Method for purifying XVII type collagen

Abstract

The invention discloses a method for purifying XVII type collagen. The method comprises the following steps: firstly, balancing a boric acid affinity chromatography column by using an alkaline solution; secondly, balancing again after loading the sample; thirdly, washing impurities with alkaline leacheate; and finally, eluting the XVII type collagen from the boric acid affinity filler by using a slightly acidic eluent. The purification effect on the XVII type collagen is good, and the purity is higher than 95%; moreover, the purification method provided by the invention is simple to operate and relatively low in cost, and has a good application prospect.

Description

Technical Field

[0001] The present invention relates to a method for purifying collagen, in particular to a method for purifying type XVII collagen. Background Art

[0002] Collagen is the most abundant and widely distributed functional protein in mammals. It has the characteristics of low immunogenicity, biodegradability and promoting cell proliferation and differentiation. It is widely used in tissue engineering, clinical medicine, food industry, packaging materials, cosmetics, medical cosmetology, biomaterials and medical devices. Type XVII collagen is a transmembrane protein that is not only related to skin diseases, but also a key factor in regulating a variety of stem cells. It has become a research hotspot in the fields of biomedicine and cosmetology.

[0003] Collagen has a wide range of sources. It can be extracted from animals or produced using genetic engineering. The obtained collagen usually needs to be purified to meet specific application standards. Patent application CN117186211A discloses a method for purifying collagen, which obtains high-purity collagen by steps such as crude product filtration, Ni affinity filler capture, dilution, filtration and cation exchange chromatography. The purification method uses Ni affinity chromatography, and needs to add a His tag that specifically binds to the Ni affinity filler, which is complicated to operate and has high cost. Patent application CN118745220A discloses an industrial production of high-purity recombinant human collagen purification process for injection, which gradually removes different types of impurities through filtration, ion exchange chromatography, affinity chromatography, molecular sieve chromatography, magnetic field separation technology and ultrafiltration, and achieves high purity and high recovery. The purification process adopts a multi-step chromatography combined method, which also has the problems of complex operation and high cost. In addition, there is no purification method for type XVII collagen in the prior art. Summary of the invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a simple, effective and low-cost method for purifying type XVII collagen.

[0005] Technical solution: The present invention provides a method for purifying type XVII collagen, comprising the following steps: Step 1: balancing; using 20-30 mM ammonium acetate + 250-300 mM NaCl, pH 8.5-9.0 as a balancing solution to balance the chromatography column; the filler of the chromatography column is a boric acid affinity filler; Step 2: loading: adding the loading solution containing type XVII collagen to the chromatography column; Step 3: Post-equilibrium: re-equilibrate the chromatography column with the equilibration solution described in step 1; Step 4: washing: use 20-30 mM ammonium acetate + 0.5-0.6 M NaCl, pH 8.5-9.0 as the washing solution for washing; Step 5: Elution: Use 20-30 mM ammonium acetate + 250-300 mM NaCl, pH 5.0-5.5 as the eluent and collect the effluent.

[0006] Boric acid affinity chromatography is a method for separating and enriching cis-diol compounds based on the specific binding of boronic acid ligands to cis-diols. Cis-diol compounds include glycoproteins. Since type XVII collagen is glycosylated, it can be captured using a boronic acid affinity filler. Therefore, the present invention first maintains an alkaline environment in the first four steps, so that type XVII collagen coordinates with the boronic acid group to form a stable structure, thereby binding to the chromatography filler; and the impurities are washed away because they cannot specifically bind to the boronic acid affinity filler, and are separated from the type XVII collagen; finally, an acidic solution is used to elute the type XVII collagen from the boronic acid affinity filler to complete the purification. The present invention can remove most of the impurities in type XVII collagen by only one chromatography, and the operation is simple, the purification effect is good, and the cost is low.

[0007] Preferably, in step 1, the boric acid affinity filler is Monomix HC60-Boronate.

[0008] Preferably, in step five, a chromatographic signal with a wavelength of UV214 and a peak cutoff range of 50-50 mAU is collected.

[0009] Preferably, in step five, the amount of the initial eluent used is 13.5-15 column volumes.

[0010] Preferably, in step 1, the amount of the equilibration solution is 3-5 column volumes.

[0011] Preferably, in step 3, the amount of the equilibration solution is 5-8 column volumes.

[0012] Preferably, in step 4, the amount of the eluent used is 3-5 column volumes.

[0013] Preferably, in step five, after the collection is completed, a second elution is performed, using 20-30 mM ammonium acetate + 250-300 mM NaCl, pH 4.0-4.5 as the secondary eluent, and the effluent is collected.

[0014] Preferably, the amount of the secondary eluent is 8-10 column volumes.

[0015] Preferably, after elution is completed, the chromatography column is cleaned with an alkaline solution, preferably 0.1 M NaOH, and the amount used is preferably 3 column volumes.

[0016] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. The present invention has a good purification effect on type XVII collagen, with a purity higher than 95%; 2. The purification method provided by the present invention is simple to operate, low in cost, and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 This is a chromatogram of purified type XVII collagen in Example 1 of the present invention; Figure 2 This is a chromatogram of purified type XVII collagen in Comparative Example 1 of the present invention; Figure 3 The electrophoretogram of the effluent collected in Example 1 of the present invention; Figure 4 This is the electrophoretogram of the effluent collected in Comparative Example 1 of the present invention. DETAILED DESCRIPTION

[0018] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0019] The type XVII collagen sample used in the examples and comparative examples was expressed by Pichia pastoris, with a molecular weight of 36 kDa and an isoelectric point of about 10. The original concentration was 15 g / L (BCA total protein), and the sample was in a gel state after thawing and clarified in a 60°C water bath for 3 min. Sample pretreatment: cells were broken, supernatant was obtained by centrifugation, and concentrated by hollow fiber ultrafiltration.

[0020] The fillers used in the examples and comparative examples were all from Suzhou Saifen Technology Co., Ltd. Other materials were commercially available.

[0021] Example 1: A method for purifying type XVII collagen This embodiment provides a method for purifying type XVII collagen, comprising the following steps: Step 1: balance; use 20 mM ammonium acetate + 250 mM NaCl, pH 8.5 as the balance solution, and use 3 column volumes of the balance solution to balance the chromatography column; the filler of the chromatography column is boric acid affinity filler Monomix HC60-Boronate, the chromatography column specification is 6.6*110 mm, and the column volume is 3.763 mL; the flow rate is 0.753 mL / min.

[0022] Step 2: Sample loading; dilute the original sample three times with the equilibration solution and filter it through a 0.22 μm filter membrane. Add the sample to the chromatography column at a sample volume of 6 mL and a sample load of 7.97 g / L. The flow rate is 0.753 mL / min.

[0023] Step 3: Post-equilibrium: re-equilibrate the column with 5 column volumes of the equilibration solution in step 1; the flow rate is 0.753 mL / min.

[0024] Step 4: Elution: Use 20 mM ammonium acetate + 0.5 M NaCl, pH 8.5 as the eluent, and use 3 column volumes of the eluent for washing; the flow rate is 0.753 mL / min.

[0025] Step 5: Elution; Use 20 mM ammonium acetate + 250 mM NaCl, pH 5.0 as the primary eluent, and use 13.5 column volumes of the primary eluent for the primary elution; collect the effluent, 45 mL per tube; the flow rate is 0.753 mL / min. After the collection is completed, use 20 mM ammonium acetate + 250 mM NaCl, pH 4.0 as the secondary eluent, and use 8 column volumes of the secondary eluent for the secondary elution; collect the effluent, 45 mL per tube; the flow rate is 0.753 mL / min.

[0026] After elution was complete, the column was washed with 3 column volumes of 0.1 M NaOH at a flow rate of 0.753 mL / min.

[0027] The chromatographic signals with a wavelength of UV214 and a peak cutoff range of 50-50 mAU were collected throughout the process, and the proteins in the effluent were analyzed by electrophoresis.

[0028] The chromatogram collected in this embodiment is Figure 1 As can be seen from the figure, impurities such as pigments in the sample flow out in the post-equilibrium stage, so the UV signal of the initial elution is higher. Figure 3 It can be seen (lane 2 is the effluent from the loading stage, lane 3 is the effluent from the primary elution stage, and lane 4 is the effluent from the secondary elution stage) that most of the impurities in the primary elution have been removed, leaving only the target protein type XVII collagen and aggregates with a molecular weight of about 75 kDa. For the secondary elution, Figure 1 The signal peak is lower than the initial elution. Figure 3 It is proved that in addition to the target protein, there are also polymers with a molecular weight of about 75 kDa and degradation fragments with a molecular weight of about 30 kDa in the secondary elution. In summary, this example uses boric acid affinity filler to purify the type XVII collagen sample, which can remove most of the impurities therein. The grayscale analysis results show that the purity of the purified type XVII collagen is greater than 95%.

[0029] Example 2: A method for purifying type XVII collagen This embodiment provides a method for purifying type XVII collagen, which differs from Embodiment 1 mainly in the concentration and amount of the solution used, and comprises the following steps: Step 1: balance; use 30 mM ammonium acetate + 300 mM NaCl, pH 9.0 as the balance solution, and use 5 column volumes of the balance solution to balance the chromatography column; the filler of the chromatography column is boric acid affinity filler Monomix HC60-Boronate, the chromatography column specification is 6.6*110 mm, and the column volume is 3.763 mL; the flow rate is 0.753 mL / min.

[0030] Step 2: Sample loading; dilute the original sample three times with the equilibration solution and filter it through a 0.22 μm filter membrane. Add the sample to the chromatography column at a sample volume of 6 mL and a sample load of 7.97 g / L. The flow rate is 0.753 mL / min.

[0031] Step 3: Post-equilibrium: re-equilibrate the column with 8 column volumes of the equilibration solution in step 1; the flow rate is 0.753 mL / min.

[0032] Step 4: Elution: Use 30 mM ammonium acetate + 0.6 M NaCl, pH 9.0 as the eluent, and use 5 column volumes of the eluent for washing; the flow rate is 0.753 mL / min.

[0033] Step 5: Elution; use 30 mM ammonium acetate + 300 mM NaCl, pH 5.5 as the primary eluent, use 15 column volumes of the primary eluent for the primary elution; collect the effluent, 45 mL per tube; the flow rate is 0.753 mL / min. After the collection is completed, use 30 mM ammonium acetate + 300 mM NaCl, pH 4.5 as the secondary eluent, use 10 column volumes of the secondary eluent for the secondary elution; collect the effluent, 45 mL per tube; the flow rate is 0.753 mL / min.

[0034] After elution was complete, the column was washed with 3 column volumes of 0.1 M NaOH at a flow rate of 0.753 mL / min.

[0035] Comparative Example 1: A method for purifying type XVII collagen The comparative example provides a method for purifying type XVII collagen, which is mainly different from Example 1 in that a hydrophobic filler is used for chromatography. Specifically, the method comprises the following steps: Step 1: Balance; use the balance solution 50 mM ammonium acetate + 2 M (NH 4 ) 2 SO 4 , pH 6.0, and equilibrate the chromatography column with 3 column volumes of equilibrium liquid; the filler of the chromatography column is the hydrophobic filler Polar MC30-HIC Phenyl, the chromatography column specification is 6.6*200 mm, the column volume is 6.842 mL; the flow rate is 1.368 mL / min.

[0036] Step 2: Sample loading; dilute the original sample 10 times with the equilibrium solution, adjust the conductivity and pH to be consistent with the equilibrium solution, load 50 mL, the sample load is 10.96 mg / mL, and the flow rate is 1.368 mL / min.

[0037] Step 3: Post-equilibrium: re-equilibrate the column with 5 column volumes of the equilibration solution in step 1; the flow rate is 1.368 mL / min.

[0038] Step 4: Washing: Use 50 mM ammonium acetate + 1.4 M (NH 4 ) 2 SO 4 , pH 6.0, and 5 column volumes of eluent were used for washing; the flow rate was 1.368 mL / min.

[0039] Step 5: Elution; use 50 mM ammonium acetate, pH 6.0, and elute with 8 column volumes of eluent; collect the effluent in 45 mL tubes; the flow rate is 1.368 mL / min.

[0040] After elution, the column was cleaned with pure water, 1 M NaOH, and pure water in sequence, with the solution dosage being 5, 3, and 5 column volumes, respectively; the flow rate was 1.368 mL / min.

[0041] The chromatographic signals with a wavelength of UV214 and a peak cutoff range of 50-50 mAU were collected throughout the process, and the proteins in the effluent were analyzed by electrophoresis.

[0042] The chromatogram collected for this comparative example is Figure 2 As can be seen from the figure, impurities such as pigments flow through during the injection stage and the post-equilibrium stage, and most of the aggregates are removed during the elution stage. Figure 4 The results showed that (lane 2 is the effluent from the loading stage, and lane 3 is the effluent from the elution stage) that although the hydrophobic filler had a removal effect on most aggregates, it had a poor removal effect on impurities with a molecular weight of about 75 kDa and fragment-type impurities.

Claims

1. A method for purifying type XVII collagen, characterized in that: The following steps are involved: Step 1: balancing; using 20-30 mM ammonium acetate + 250-300 mM NaCl, pH 8.5-9.0 as a balancing solution to balance the chromatography column; the filler of the chromatography column is a boric acid affinity filler; Step 2: loading: adding the loading solution containing type XVII collagen to the chromatography column; Step 3: Post-equilibrium: re-equilibrate the chromatography column with the equilibration solution described in step 1; Step 4: washing: use 20-30 mM ammonium acetate + 0.5-0.6 M NaCl, pH 8.5-9.0 as the washing solution for washing; Step 5: Elution: Use 20-30 mM ammonium acetate + 250-300 mM NaCl, pH 5.0-5.5 as the eluent and collect the effluent.

2. The method according to claim 1, characterized in that In step 1, the boric acid affinity filler is Monomix HC60-Boronate.

3. The method according to claim 1, characterized in that In step 5, the chromatographic signal with a wavelength of UV214 and a peak cutoff range of 50-50mAU is collected.

4. The method according to claim 1, characterized in that: In step 5, the amount of the eluent used is 13.5-15 column volumes.

5. The method according to claim 1, characterized in that In step 1, the amount of the equilibration solution is 3-5 column volumes.

6. The method according to claim 1, characterized in that In step 3, the amount of the equilibration solution is 5-8 column volumes.

7. The method according to claim 1, characterized in that In step 4, the amount of the eluent used is 3-5 column volumes.

8. The method according to claim 1, characterized in that In step 5, after the collection is completed, a second elution is performed, using 20-30 mM ammonium acetate + 250-300 mM NaCl, pH 4.0-4.5 as the secondary eluent, and the effluent is collected.

9. The method according to claim 8, characterized in that The amount of the secondary eluent used is 8-10 column volumes.

10. The method according to claim 1, characterized in that After elution is completed, the column is cleaned with alkaline solution.

Images