Purification process of OAS1-Cas13aREC fusion protein

Abstract

The invention relates to the technical field of biology, and discloses a purification process of an OAS1-Cas13aREC fusion protein. According to optimization of reagents in a conventional protein purification process, a purification process of the OAS1-Cas13aREC fusion protein expressed in escherichia coli is provided, a bacterial lysis solution and an affinity equilibrium solution and an affinity eluent in a chromatographic column are improved and optimized, the yield and the protein purity of the OAS1-Cas13aREC fusion protein are improved, the purity of the OAS1-Cas13aREC fusion protein is improved, the purity of the OAS1-Cas13aREC fusion protein is improved, and the purity of the OAS1-Cas13aREC fusion protein is improved. And protein oxidation is prevented, and the protein structure is stabilized, so that the protein is not easy to denature.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and in particular to a purification process for an OAS1-Cas13aREC fusion protein. Background Art

[0002] Fusion proteins, products of recombinant DNA technology, have been developed as a new class of biomolecules with multifunctional properties. By functionally fusing two or more protein domains, fusion proteins can acquire a variety of functions derived from their individual components. In addition to their widespread application in biological research, such as protein purification and imaging, recombinant fusion proteins have also become an important category of biopharmaceuticals.

[0003] In order to combine the direct response ability of OAS1 to dsRNA with the high-precision targeting ability of the CRISPR system to specific nucleotides, the preliminary research of this application recombined the Cas-13aREC domain in the CRISPR-Cas system with the OAS1 protein to construct a fusion protein. Through the understanding of the REC domain in Cas13a, a fusion protein can be constructed to use REC as a crRNA recognizer, and the raised stem-loop structure in the secondary structure of crRNA binds to REC as a direct repeat sequence. At the same time, the target RNA-specific sequence at the tail end binds to the target chain of the test sample to form an RNA double-stranded activation OAS1 portion. The PPi generated by the OAS1 protein under the combination of short dsRNA and ATP can be measured by colorimetric analysis, so that the target RNA sequence added to the system can be quantitatively determined. Among them, the OAS1-Cas13aREC fusion protein needs to be prepared and purified by an expression vector before being applied to the above-mentioned research scheme of this application. However, after research, it was found that the existing commonly used expression and purification reagents are not suitable for the OAS1-Cas13aREC fusion protein, so that its purity and expression level are not high. Therefore, it is necessary to provide a purification process suitable for the OAS1-Cas13aREC fusion protein to improve its expression level and purity. Summary of the Invention

[0004] In view of this, the object of the present application is to provide a purification process for OAS1-Cas13aREC fusion protein, so that the purification process can improve the purification yield and protein purity of OAS1-Cas13aREC fusion protein;

[0005] Another object of the present application is to provide the use of the bacterial lysate in the purification process in purifying the OAS1-Cas13aREC fusion protein or preparing a purification reagent product, as well as a reagent product for purifying the OAS1-Cas13aREC fusion protein.

[0006] In order to solve the above technical problems / achieve the above objectives or at least partially solve the above technical problems / achieve the above objectives, as a first aspect of the present application, a purification process of the OAS1-Cas13aREC fusion protein is provided, comprising:

[0007] S1. The expression vector of the OAS1-Cas13aREC fusion protein is transformed into Escherichia coli competent cells; the protein sequence of the OAS1-Cas13aREC fusion protein is shown in SEQ ID No.1;

[0008] S2. Inducing the competent E. coli cells with IPTG for expression, and harvesting the cells after the induction expression is completed;

[0009] S3. The cells were resuspended in bacterial lysis buffer and disrupted, and the supernatant was filtered to obtain the affinity chromatography sample; the components of the bacterial lysate were as follows:

[0010] 20mmol / L±5mmol / L HEPES, 400mmol / L±50mmol / L sodium chloride, 30mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, pH 7.5±0.2;

[0011] S4. The loaded sample is subjected to affinity chromatography to obtain the purified OAS1-Cas13aREC fusion protein.

[0012] Optionally, the expression vector is a commercial pET plasmid comprising a histidine tag, a multiple cloning site, a promoter, a replication origin, a transcription termination signal, and a resistance gene, and the expression sequence of the OAS1-Cas13aREC fusion protein is inserted into the multiple cloning site.

[0013] Optionally, step S2 includes:

[0014] The Escherichia coli competent cells are inoculated into LB medium for expansion, and corresponding antibiotics are added to the medium according to the resistance gene carried by the expression vector of the OAS1-Cas13aREC fusion protein;

[0015] After expansion, IPTG was added to the LB medium at a final concentration of 0.5 mM ± 0.2 mM, and the culture was continued at 30°C and 120 RPM for 20 h. After the induction expression was completed, the bacteria were collected by centrifugation.

[0016] Optionally, step S3 includes:

[0017] The bacteria were resuspended in a bacterial lysis solution 10 times their weight, and the bacteria were disrupted by ultrasound. The ultrasound time and the volume ratio of the bacterial lysis solution were 1.5 min:1 mL. After filtration, the supernatant was obtained as the affinity chromatography sample.

[0018] Optionally, step S4 includes:

[0019] S4.1. Equilibrate the chromatography column with an affinity equilibration solution consisting of 20 mmol / L ± 5 mmol / L HEPES, 1 mol / L ± 50 mmol / L sodium chloride, 30 mmol / L ± 5 mmol / L imidazole, 5 mmol / L ± 2 mmol / L β-mercaptoethanol, and 10% ± 2% glycerol, with a pH of 7.5 ± 0.2.

[0020] S4.2. Load the sample onto the equilibrated chromatography column. After loading, use the affinity equilibration solution to wash away proteins that are not bound to the chromatography column.

[0021] S4.3. Use affinity eluent to elute the chromatography column to obtain the purified OAS1-Cas13aREC fusion protein; the affinity eluent components are 20mmol / L±5mmol / LHEPES, 500mmol / L±50mmol / L sodium chloride, 300mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, and pH is 7.5±0.2.

[0022] Further optionally, step S4.3 includes:

[0023] The affinity elution solution is diluted with the affinity balance solution to a concentration of 1-5%, and the affinity elution solution with a diluted concentration of 1-5% is added to the chromatography column to wash the weakly bound impurities;

[0024] The affinity elution solution was diluted with the affinity equilibration solution to a concentration of 70±5%, and the affinity elution solution with a diluted concentration of 70±5% was added to the chromatography column several times to elute the OAS1-Cas13aREC fusion protein.

[0025] Optionally, the method further comprises: ultrafiltration of the OAS1-Cas13aREC fusion protein to remove imidazole, and adding a protein preservation solution for preservation.

[0026] As a second aspect of the present application, provided is the use of a bacterial lysate in purifying an OAS1-Cas13aREC fusion protein or in preparing a reagent product for purifying an OAS1-Cas13aREC fusion protein;

[0027] The bacterial lysate components are as follows:

[0028] 20mmol / L±5mmol / L HEPES, 400mmol / L±50mmol / L sodium chloride, 30mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, pH 7.5±0.2.

[0029] As a third aspect of the present application, a reagent product for purifying the OAS1-Cas13aREC fusion protein is provided, comprising a bacterial lysate, an affinity equilibration solution and / or an affinity elution solution, wherein the components of the bacterial lysate are as follows:

[0030] 20mmol / L±5mmol / L HEPES, 400mmol / L±50mmol / L sodium chloride, 30mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, pH 7.5±0.2.

[0031] Optionally, the affinity balancing solution comprises 20 mmol / L±5 mmol / L HEPES, 1 mol / L±50 mmol / L sodium chloride, 30 mmol / L±5 mmol / L imidazole, 5 mmol / L±2 mmol / L β-mercaptoethanol, 10%±2% glycerol, and a pH of 7.5±0.2;

[0032] The affinity eluent comprises 20 mmol / L±5 mmol / L HEPES, 500 mmol / L±50 mmol / L sodium chloride, 300 mmol / L±5 mmol / L imidazole, 5 mmol / L±2 mmol / L β-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, and has a pH of 7.5±0.2.

[0033] This application optimizes the reagents in the conventional protein purification process and provides a purification process for the OAS1-Cas13aREC fusion protein expressed in Escherichia coli. The bacterial lysate and the affinity balance solution and affinity elution solution in the chromatography column are improved and optimized, which not only improves the yield and protein purity of the OAS1-Cas13aREC fusion protein, but also prevents protein oxidation and stabilizes the protein structure, making it less susceptible to denaturation. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] The drawings constituting a part of the present application are used to provide a further understanding of the present application. The exemplary embodiments and descriptions of the present application are used to explain the present application and do not constitute an improper limitation on the present application.

[0035] Figure 1 Shown is a map of the OAS1-Cas13aREC fusion protein expression vector;

[0036] Figure 2 Shown is a gel image of protein gravity column purification in the purification process of this application;

[0037] Figure 3 Shown are the gel detection results of the target fusion protein after lysis with different bacterial lysates;

[0038] Lane 1: Induction conditions: 1.0 mM IPTG, 30°C, 180 rpm, 12 h induction time, using conventional bacterial lysis buffer;

[0039] Lane 2: Induction conditions: 0.5 mM IPTG, 30°C, 120 rpm, induction time 20 h, using conventional bacterial lysis buffer;

[0040] Lane 3: Induction conditions: 0.5 mM IPTG, 30°C, 120 rpm, induction time 20 h, using the bacterial lysis solution of this application;

[0041] Figure 4 Shown is a comparison of the actual results of the target fusion protein purified by different purification reagents; the left tube shows the results using conventional bacterial lysis buffer, affinity equilibration buffer, and affinity elution buffer; the right tube shows the results using the bacterial lysis buffer, affinity equilibration buffer, and affinity elution buffer of the present application. DETAILED DESCRIPTION

[0042] The present application discloses a purification process for an OAS1-Cas13aREC fusion protein. Those skilled in the art can refer to the contents of this article and appropriately improve the process parameters for implementation. It should be noted in particular that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The products, processes and applications described in this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods described herein without departing from the content, spirit and scope of this application to implement and apply the technology of this application. Obviously, the described embodiments are part of the embodiments of this application, not all of the embodiments. Based on the embodiments in this application, all other embodiments obtained by those of ordinary skill in the art without making creative work are within the scope of protection of this application.

[0043] It should be noted that, in this document, if relational terms such as "first" and "second", "step 1" and "step 2", and "(1)" and "(2)" appear, they are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply that there is any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device. In the absence of further restrictions, the elements defined by the sentence "including a..." do not exclude the presence of other identical elements in the process, method, article or device comprising the elements. At the same time, the embodiments in this application and the features in the embodiments can be combined with each other in the absence of conflict.

[0044] In a first aspect of the present application, a purification process for the OAS1-Cas13aREC fusion protein is provided, comprising:

[0045] S1. The expression vector of the OAS1-Cas13aREC fusion protein is transformed into Escherichia coli competent cells; the protein sequence of the OAS1-Cas13aREC fusion protein is shown in SEQ ID No.1;

[0046] S2. Inducing the competent E. coli cells with IPTG for expression, and harvesting the cells after the induction expression is completed;

[0047] S3. The cells were resuspended in bacterial lysis buffer and disrupted, and the supernatant was filtered to obtain the affinity chromatography sample; the components of the bacterial lysate were as follows:

[0048] 20mmol / L±5mmol / L HEPES, 400mmol / L±50mmol / L sodium chloride, 30mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, pH 7.5±0.2;

[0049] S4. The loaded sample is subjected to affinity chromatography to obtain the purified OAS1-Cas13aREC fusion protein.

[0050] The sodium chloride concentration of each solution used in conventional protein purification is generally 300mmol / L, but the OAS1-Cas13aREC fusion protein structure is unstable and protein denaturation is prone to occur in conventional solution systems, so the sodium chloride concentration of each solution used in the protein purification is increased in this application. Taking into account the particularity of the structure and properties of the OAS1-Cas13aREC fusion protein, this application adjusts the components of the bacterial lysate, affinity balance solution, and affinity eluate in the purification process, adds glycerol and Tween 20, and replaces the buffer type. The pH value of each reagent is also optimized to 7.5±0.2 to prevent protein aggregation and denaturation.

[0051] In certain embodiments of the present application, the expression vector is a commercial pET plasmid comprising a histidine tag, a multiple cloning site, a promoter, a replication origin, a transcription termination signal, and a resistance gene, and the expression sequence of the OAS1-Cas13aREC fusion protein is inserted into the multiple cloning site. In other embodiments of the present application, the plasmid map of the expression vector of the OAS1-Cas13aREC fusion protein is shown in FIG. Figure 1 After the OAS1-Cas13aREC fusion protein was induced and expressed and purified, since the plasmid carried a histidine tag and a T7 promoter, its protein sequence was increased by 6×His and LE on the basis of SEQ ID No. 1, as shown in SEQ ID No. 2:

[0052] HMELRHTPARDLDKFIEDHLLPNTCFRTQVKEAIDIVCRFLKERCFQGTADPVRVSKVVKGGSSGKGTTLRGRSDADLVVFLTKLTSFEDQLRRRGEFIQEIRRQLEACQREQKFKVTFEVQSPRRENPRALSFVLSSPQLQQEVEFDVLPAFDALGQWTPGYKPNPEIYVQLIKECKSRGKEGEFSTCFTELQRD FLRNRPTKLKSLIRLVKHWYQTCKKTHGNKLPPQYALELLTVYAWEQGSRKTDFSTAQGFQTVLELVLKHQKLCIFWEAYYDFTNPVVGRCMLQQLKKPRPVILDPADPTGNVGGGDTHSWQRLAQEARVWLGYPCCKNLDGSLVGAWTMLQKI (Cas13a REC domain) GGGGSGGGGSGGGGS (flexible protein linker) MKV TKVDGISHKKYIEEGKLVKSTSEENRTSERLSELLSIRLDIYIKNPDNASEEENRIRRENLKKFFSNKVLHLKDSVLYLKNRKEKNAVQDKNYSEEDISEYDLKNKNSFSVLKKILLNEDVNSEELEIFRKDVEAKLNKINSLKYSFEENKANYQKINENNVEKVGGKSKRNIIYDYYRESAKRNDYINNVQEAFD KLYKKEDIEKLFFLIENSKKHEKYKIREYYHKIIGRKNDKENFAKIIYEEIQNVNNIKELIEKIPDMSELKKSQVFYKYYLDKEELNDKNIKYAFCHFVEIEMSQLLKNYVYKRLSNISNDKIKRIFEYQNLKKLIENKLLNKLDTYVRNCGKYNYYLQVGEI (OAS1 protein part) HHHHHH (histidine tag) LE (T7 promoter).

[0053] In certain embodiments of the present application, step S2 includes:

[0054] The Escherichia coli competent cells are inoculated into LB medium for expansion, and corresponding antibiotics are added to the medium according to the resistance gene carried by the expression vector of the OAS1-Cas13aREC fusion protein;

[0055] After expansion, IPTG was added to the LB medium at a final concentration of 0.5 mM ± 0.2 mM, and the culture was continued at 30°C and 120 RPM for 20 h. After the induction expression was completed, the bacteria were collected by centrifugation.

[0056] In some other embodiments of the present application, the Escherichia coli competent cells are DH5α competent cells, and kanamycin is added to the LB medium to screen Escherichia coli transformed with the expression vector (containing the kanamycin resistance gene) of the OAS1-Cas13aREC fusion protein.

[0057] In certain embodiments of the present application, step S3 includes:

[0058] The bacteria were resuspended in a bacterial lysis solution 10 times their weight, and the bacteria were disrupted by ultrasound. The ultrasound time and the volume ratio of the bacterial lysis solution were 1.5 min:1 mL. After filtration, the supernatant was obtained as the affinity chromatography sample.

[0059] In some other embodiments of the present application, the ultrasound program is set to ultrasound for 5 seconds and stop for 9 seconds.

[0060] In certain embodiments of the present application, step S4 includes:

[0061] S4.1. Equilibrate the chromatography column with an affinity equilibration solution consisting of 20 mmol / L ± 5 mmol / L HEPES, 1 mol / L ± 50 mmol / L sodium chloride, 30 mmol / L ± 5 mmol / L imidazole, 5 mmol / L ± 2 mmol / L β-mercaptoethanol, and 10% ± 2% glycerol, with a pH of 7.5 ± 0.2.

[0062] S4.2. Load the sample onto the equilibrated chromatography column. After loading, use the affinity equilibration solution to wash away proteins that are not bound to the chromatography column.

[0063] S4.3. Use affinity eluent to elute the chromatography column to obtain the purified OAS1-Cas13aREC fusion protein; the affinity eluent components are 20mmol / L±5mmol / LHEPES, 500mmol / L±50mmol / L sodium chloride, 300mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, and pH is 7.5±0.2.

[0064] In some other embodiments of the present application, the chromatography column is a gravity column filled with NI NTA beads.

[0065] In certain embodiments of the present application, step S4.1 includes: adding pure water to wash the chromatography column, and then rinsing the chromatography column with the affinity equilibration solution, 10 ml / time, for two or three times.

[0066] In certain embodiments of the present application, step S4.3 includes:

[0067] Use the affinity balancing solution to dilute the affinity elution solution to a concentration of 1-5%, for example, 2%, and add the affinity elution solution with a diluted concentration of 1-5% to the chromatography column to clean the weakly bound impurities, usually adding 10 mL;

[0068] The affinity elution solution was diluted with the affinity equilibration solution to a concentration of 70±5%, and the affinity elution solution with a diluted concentration of 70±5% was added to the chromatography column several times to elute the OAS1-Cas13aREC fusion protein, usually adding 15mL±2mL of affinity elution solution in multiple times.

[0069] In certain embodiments of the present application, the method further comprises: ultrafiltration of the OAS1-Cas13aREC fusion protein to remove imidazole, and adding a protein preservation solution for preservation.

[0070] In the second aspect of the present application, a bacterial lysis test was performed with a conventional bacterial lysis solution, and the protein gel results showed that more OAS1-Cas13aREC fusion protein could be obtained using the bacterial lysate optimized in the present application; based on this, the present application provides the use of bacterial lysate in purifying OAS1-Cas13aREC fusion protein or in preparing a reagent product for purifying OAS1-Cas13aREC fusion protein;

[0071] The bacterial lysate components are as follows:

[0072] 20mmol / L±5mmol / L HEPES, 400mmol / L±50mmol / L sodium chloride, 30mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, pH 7.5±0.2.

[0073] In the third aspect of the present application, a reagent product for purifying the OAS1-Cas13aREC fusion protein is also provided, comprising a bacterial lysate, and an affinity balance solution and / or an affinity elution solution, wherein the components of the bacterial lysate are as follows:

[0074] 20mmol / L±5mmol / L HEPES, 400mmol / L±50mmol / L sodium chloride, 30mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, pH 7.5±0.2.

[0075] In certain embodiments of the present application, the affinity balancing solution comprises 20 mmol / L±5 mmol / L HEPES, 1 mol / L±50 mmol / L sodium chloride, 30 mmol / L±5 mmol / L imidazole, 5 mmol / L±2 mmol / L β-mercaptoethanol, 10%±2% glycerol, and a pH of 7.5±0.2;

[0076] The affinity eluent comprises 20 mmol / L±5 mmol / L HEPES, 500 mmol / L±50 mmol / L sodium chloride, 300 mmol / L±5 mmol / L imidazole, 5 mmol / L±2 mmol / L β-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, and has a pH of 7.5±0.2.

[0077] The protein purified using the bacterial lysate, affinity equilibration solution, and affinity elution solution provided in this application has a higher yield of OAS1-Cas13aREC fusion protein than conventional purification processes and reagents, and the protein liquid is clear and turbid, indicating higher stability.

[0078] In the comparative experiments provided in this application, unless otherwise specified, all experimental conditions and materials, except for the differences noted in each group, were kept consistent to ensure comparability. In addition, all materials used in this application can be purchased from commercial sources.

[0079] In the sequence information provided in this application, unless otherwise specified, the sequence is in 5'-3' order, that is, the default writing rules in the biological field are adopted.

[0080] The following further describes the purification process of an OAS1-Cas13aREC fusion protein provided in this application.

[0081] Example 1:

[0082] By using gene recombination technology, the OASI and Cas13aREC domain target genes, his tag, and anti-kana gene were integrated into the pET28a vector and introduced into E. coli. The target protein was expressed in E. coli by propagation of E. coli and induction with IPTG. The expression plasmid of the successfully constructed strain is shown in the attached figure. Figure 1 shown.

[0083] 1. Use E. coli culture to induce the production of OAS1-Cas13aREC fusion protein

[0084] The culture medium used in the experiment was ordinary LB liquid culture medium, that is, 10 g of tryptone, 5 g of yeast extract, and 10 g of sodium chloride were added to each 1 L of culture medium, and kanamycin was added to make the final concentration 50 μg / mL.

[0085] The construction operation of the expression strain is as follows: 5 μL of the synthesized OAS1-Cas13aREC plasmid (10 ng / μL) is added to 50 μL of DH5α competent cells and flicked to mix, and quickly placed on ice for 30 minutes, and then immediately transferred to ice after heat shock in a 42 ° C water bath for 90 seconds. The obtained competent cells are added to 500 μL of LB medium and cultured for 1 hour under the conditions of 37 ° C and 200 RPM to recover bacteria. After the culture is completed, the bacterial solution is spread on LB solid medium containing 50 μg / mL kanamycin (Kana), and the single colony is picked for amplification after overnight culture. The bacterial solution after expansion is sequenced. When the sequencing result is consistent with the OAS1-Cas13a plasmid, the expression strain is successfully constructed. The successfully constructed strain is stored at -80 ° C by adding sterilized 50% glycerol after passage.

[0086] Glycerol culture recovery: Add 5 μL of 50 μg / mL kanamycin solution to 5 mL of LB medium, then inoculate 50 μL of glycerol culture and recover at 37°C and 180 rpm for about 12 h.

[0087] E. coli expansion and induced expression: 500 μL of glycerol bacteria recovery solution was inoculated into 500 mL of LB liquid medium containing 50 μg / mL Kana, and cultured at 37°C and 180 rpm to an OD value of 0.8. Then, isopropyl-β-D-thiogalactopyranoside (IPTG) inducer was added at a final concentration of 0.5 mM ± 0.2 mM, and cultured at 30°C and 120 RPM for 20 h.

[0088] 2. Bacterial culture fluid treatment and protein purification by NI gravity column affinity chromatography

[0089] Lysis of bacterial culture: After induction, centrifuge the culture at 9800 RPM and 4°C for 5 minutes to collect the cells. Remove the supernatant and resuspend the cells in approximately 10 times the cell mass in lysis buffer. Use a pipette to repeatedly disperse the pellet during resuspension. Disrupt the cells using ultrasound. Use an ultrasonic cell disruptor at 25% power, program for 5 seconds on, 9 seconds off, and a total sonication time of 1.5 times the volume of lysis buffer (e.g., for a 30 mL disruption volume, the sonication time is 45 minutes). Once the cells are fully disrupted, the turbid suspension will turn translucent. Centrifuge the disrupted culture again at 9800 RPM and 4°C for 30 minutes. Filter the supernatant through a 0.22 μm filter and harvest the resulting suspension for affinity chromatography. It is recommended that all steps above, except centrifugation, be performed in an ice bath at 4°C to prevent protein denaturation.

[0090] The bacterial lysate components are as follows:

[0091] 20mmol / L±5mmol / L HEPES, 400mmol / L±50mmol / L sodium chloride, 30mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, pH 7.5±0.2.

[0092] 3. Gravity column affinity chromatography operation

[0093] Reagent preparation: pure water, 20% ethanol, affinity equilibration solution, affinity elution solution.

[0094] The affinity balance solution consists of 20 mmol / L ± 5 mmol / L HEPES, 1 mol / L ± 50 mmol / L sodium chloride, 30 mmol / L ± 5 mmol / L imidazole, 5 mmol / L ± 2 mmol / L β-mercaptoethanol, and 10% ± 2% glycerol, with a pH of 7.5 ± 0.2.

[0095] The affinity eluent components are 20mmol / L±5mmol / L HEPES, 500mmol / L±50mmol / L sodium chloride, 300mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, and the pH is 7.5±0.2.

[0096] (1) First, fill the column. Take about 5 mL of NI NTA beads filler and place it in the column for stratification and let it stand. Open the flow-through outlet. When about 1 mL of supernatant remains, cover and press the gasket until a solid is formed (pressing too tightly or adding too much material will result in a slow flow rate).

[0097] (2) After filling the column, add pure water to clean the column, 10 ml / time, and wash twice.

[0098] (3) Rinse the filler with affinity balancing solution, 10 ml / time, two or three times.

[0099] (4) Add the affinity chromatography sample obtained in the previous operation to the column. Do not load too much sample at a time and keep the flow-through solution dripping slowly. The target protein has a His tag. The histidine (His) residue has an imidazole group that can react with Ni 2+ 、Co 2+ Transition metal ions such as His-tagged proteins selectively bind to metal ions by forming coordination bonds. These metal ions can be immobilized on the chromatographic medium using chelating ligands. Therefore, His-tagged proteins can selectively bind to the chromatographic medium when passing through the metal ion-equipped medium, while other impurity proteins cannot bind or can only bind weakly. After approximately one column volume of flow-through has flowed through, a separate 2 mL of flow-through can be collected and subsequently run on the gel to observe the binding of the target protein to the column.

[0100] (5) After loading, add 10 mL of equilibration solution to wash away proteins that are not bound to the column, and collect 2 mL of flow-through solution to run the gel for observation.

[0101] (6) Dilute the affinity elution buffer to a concentration of 2% with affinity equilibration buffer, add 10 mL of 2% affinity elution buffer to wash away weakly bound impurities, and collect 2 mL of flow-through buffer for gel run and observation.

[0102] (7) Dilute the affinity elution buffer to a concentration of 70% with affinity equilibration buffer. Add approximately 15 mL of 70% affinity elution buffer several times to elute the target protein. Collect all the flow-through and run the gel for observation.

[0103] (8) After the sample is loaded, 10 mL of undiluted affinity eluent is used to wash away the residual protein in the column, and 2 mL of flow-through is collected and run on the gel for observation.

[0104] (9) Wash the column twice with 10 mL of pure water.

[0105] (10) Wash the column twice with 20% ethanol (10 mL each time). Finally, fill the column with 20% ethanol and store it at 4°C.

[0106] All liquids on the column need to be filtered with a 0.22μm filter membrane to avoid clogging of the column. Ni affinity chromatography gravity column needs to be regenerated after three uses to avoid weakening the binding force between the target protein and metal ions and affecting protein yield. The gel images of the protein gel of each step of the gravity column purification process are shown in the attached figure. Figure 2 As shown. Figure 2It can be seen that after the purification process of the present application, a fusion protein with the expected molecular weight (85KD) and high purity can be obtained.

[0107] 4. Protein ultrafiltration and preservation

[0108] The experiment used a 30kd ultrafiltration column. First, pour out 20% of the ethanol in the ultrafiltration tube, add ultrapure water to the ultrafiltration tube, and centrifuge at 3800g for 20 minutes at 4°C; then pour out the water filtered at the bottom of the centrifuge tube, add ultrapure water to the scale line, and centrifuge again at 3800g for 20 minutes at 4°C; the ultrafiltration tube was washed twice with filtered heparin column equilibrium solution; the protein solution purified by Ni column affinity chromatography was added to the scale line, and centrifuged at 3800g for 20 minutes at 4°C, and the operation was repeated until all samples were transferred; then the protein preservation solution filtered through a 0.22μm filter membrane was used to replace the high concentration of imidazole in the protein solution 3 times (retaining the supernatant protein solution), and finally 10ml of protein concentrate was retained; after taking out the sample, the ultrafiltration tube was washed with pure water several times, and finally the tube was filled with 20% ethanol and stored in a 4°C refrigerator. Use protein preservation solution to replace the elution solution containing high concentration of imidazole in the protein solution and remove some impurities. The obtained protein is packaged and stored at -80℃. It can still maintain good activity after long-term storage.

[0109] Example 2:

[0110] 1. Comparison of different bacterial lysates

[0111] The composition of conventional bacterial lysis buffer is:

[0112] 50mmol / L Tris-HCl, 300mmol / L sodium chloride, 20mmol / L imidazole, 5mmol / L β-mercaptoethanol, pH 8.0;

[0113] The bacterial lysis solution of this application is composed of:

[0114] 20mmol / L HEPES, 400mmol / L sodium chloride, 30mmol / L imidazole, 5mmol / L β-mercaptoethanol, 10% glycerol, 2% Tween 20, pH 7.5;

[0115] Prepare affinity chromatography sample according to steps 1-2 of Example 1, and then perform protein gel detection. The results are shown in Figure 3 ;according to Figure 3 It shows that more fusion protein can be obtained after 20 h of induction at 0.5 mM IPTG, 30°C, 120 rpm, and further using the bacterial lysate of the present application, the fusion protein yield is higher, and the color of the target protein band in lane 3 is darker than that in lanes 1 and 2 using conventional bacterial lysate.

[0116] 2. Comparison without bacterial lysis buffer, affinity balance buffer, or affinity elution buffer

[0117] The composition of conventional bacterial lysis buffer is:

[0118] 50mmol / L Tris-HCl, 300mmol / L sodium chloride, 20mmol / L imidazole, 5mmol / L β-mercaptoethanol, pH 8.0;

[0119] The conventional affinity balance solution is composed of:

[0120] 50mmol / L Tris-HCl, 300mmol / L sodium chloride, 5mol / L β-mercaptoethanol, pH 7.4;

[0121] The conventional affinity elution buffer composition is:

[0122] 50mmol / L Tris-HCl, 300mmol / L sodium chloride, 300mmol / L imidazole, 5mmol / L β-mercaptoethanol, pH 7.4;

[0123] The bacterial lysis solution of this application is composed of:

[0124] 20mmol / L HEPES, 400mmol / L sodium chloride, 30mmol / L imidazole, 5mmol / L β-mercaptoethanol, 10% glycerol, 2% Tween 20, pH 7.5;

[0125] The affinity balance solution components of this application are:

[0126] 20mmol / L HEPES, 1mol / L sodium chloride, 30mmol / L imidazole, 5mmol / L β-mercaptoethanol, 10% glycerol, pH 7.5;

[0127] The affinity eluent of the present application comprises 20 mmol / L HEPES, 500 mmol / L sodium chloride, 300 mmol / L imidazole, 5 mmol / L β-mercaptoethanol, 10% glycerol, 2% Tween 20, and a pH of 7.5.

[0128] Refer to step 1 to step 3 of Example 1 to obtain the target protein after elution. Figure 4 ; Figure 4 It shows that the protein in the left tube is the purified product eluted with conventional purification reagents, and there is obvious protein turbidity and denaturation; the protein in the right tube is the purified product eluted with the purification reagent of the present application, which is a clear and slightly viscous liquid, and its stability is significantly higher than that of conventional purification reagents.

[0129] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand or implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments shown herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. A purification process for OAS1-Cas13aREC fusion protein, characterized in that, include: S1. The expression vector of the OAS1-Cas13aREC fusion protein is transformed into Escherichia coli competent cells; the protein sequence of the OAS1-Cas13aREC fusion protein is shown in SEQ ID No.1; S2. inducing the competent E. coli cells to express using IPTG, and harvesting the cells after the induction expression is completed; S3. The bacterial cells are added to the bacterial lysis solution, resuspended and broken, and the supernatant is obtained after filtration as the affinity chromatography loading sample; the components of the bacterial lysis solution are as follows: 20mmol / L±5mmol / L HEPES, 400mmol / L±50mmol / L sodium chloride, 30mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, pH 7.5±0.2; S4. The loaded sample is subjected to affinity chromatography to obtain the purified OAS1-Cas13aREC fusion protein.

2. The purification process according to claim 1, characterized in that The expression vector is a commercial pET plasmid comprising a histidine tag, a multiple cloning site, a promoter, a replication start point, a transcription termination signal, and a resistance gene, and the expression sequence of the OAS1-Cas13aREC fusion protein is inserted into the multiple cloning site.

3. The purification process according to claim 1, characterized in that Step S2 includes: The E. coli competent cells are inoculated into LB medium for expansion, and corresponding antibiotics are added to the medium according to the resistance gene carried by the expression vector of the OAS1-Cas13aREC fusion protein; After expansion, add IPTG with a final concentration of 0.5 mM ± 0.2 mM to the LB medium, and continue culturing at 30°C and 120 RPM for 20 h. After the induction expression is completed, collect the bacteria by centrifugation.

4. The purification process according to claim 1, characterized in that Step S3 includes: The bacterial cells were resuspended in a bacterial lysis solution with a volume of 10 times the mass of the bacterial cells, and the bacterial cells were broken by ultrasound, with the ultrasound time and the volume ratio of the bacterial lysis solution being 1.5 min:1 mL. After filtration, the supernatant was obtained as an affinity chromatography loading sample.

5. The purification process according to claim 1, characterized in that Step S4 includes: S4.1, using affinity balancing solution to balance the chromatography column; the affinity balancing solution comprises 20mmol / L±5mmol / L HEPES, 1mol / L±50mmol / L sodium chloride, 30mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / L β-mercaptoethanol, 10%±2% glycerol, and pH 7.5±0.2; S4.2, adding the sample to the equilibrated chromatography column for loading, and after loading, using the affinity equilibration solution to wash away the protein not bound to the chromatography column; S4.

3. Use affinity eluent to elute the chromatography column to obtain the purified OAS1-Cas13aREC fusion protein; the affinity eluent components are 20mmol / L±5mmol / LHEPES, 500mmol / L±50mmol / L sodium chloride, 300mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, and the pH is 7.5±0.

2.

6. The purification process according to claim 5, characterized in that Step S4.3 includes: The affinity elution solution is diluted with the affinity balance solution to a concentration of 1-5%, and the affinity elution solution with a diluted concentration of 1-5% is added to the chromatography column to wash the weakly binding impurities; The affinity elution solution was diluted with the affinity balance solution to a concentration of 70±5%, and the affinity elution solution with a diluted concentration of 70±5% was added to the chromatography column several times to elute the OAS1-Cas13aREC fusion protein.

7. The purification process according to any one of claims 1 to 6, characterized in that: Also includes: The OAS1-Cas13aREC fusion protein was ultrafiltered to remove imidazole, and protein preservation solution was added for storage.

8. Use of bacterial lysate in purifying OAS1-Cas13aREC fusion protein or in preparing reagent products for purifying OAS1-Cas13aREC fusion protein; The components of the bacterial lysate are as follows: 20mmol / L±5mmol / L HEPES, 400mmol / L±50mmol / L sodium chloride, 30mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, pH 7.5±0.

2.

9. A reagent product for purifying OAS1-Cas13aREC fusion protein, characterized in that, It includes bacterial lysate, affinity balance solution and / or affinity elution solution, and the components of the bacterial lysate are as follows: 20mmol / L±5mmol / L HEPES, 400mmol / L±50mmol / L sodium chloride, 30mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, pH 7.5±0.

2.

10. The reagent product according to claim 9, characterized in that: The affinity balance solution comprises 20 mmol / L±5 mmol / L HEPES, 1 mol / L±50 mmol / L sodium chloride, 30 mmol / L±5 mmol / L imidazole, 5 mmol / L±2 mmol / Lβ-mercaptoethanol, 10%±2% glycerol, and a pH of 7.5±0.2; The affinity eluent comprises 20mmol / L±5mmol / L HEPES, 500mmol / L±50mmol / L sodium chloride, 300mmol / L±5mmol / L imidazole, 5mmol / L±2mmol / Lβ-mercaptoethanol, 10%±2% glycerol, 2%±1% Tween 20, and the pH is 7.5±0.2.

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