute. The original CD signal was normalized to the average residue ellipticity, depending on the length and concentration of the peptide. Average residue ellipticity (MRE, [ theta ]]) Using the formula [ theta ]]Calculation = (θ×m)/(c×l×n), where θ is the measured ovality (mdeg), m is the molecular weight (g/mol), c is the concentration (mg/mL), l is the path length (mm) of the cuvette, and n is the number of amino acid residues in the peptide. The derivative of the melting curve was constructed using JASCO Spectra Manager software (version 2.10.05) and the temperature at which the derivative curve was lowest was defined as the melting temperature (T m ). Each T given in this study m Values were all from the average of three replicates.
Referring to FIG. 1, CMP6 in pure water solution showed a characteristic collagen-like CD spectrum, showing a strong positive peak around 225nm, indicating a complete triple helix structure; the CD thermal expansion curve monitored at this wavelength shows that CMP6 transitions from a triple helix structure to a single chain during the melt transition, the minimum of the derivative of the thermal expansion curve, T m Is 30 ℃. While 2M guanidine hydrochloride and 6M urea can make the CD spectrum value of CHP6 displayThe decrease was significant and no CD thermal conversion curves were shown in the 2M guanidine hydrochloride and 6M urea solutions, indicating that the CHP6 triple helix could be fully extended in the 2M guanidine hydrochloride and 6M urea solutions.
Example 1
A method for inducing collagen hybridizing peptide to maintain single-chain collagen hybridizing ability.
CHP9, i.e. GPO 9 Dissolving the lyophilized powder into urea with concentration of 8M to obtain CHP9 polypeptide solution with volume of 200 μL and concentration of 400 μM, making polypeptide structure reach its final stable single chain state, and adding 1×PBS solution to dilute polypeptide solution during activation.
Example 2
A method for inducing collagen hybridizing peptide to maintain single-chain collagen hybridizing ability.
CHP9, i.e. GPO 9 Dissolving the lyophilized powder into 8M guanidine hydrochloride to obtain CHP9 polypeptide solution with volume of 200 μL and concentration of 400 μM, making polypeptide structure reach its final stable single chain state, and adding 1×PBS solution to dilute polypeptide solution during activation.
Example 3
A method for inducing collagen hybridizing peptide to maintain single-chain collagen hybridizing ability.
CHP9, i.e. GPO 9 The lyophilized powder is dissolved in 6M guanidine isothiocyanate to prepare CHP9 polypeptide solution with the volume of 200 mu L and the concentration of 400 mu M, so that the polypeptide structure reaches the final stable single-chain state, and 1X PBS solution is added to dilute the polypeptide solution during activation.
Gelatin result detection:
porcine gelatin (Sigma, V900863-100G) was dissolved in 1 XPBS (10% w/V) at 70℃and approximately 6. Mu.L gelatin solution was plated per well in 96-well plates, followed by incubation at 4℃for 10min to gel the gelatin. The hydrogel film of gelatin was shaken overnight at room temperature with 2mM N-hydroxysuccinimide (NHS) and 10mM 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in 2-morpholinoethanesulfonic acid (MES) buffer (0.1M, pH 4.7, 100. Mu.L/well). The crosslinked gelatin film was washed with 150. Mu.L/well of 1 XPBS at 4℃for 10min 3 times. 400. Mu.M of the CHP9 stock solution prepared in examples 1-3 was diluted to a final concentration of 10. Mu.M in 1 XPBS, added to the cross-linked gelatin wells (50. Mu.L/well) and allowed to bind overnight at 4 ℃. The peptide solution of the "heating group" was heated at 85℃for 10min, then cooled in ice water for 10 seconds and added to the gelatin well. The "unheated" set of polypeptide solutions was added directly to gelatin wells at room temperature without any pretreatment. After binding, the plates were washed 3 times with 1 XPBS at 4℃and fluorescence was measured with a microplate reader (Ex: 540nm, em:590 nm). Binding experiments for each peptide were performed four times (n=4). Data were analyzed using one-way ANOVA followed by Tukey HSD test. * Significant differences in mean (P < 0.01), mean differences (P < 0.05), sign mean statistically significant compared to water (heated group).
Referring to FIG. 2, three high concentration protein denaturants have good denaturation effect on three-helix form CHP9, 8M urea can partially unwind three helices of CHP9, and CHP9 prepared from 6M guanidine isothiocyanate and 8M guanidine hydrochloride has fluorescence signals similar to those of a positive control group (heating group), which indicates that CHP9 prepared from 6M guanidine isothiocyanate and 8M guanidine hydrochloride and the control group (heating group) can completely open three helices of CHP9, and the polypeptide structure reaches the final stable state.
Example 4
A method for inducing collagen hybridizing peptide to maintain single-chain collagen hybridizing ability.
CHP9, i.e. GPO 9 Dissolving the lyophilized powder into 6M guanidine hydrochloride to obtain 400 μL CHP9 polypeptide solution with 400 μM concentration, to make polypeptide structure reach final stable single chain state, and adding 1×PBS solution to dilute polypeptide solution during activation.
Example 5
A method for inducing collagen hybridizing peptide to maintain single-chain collagen hybridizing ability.
CHP9, i.e. GPO 9 The lyophilized powder is dissolved in 4M guanidine isothiocyanate to prepare 400 mu L CHP9 polypeptide solution with 400 mu M concentration, so that the polypeptide structure reaches the final stable single-chain state, and 1 XPBS solution is added to dilute the polypeptide solution during activation.
Tissue staining detection:
for frozen sections of mouse myocardial infarction heart 14 days from left anterior descending branch artery occlusion, OCT was removed by washing with PBS for 5min after the tissue slide was returned to room temperature. Without antigen retrieval, tissues were incubated with 5% goat serum for 20min at room temperature to block non-specific binding. Prior to addition of the CHP9 staining solution to the tissue, the denaturing agent treated Cy3-CHP9 stock solution (400. Mu.M) was diluted 40-fold to working concentration (10. Mu.M) with 1 XPBS, then anti-type I collagen antibodies (Abcam, ab34710,1:200 dilution) were rapidly mixed with the CHP9 solution and added to the tissue. For Cy3-CHP9 stock solution in water (400. Mu.M), after 40-fold dilution with 1 XPBS, heat at 80℃for 10min to completely dissociate CHP9 into single strands, then immediately incubate in ice water for 10-15 seconds, quench the hot solution to room temperature, and then dilute the antibody into the CHP solution returning to room temperature for co-staining. After incubation of tissue samples overnight in a wet box at 4 ℃, primary antibodies were detected by incubation with AlexaFluor 647-labeled secondary antibodies (goat anti-rabbit IgG H & L, abcam, ab150079, diluted to 5 μg/mL in PBS) for 1 hour at room temperature. After three washes (5 min/time) in PBS, nuclei were stained with DAPI (1:1000 dilution in PBS). Finally, all tissue sections were imaged using an EVOS M7000 imaging system (Thermo Fisher).
For paraffin tissue (i.e., multi-tumor tissue chip, purchased from chinese light), paraffin was removed by first rinsing with xylene (10 min×3), 100% ethanol (5 min×2), and 95% ethanol, 80%, 70%, 50% ethanol, and deionized water in sequential order, each solvent for 3 min. After deparaffinization, cy3-CHP9 stock solution (400. Mu.M) prepared from 6M guanidine hydrochloride was directly diluted 40-fold with PBS buffer, added dropwise to the tissue, and incubated overnight in a wet box at 4 ℃. After CHP staining, the slides were imaged with an EVOS M7000 imaging system (Thermo Fisher). CHP imaging was followed by further counterstaining with type I collagen antibodies. The sections were immersed in sodium citrate buffer (MVS-0066, pH: 6.0) and heated at 110℃for 10min for antigen retrieval. After blocking any endogenous biotin in the tissue with an endogenous biotin blocking kit (BLK-0002, michaelis chemical Co., ltd.) the tissue was incubated with 10% goat serum at room temperature for 30min to block non-specific binding. Subsequently, the slides were stained overnight with anti-type I collagen antibodies (Abcam, ab24821,1:200 dilution) at 4 ℃. The next day, alexaFluor 647-labeled secondary antibodies (goat anti-rabbit IgG H & L, abcam, ab150079, diluted to 5 μg/mL in PBS) were incubated at room temperature for 1 hour to detect primary antibodies. After PBS washing and DAPI staining, all tissue sections were imaged using an EVOS M7000 imaging system (Thermo Fisher).
Referring to FIG. 3,4M, guanidine isothiocyanate and 6M guanidine hydrochloride unwinding 400. Mu.M CHP9 can be successfully applied to the unwinding collagen staining in heart tissue of 14 day myocardial mice, and the staining effect is consistent with that of single-chain CHP9 obtained by preheating. In addition, the concentration of denaturing agent remaining (0.1-0.2M) after the mother liquor is diluted to working concentration by 1 XPBS does not affect the structure and function of Col I antibodies and the structure of type I collagen in tissues (epitope of Col I), and Col I antibodies can work normally in both non-myocardial infarction regions and myocardial infarction regions enriched with unwinding collagen.
Referring to fig. 4, single-chain CHP9, which was unwound by 6M guanidine hydrochloride, was successfully applied to section staining of the unwound collagen in different types of tumor tissue study samples.
Thus, it was found from the experiments of examples 4 and 5 that the final stable single-stranded state of 4M guanidine isothiocyanate and 6M guanidine hydrochloride unwound single-stranded CHP9 was achieved, and that the CHP9 single-stranded was restored to its original folding and collagen hybridization ability after PBS dilution.
Example 6
A method for inducing collagen hybridizing peptide to maintain single-chain collagen hybridizing ability.
CHP7, i.e. GPO 7 Dissolving the lyophilized powder into urea with concentration of 8M to obtain CHP7 polypeptide solution with volume of 200 μL and concentration of 400 μM, making polypeptide structure reach its final stable single chain state, and adding 1×PBS solution to dilute polypeptide solution during activation.
Animal live imaging detection:
prior to tail vein administration, 400. Mu.M CHP7 stock solution of 8M urea was diluted into pre-filter sterilized 1 XPBS to a final concentration of 10. Mu.M, and 100. Mu.L of the diluted probe solution was injected into normal female BALB/c nude mice (8-15 weeks) via the tail vein, each of which had an administration amount of 1nmol of CHP7. Wherein, the polypeptide solution of the "water (heating)" group is heated at 85 ℃ for 10min, then cooled in ice water for 10s, and then injected into tail vein; the polypeptide solution of the "water (unheated)" group was directly administered by tail vein injection without preheating treatment. Mice were imaged using an IVIS spectrum (PerkinElmer) imager. At 2 hours post injection, each mouse was euthanized and the skin of the mouse was stripped to image deep tissues while the major organs and bones were harvested for imaging. This experiment was repeated three times, with consistent results each time.
From fig. 5-8, fig. 5 shows that the single-stranded CHP7 of example 6 shows substantially the same targeting sites and fluorescence signal intensity as the conventionally unwound single-stranded CHP7 probe, both of which demonstrate the targeting of CHP7 to musculoskeletal soft tissues of normal mice, including intervertebral discs, cartilage in rib cage, joints (knee, ankle and wrist) and mandible, which are believed to contain some level of denatured collagen molecules due to heavy tissue or daily mechanical wear; whereas pure water formulated trimeric CHP7 did not show any in vivo tissue targeting without preheating. Fluorescence imaging of isolated organs (heart: H liver: lv lung: ln spleen: S kidney: K) showed that CHP7 is primarily likely to be metabolized by the kidneys and liver. These results indicate that CHP7, which is monomeric from urea, can effectively target denatured collagen in vivo, allowing direct administration without preheating.
From the above, it is known from examples 1 to 6 that three denaturants (urea, guanidine isothiocyanate and guanidine hydrochloride) can open the triple helix structure of CHP, wherein the concentration of urea is 4 to 10M; the concentration of the guanidine isothiocyanate is 1-8M; guanidine hydrochloride is present at a concentration of 2-10M and all three denaturants maintain the open CHP triple helix structure in a single-stranded state without self-aggregation, and the CHP single strand regains its collagen hybridization ability upon dilution with 1 XPBS solution.
Comparative example 1
A method for inducing collagen hybridizing peptide to maintain single-chain collagen hybridizing ability.
CHP9, i.e. GPO 9 Dissolving the lyophilized powder in 3M guanidine hydrochloride to obtain CHP9 polypeptide solution with volume of 200 μL and concentration of 400 μM, and coagulatingThe final stable conformation was achieved and the polypeptide solution was diluted by the addition of 1 XPBS solution upon activation.
Comparative example 2
A method for inducing collagen hybridizing peptide to maintain single-chain collagen hybridizing ability.
Comparative example 2 differs from comparative example 1 in that comparative example 2 uses 3M guanidine isothiocyanate instead of 3M guanidine hydrochloride of comparative example 1.
Comparative example 3
A method for inducing collagen hybridizing peptide to maintain single-chain collagen hybridizing ability.
Comparative example 3 differs from comparative example 1 in that comparative example 3 uses 3M urea instead of 3M guanidine hydrochloride of comparative example 1.
As can be seen from FIG. 9, wherein the heating R-CHP group is a water treatment heating CHP9 treatment group, heating at 85℃for 10min, and then cooling in ice water for 10 seconds; the results of gelatin binding showed (gelatin detection, see example 3) that 3M guanidine hydrochloride and urea hardly opened the triple helix of CHP9 and 3M guanidine isothiocyanate partially opened the triple helix of CHP 9. Indicating that lower concentrations of denaturing agents do not fully open the triple helix structure of CHP9 and do not ensure maintenance of the single stranded state of CHP 9.
Claims (10)
1. A method of inducing a collagen hybridizing peptide to retain single-chain collagen hybridizing ability, comprising the steps of:
(1) Dissolving triple-helical collagen hybrid peptide in a denaturant solution to prepare a polypeptide solution;
(2) And adding the polypeptide solution into an aqueous phase buffer solution and/or pure water for dilution and activation.
2. The method of claim 1, wherein in step (1), the triple-helical collagen hybridizing peptide is (GPO) n Is a positive integer.
3. The method of claim 1, wherein in step (1), the concentration of the polypeptide solution is 50 to 600 μm.
4. The method of claim 1, wherein in step (1), the denaturant solution comprises at least one of urea, guanidine hydrochloride, guanidine isothiocyanate.
5. The method according to claim 4, wherein the urea concentration is 4-10M.
6. The method of claim 4, wherein the guanidine hydrochloride is present at a concentration of 2-10M.
7. The method of claim 4, wherein the concentration of guanidine isothiocyanate is 1-8M.
8. The method of claim 1, wherein in step (2), the aqueous buffer solution comprises a phosphate buffer salt solution.
9. The method of claim 1, wherein in step (2), the dilution factor is 30-60.
10. Use of the method of any one of claims 1-9 for the preparation of a targeted denatured collagen biosensor, a detection kit, pathological tissue staining, in vivo imaging.
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