Method for purifying collagen by using concanavalin A

Abstract

The invention discloses a method for purifying collagen by using concanavalin A. The method adopts a chromatographic column containing the concanavalin A to purify the collagen. The filler of the chromatographic column containing the concanavalin A is porous polystyrene / divinyl benzene microspheres with high crosslinking degree, a hydrophilic coating is bonded on the surface of the filler, and the concanavalin A is bonded on the surface of the coating. According to the method for purifying the type III collagen, most glycosylation can be removed through one-step purification, and the quality of the type III collagen is improved. And the glycosyl removal rate is up to 95%.

Description

Technical Field

[0001] The invention belongs to the technical field of collagen purification, and particularly relates to a method for purifying collagen by using concanavalin A. Background Art

[0002] Collagen is one of the key proteins in the extracellular matrix of mammalian tissues, accounting for about 25% to 35% of the body's protein. It is mainly found in fibrous tissues, such as tendons, ligaments, cartilage and skin. Collagen is a white, opaque, unbranched fibrous protein. The molecular weight is about 300kDa, and it has a triple helix structure. There are 28 known types of collagen, with type I & III collagen having the highest content, accounting for more than 90% of the collagen in the human body. Tropocollagen is the basic structural unit of collagen, and each tropocollagen chain contains about 1,000 amino acids. The quaternary structure of collagen, primary structure: (Gly-XY)n repeating units, where X and Y are often proline and hydroxyproline, and a few are lysine and hydroxylysine (UV220-230nm); secondary structure: left-handed α-helical peptide chain; tertiary structure: 3 polypeptide chains are entangled, and the right-handed helix constitutes the original collagen; quaternary structure: the original collagen is arranged in parallel into bundles, covalently cross-linked into fibrils, and aggregated into bundles of collagen. It can be divided into different types according to its structure and function, mainly type I, type II, type III and type V collagen. Among them, type I collagen is the most common type, which is widely found in the skin, bones, tendons and ligaments, and is mainly responsible for providing strength and support. Type II collagen is mainly found in cartilage, has good buffering and supporting effects, and is often used in joint health products. Type III collagen is mainly found in the skin, blood vessels and internal organs, and is responsible for the elasticity and flexibility of the tissue. Type V collagen is more abundant in the placenta and hair follicles, and is involved in the development and regeneration of cells and tissues.

[0003] Concanavalin A is abbreviated as ConA or Con A. Its Chinese name is Concanavalin A, also known as Concanavalin A, Concanavalin A or Concanavalin A. It is a mannose / glucose binding lectin isolated from Jack beans (Canavalia ensiformis). It is one of the most widely used lectins. Its CAS number is 11028-71-0. Its monomer molecular weight is about 25.5kDa and can bind to a Ca 2+ and a Mn 2+ , contains a glycosyl binding site with high affinity for terminal α-D-mannosyl and α-D-glucosyl residues.

[0004] Since the glycosylation is difficult to remove during the current type III collagen purification process, this is also the focus of the present invention during the type III collagen purification process. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a one-step method for purifying collagen using a concanavalin A bonded affinity chromatography column, which can achieve a glycosyl removal rate of 95%.

[0006] Technical solution: To achieve the above technical objectives, the present invention provides a method for purifying collagen using concanavalin A, wherein the method uses a chromatographic column containing concanavalin A to purify collagen.

[0007] The filler of the chromatographic column containing concanavalin A is porous polystyrene / divinylbenzene microspheres with a high degree of cross-linking, a hydrophilic coating is bonded to the surface of the filler, and the concanavalin A is bonded to the surface of the coating.

[0008] The average particle size of the filler is 20 μm, the narrow particle size distribution is D90 / D10<1.3, and the pore size of the filler is

[0009]

[0010] Wherein, the chromatographic column containing concanavalin A includes but is not limited to the Proteomix POR20-ConA chromatographic column.

[0011] Wherein, the collagen includes but is not limited to type III collagen.

[0012] Wherein, the method for purifying collagen using concanavalin A specifically comprises the following steps:

[0013] 1) Pretreatment of type III collagen:

[0014] 2) Purify the pretreated type III collagen using a chromatographic column containing concanavalin A.

[0015] Among them, the pretreatment step of type III collagen in step 1) is specifically: adding a sample containing type III collagen to a balanced solution to dissolve and filter to obtain a solution containing type III collagen, wherein the balanced solution contains 50-60mM NaAc, 0.2-0.3M NaCl, 1-2mM CaCl2, 1-2mM MgCl2, and pH 5.3-5.5.

[0016] Wherein, the purification step in step 2) includes equilibration, loading, post-equilibration, elution and equilibration steps.

[0017] Wherein, the balancing solution in the balancing step contains 50-60 mM NaAc, 0.2-0.3 M NaCl, 1-2 mM CaCl2, 1-2 mM MgCl2, pH 5.3-5.5.

[0018] The elution solution in the elution step contains 50-60 mM NaAc, 0.2-0.3 M NaCl, 1-2 mM CaCl2, 1-2 mM MgCl2, 100-120 mM α-methyl-D-mannoside, pH 5.3-5.5

[0019] Beneficial effects: Compared with the prior art, the present invention has the following advantages: The present invention provides a one-step method for purifying collagen using a concanavalin A bonded affinity chromatography column, specifically, purification is performed by using a Proteomix POR20-ConA chromatography column. The purification method can remove the sugar groups in collagen by up to 95%. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is the purification map of Proteomix POR20-ConA.

[0021] Figure 2 This is the purification map of Monomix HC60-Boronate. DETAILED DESCRIPTION

[0022] The present invention is further described in detail below in conjunction with embodiments so that those skilled in the art can implement the invention with reference to the description.

[0023] Preparation of type III collagen used in the examples of the present invention:

[0024] 1. The gene sequence of human type III collagen α1 chain was obtained from the NCBI website (GenBank: AB384877.1), and the 1023-2523 bases (shown in SEQ ID NO.1) were intercepted as the expression fragment, i.e., the 341-841 amino acid sequence (SEQ ID NO.2). The restriction site XhoI was added to the 5' end of the gene, and the restriction site SacII was added to the 3' end. The gene fragment was synthesized by a company, and then the gene fragment was connected with the linearized pPIC9K expression vector that was also digested with the same restriction site to obtain a recombinant expression vector, and then the recombinant expression vector was transformed into Pichia pastoris GS115 competent cells to obtain recombinant human type III collagen engineering bacteria.

[0025] 2. Inoculate the recombinant human type III collagen engineering bacteria into 200 mL YPD liquid culture medium, culture at 30°C and 300 r / min for 24 h to obtain the recombinant human collagen engineering bacteria seed solution, wherein the components of the YPD liquid culture medium include: 1 g yeast powder, 2 g peptone, 2 g glucose, dilute to 100 mL, and sterilize at 110°C for 20 min.

[0026] 3. Inoculate the recombinant human collagen engineering bacteria seed liquid into 2.5L BSM liquid culture medium at a volume ratio of 1:25, and culture it at 30℃ and 200 r / min overnight to obtain the recombinant human collagen engineering bacteria activation liquid. The components of BSM liquid culture medium include: 10 mL 100 mmol / L phosphate buffer, 1.26 g glycerol, 1.63 g (NH4)2SO4, 1.49 g MgSO4·7H2O, 0.093 g CaSO4·2H2O, make up to 100 mL, sterilize at 121℃ for 15 min, and add 0.44 mL PTM1. PTM1 includes: 6 g CuSO4·5 H2O, 0.088 g KI, 3 g MnSO4·H2O, 0.2 g Na2MoO4·2H2O, 0.02 g H3BO3, 0.5 g CoCl2·6 H2O, 20 g ZnCl2, 65 g FeSO4·7 H2O, 0.2 g biotin, and 5 mL concentrated H2SO4. The volume was made up to 1 L and filtered for sterilization.

[0027] 4. The recombinant human collagen engineering bacteria activated liquid was inoculated into 50L BSM liquid culture medium at a volume ratio of 1:25. During this process, methanol was continuously added to a final concentration of 1.5% to induce fermentation. The fermentation was induced at 30°C and 300 r / min for 80 hours. After the fermentation was completed, the supernatant was collected by centrifugation at 8000 r / min for 20 minutes to obtain a fermentation liquid containing recombinant human type III collagen. The amino acid sequence of the recombinant human type III collagen is shown in SEQ ID NO.2. The concentration of the recombinant human type III collagen fermentation liquid is 15g / L.

[0028] SEQ ID NO.1:

[0029]

[0030]

[0031] SEQ ID NO.2

[0032] PRGAPGERGRPGLPGAAGARGNDGARGSDGQPGPPGPPGTAGFPGSPGAKGEVGPAG

[0033] SPGSNGAPGQRGEPGPQGHAGAQGPPGPPGINGSPGGKGEMGPAGIPGAPGLMGARG

[0034] PPGPAGANGAPGLRGGAGEPGKNGAKGEPGPRGERGEAGIPGVPGAKGEDGKDGSP

[0035] GEPGANGLPGAAGERGAPGFRGPAGPNGIPGEKGPAGERGAPGPAGPRGAAGEPGRD

[0036] GVPGGPGMRGMPGSPGGPGSDGKPGPPGSQGESGRPGPPGSGPRGQPGVMGFPGPK

[0037] GNDGAPGKNGERGGPGGPGPQGPPGKNGETGPQGPPGPTGPGGDKGDTGPPGPQGL

[0038] QGLPGTGGPPGENGKPGEPGPKGDAGAPGAPGGKGDAGAPGERGPPGLAGAPGLRG

[0039] GAGPPGPEGGKGAAGPPGPPGAAGTPGLQGMPGERGGLGSPPGPKGDKGEPGGPGAD

[0040] GVPGKDGPRGPTGPIGPPGPAGQPGDKGEGGAPLPGIAGPRGSPGER

[0041] Example 1 Deglycosylation of recombinant human type III collagen

[0042] The experimental chromatography column specification is 4.6*50mm, the column volume is 0.83mL, and the filler used is Proteomix POR20-ConA chromatography column produced by Suzhou Saifen Technology Co., Ltd., item number 271320980P-4605.

[0043] The Proteomix POR20-ConA affinity chromatography column is specially suitable for rapid quantitative analysis and purification of carbohydrate compounds. Its chromatographic filler is a highly cross-linked porous polystyrene / divinylbenzene (PS / DVB) microsphere with a uniform hydrophilic coating on the surface. Concanavalin A (Con A) is bonded to the coating surface. The average particle size is 20μm, with a narrow particle size distribution of D90 / D10<1.3, and a pore size of 2000A. The Con A protein bonded to the surface of the column can bind to α-mannose and a-glucose, and divalent metal ions such as Ca are required during use. 2+ ,Mg 2+To maintain the affinity activity of Con A with carbohydrate compounds. It is mainly suitable for affinity purification of glycoconjugates, such as glycoproteins, glycopeptides and polysaccharides. The chromatographic filler has good mechanical strength and can withstand high flow rate and high pressure operation. It can be applied to HPLC and FPLC systems at a higher flow rate.

[0044] The specific steps for purifying type III collagen solution using Proteomix POR20-ConA affinity chromatography are as follows:

[0045] 1. Balance: Use balance solution (50mM NaAc, 0.2M NaCl, 1mM CaCl2, 1mM MgCl2, pH 5.3), balance for 2min, flow rate is 0.5mL / min.

[0046] 2. Loading: Dilute the type III collagen fermentation broth three times with the equilibrium solution to a concentration of 5 mg / mL. Filter the sample solution with a 0.22 μm filter membrane before loading. The loading volume is 5 μL, the flow rate is 0.5 mL / min, and the sample is manually collected after the peak appears.

[0047] 3. Post-equilibrium: Use equilibration solution (50mM NaAc, 0.2M NaCl, 1mM CaCl2, 1mM MgCl2, pH5.3) and 0-100% elution solution (50mM NaAc, 0.2M NaCl, 1mM CaCl2, 1mM MgCl2, 100mM α-methyl-D-mannoside, pH5.3) for linear equilibrium for 0.5min, with a flow rate of 0.5mL / min.

[0048] 4. Elution: Use eluent (50mM NaAc, 0.2M NaCl, 1mM CaCl2, 1mM MgCl2, 100mM α-methyl-D-mannoside, pH 5.3) for 5.5min, with a flow rate of 0.5mL / min, and collect the wavelength UV214. Manually collect the sample after the peak appears.

[0049] 5. Balance: Use balance solution (50mM NaAc, 0.2M NaCl, 1mM CaCl2, 1mM MgCl2, pH5.3), balance for 8min, flow rate is 0.5mL / min. Figure 1 The peak with a retention time of 1.227 min is the post-equilibrium peak, which is the non-glycosylated sample, and the peak with a retention time of 7.294 min is the elution peak, which is the glycosylated sample.

[0050] 6. Through the above steps, the total sugar content of the sample solution and the post-equilibrium solution is tested.

[0051] The total sugar content detection method is as follows:

[0052] S1. Instruments and supplies: total sugar content detection kit (name: Nominkoda, item number: NM-W-0206 micro-method 100T, 96S), ELISA reader (company name: Thermo Fisher, model: MULTISKAN GO), 96-well plate, desktop centrifuge, constant temperature water bath / incubator, homogenizer, balance, adjustable pipette, distilled water.

[0053] S2. Experimental steps:

[0054] Preparation of standard solution: dilute the standard stock solution in the 10 mg / mL total sugar detection kit with distilled water to make 4, 2, 1, 0.8, 0.6, 0.4, and 0.2 mg / mL standard solutions for use.

[0055] Table 1 Volume and concentration of standard stock solution before and after dilution

[0056]

[0057] Sample dilution: Take 0.05 mL of the sample solution and the post-equilibrium solution respectively, add 750 μL of distilled water, mix well, add 500 μL of reagent 1 in the total sugar detection kit, seal and heat in a boiling water bath for 30 minutes, cool to room temperature with cold water, add 500 μL of reagent 2 in the total sugar detection kit, make up to 2 mL with distilled water, vortex mix, centrifuge at 12000 rpm, 25°C for 10 minutes, and take the supernatant for later use.

[0058] Pre-experimental operation: Preheat the microplate reader for more than 30 minutes and adjust the wavelength to 540nm.

[0059] Table 2 Operation table of specific determination

[0060]

[0061] According to the above operation method, the ΔA determination and ΔA standard of the loading solution and the post-equilibrium solution were obtained. By reading the data on the microplate reader, the total sugar contents of the loading solution and the post-equilibrium solution were 10% and 0.5%, respectively. Therefore, the glycosylation removal rate in this example was 95%.

[0062] Comparative Example 1

[0063] The experimental chromatography column of this comparative example has a specification of 6.6*150 mm and a column volume of 5.132 mL. The filler used is the boric acid affinity filler Monomix HC60-Boronate produced by Suzhou Saifen Technology Co., Ltd., with a product number of 283460950.

[0064] The steps of using the boric acid affinity filler Monomix HC60-Boronate to purify the type III collagen solution prepared in Example 1 are as follows:

[0065] 1. Equilibration: Use equilibration solution (100 mM Gly-NaOH, pH 8.5) to equilibrate the boric acid affinity filler for 4 CV at a flow rate of 1.026 mL / min.

[0066] 2. Sample loading: dilute the sample 3 times with the equilibrium solution, adjust the conductivity and pH to be consistent with the equilibrium solution, load 2 mL, and the flow rate is 1.026 mL / min.

[0067] 3. Post-equilibrium: Use equilibration solution (100 mM Gly-NaOH, pH 8.5) for 5 CV at a flow rate of 1.026 mL / min.

[0068] 4. Elution: Use balancing solution (100mM Gly-NaOH, pH8.5) and eluent (100mM Gly-NaOH, pH3.0) to perform linear elution for 12CV, which is a linear elution of 0-100% eluent, and then use 100% eluent for 5CV, with a flow rate of 1.026mL / min, collect the eluent, and the collected signal is 215mAu.

[0069] 5. CIP: Use (0.1 M NaOH) to perform CIP on the chromatography column, 5CV, flow rate is 1.026mL / min.

[0070] 6. Wash: Wash with pure water for 5CV at a flow rate of 1.026mL / min. Figure 2 As can be seen from the figure, some pigments flowed out when the sample was loaded. All samples were combined on the column and the eluate was collected to detect the total sugar content. The total sugar content dropped from 10% to 5%.

[0071] 7. The collected eluate was tested for total sugar content using the total sugar determination method of Example 1. The total sugar content of this comparative example was 5%, and the glycosylation removal rate was 50%.

[0072] The above experimental results show that the glycosylation content of the original sample is 10%. After purification by Proteomix POR20-ConA, the glycosylation ratio is 0.5%, and the glycosylation removal rate is 95%. After boric acid affinity purification, the glycosylation ratio dropped from the original 10% to 5%. In summary, Proteomix POR20-ConA has higher specificity and can better remove glycosylation.

[0073] Example 2 Purification of Type III Collagen

[0074] The method is basically the same as Example 1, except that in the balancing step, the balancing solution used is (60mM NaAc, 0.3M NaCl, 2mM CaCl2, 2mM MgCl2, pH 5.5). In the post-balancing step, the balancing solution is (60mM NaAc, 0.3M NaCl, 2mM CaCl2, 2mM MgCl2, pH 5.5). In the elution step, the elution solution is (60mM NaAc, 0.3M NaCl, 2mMCaCl2, 2mM MgCl2, 120mM α-methyl-D-mannoside, pH 5.5). The experimental results show that the glycosylation content is 0.68% and the removal rate is 93.2%.

[0075] Example 3 Purification of Type III Collagen

[0076] The method is basically the same as Example 1, except that in the balancing step, the balancing solution used is (55mM NaAc, 0.25M NaCl, 1.5mM CaCl2, 1.5mM MgCl2, pH 5.4). In the post-balancing step, the balancing solution is (55mM NaAc, 0.25M NaCl, 1.5mM CaCl2, 1.5mM MgCl2, pH 5.4). In the elution step, the elution solution is (55mM NaAc, 0.25M NaCl, 1.5mM CaCl2, 1.5mM MgCl2, 110mM α-methyl-D-mannoside, pH 5.4). The experimental results show that the glycosylation content is 0.6% and the removal rate is 94%.

Claims

1. A method for purifying collagen using concanavalin A, characterized in that: The method adopts a chromatographic column containing concanavalin A to purify collagen.

2. The method for purifying collagen using concanavalin A according to claim 1, characterized in that: The filler of the chromatographic column containing concanavalin A is porous polystyrene / divinylbenzene microspheres with a high degree of cross-linking, a hydrophilic coating is bonded to the surface of the filler, and the concanavalin A is bonded to the surface of the coating.

3. The method for purifying collagen using concanavalin A according to claim 2, characterized in that: The average particle size of the filler is 20 μm, the narrow particle size distribution of D90 / D10<1.3, and the pore size of the filler is 4. The method for purifying collagen using concanavalin A according to claim 1, characterized in that: The chromatographic column containing concanavalin A is a Proteomix POR20-ConA chromatographic column.

5. The method for purifying collagen using concanavalin A according to claim 1, characterized in that: The collagen includes type III collagen.

6. The method for purifying collagen using concanavalin A according to claim 1, characterized in that: The specific steps include: 1) Pretreatment of type III collagen: 2) Purify the pretreated type III collagen using a chromatographic column containing concanavalin A.

7. The method for purifying collagen using concanavalin A according to claim 6, characterized in that: The pretreatment step of type III collagen in step 1) is specifically as follows: adding a sample containing type III collagen to a balanced solution, dissolving and filtering to obtain a type III collagen solution with a concentration of 15 g / L, wherein the balanced solution contains 50-60 mM NaAc, 0.2-0.3 M NaCl, 1-2 mM CaCl2, 1-2 mM MgCl2, and pH 5.3-5.

5.

8. The method for purifying collagen using concanavalin A according to claim 6, characterized in that: The purification step in step 2) includes equilibration, loading, post-equilibration, elution and equilibration steps.

9. The method for purifying collagen using concanavalin A according to claim 8, characterized in that: The equilibration solution in the equilibration step contains 50-60 mM NaAc, 0.2-0.3 M NaCl, 1-2 mM CaCl2, 1-2 mM MgCl2, pH 5.3-5.

5.

10. The method for purifying collagen using concanavalin A according to claim 8, characterized in that: The eluent in the elution step contains 50-60 mM NaAc, 0.2-0.3 M NaCl, 1-2 mM CaCl2, 1-2 mM MgCl2, 100-120 mM α-methyl-D-mannoside, pH 5.3-5.5.

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