TWI712691B - Dextran affinity tag and application thereof - Google Patents

Abstract

The present disclosure is related to an affinity tag, which is a segment of dextran binding domain and is able to purify target protein by taking the advantage of the affinity to dextran. The affinity tag of the present disclosure is more effective from the perspective of manufacture and purification process; thus, it can be widely used in any industrial applications involving protein purification.

Description

Dextran affinity label and its application

The present disclosure relates to affinity chromatography for purified protein, especially affinity tags used in affinity chromatography.

The recombinant protein produced by recombinant DNA technology has been widely used in the food, cosmetics and pharmaceutical industries. The production process of recombinant protein can be divided into upper, middle and downstream stages. The upstream stage is the research and development stage, mainly for gene selection and establishment of performance systems. The midstream stage is mainly for fermentation process development and process amplification. The downstream stage is for protein separation and purification, quality control and product packaging.

In the downstream stage of the recombinant protein production process, the efficiency of purification is critical to the manufacturing cost. In order to save manufacturing costs, it is necessary to explore the best purification strategy through repeated experiments. Commonly used purification methods include ion exchange chromatography, hydrophobic-interaction chromatography, gel filtration chromatography, and affinity chromatography. Affinity chromatography uses specific immobilized ligands (ligand) and the protein itself or the protein N-terminal or C-terminal affinity tag (affinity tag) to produce a specific interaction to achieve the purpose of protein purification, with high recovery rate And the advantages of simplified purification steps.

At present, a variety of affinity tags have been used in the purification of recombinant proteins, such as histidine tag (His tag), cellulose binding domain (cellulose binding domain) and chitin binding domain (chitin binding domain). Although affinity tags can improve the convenience of protein purification, the commercial affinity resins commonly used with affinity tags still have the problem of higher prices. Accordingly, there is still a need for more affinity tags to be used in the field.

One purpose of the present disclosure is to provide a novel recombinant protein that can be used as an affinity tag, providing more options for affinity chromatography in the field.

Another objective of the present disclosure is to provide a method for purifying proteins, which uses the affinity tag of the present disclosure, and has the advantage of reducing production costs.

In order to meet the above objective, the present disclosure provides an affinity tag whose amino acid sequence is SEQ ID NO: 02; the restriction condition is that the amino acid sequence is not SEQ ID NO: 01.

The present disclosure also provides an affinity tag whose amino acid sequence is SEQ ID NO: 10; the restriction condition is that its amino acid sequence is not SEQ ID NO: 01.

The present disclosure also provides an isolated polynucleotide, which encodes an affinity tag; wherein the amino acid sequence of the affinity tag is SEQ ID NO: 02; the limitation is that the amino acid of the affinity tag The sequence is not SEQ ID NO: 01.

The present disclosure further provides an isolated polynucleotide, which encodes an affinity tag; wherein the amino acid sequence of the affinity tag is SEQ ID NO: 10; the restriction condition is that the amino acid of the affinity tag The sequence is not SEQ ID NO: 01.

The present disclosure further provides a recombinant protein, which has the aforementioned affinity tag.

The present disclosure provides a method for purifying protein, which comprises: (a) calibrating a target protein with the affinity tag of the disclosure; wherein the calibrated target protein is stored in a fluid; (b) mixing the fluid with a resin To form a mixture and pass the mixture through a tube column; wherein the resin contains dextran; (c) flow a washing liquid through the tube column to obtain the target protein from the resin; wherein the washing liquid contains Dextran.

The description in this article is only exemplary and explanatory, and is not intended to limit the disclosure. The technical and scientific terms used herein should be understood as meanings commonly understood by those of ordinary skill in the art, unless clearly defined otherwise.

Unless the context clearly indicates otherwise, the singular form "一 (a or an)" in the description of this document and the scope of the patent application includes the plural meaning. Thus, for example, "a protein" refers to one or more (a) proteins, and "a compound" refers to one or more (a) compounds. The use of "comprise", "comprises", "comprising", "include", "includes" and "including" are interchangeable, and Not restrictive. It should be understood that in the description of each specific embodiment, when the term "comprising" is used, those skilled in the art will understand that in some specific cases, the language "basically composed of... .. constitute" or "consisting of" instead.

When a certain range of values is provided, unless the context clearly dictates otherwise, it should be understood that the integers in the numerical range and one tenth of each integer in the numerical range are between the upper and lower limits of the range, and in Any other stated values or intermediate values within this range are covered by this disclosure.

All documents, patents, patent applications, and other documents cited in this disclosure are fully incorporated herein as reference materials, and their contents are as pointed out in each independent document, patent, patent application or other document, and are incorporated herein. For reference purposes.

definition:

The "tag/affinity tag" mentioned in this article refers to a molecule that can be bound to a target protein for purification of the target protein. In a feasible embodiment, the tag is combined with the target protein to form a recombinant protein. The “label” mentioned herein is used to describe the binding relationship between a label and a target protein. In a feasible embodiment, the tag is labeled (ie, bound to) the N-terminal of the target protein; in another feasible embodiment, the tag is labeled (ie, bound to) the C-terminus of the target protein.

As used herein, "dextran binding ability" means that the substance in question has the affinity to form a bond with dextran. The "dextran binding domain (DBD)" mentioned in this article refers to a block that can form a bond with dextran in the dextransucrase structure. As used herein, "a segment of DBD" refers to a polypeptide chain or a protein that has the affinity to form a bond with dextran, but it is not known in the art. The full length of the glycan binding functional block. The full length of the dextran binding functional block is shown in SEQ ID NO: 01, for example.

As used herein, "polynucleotide (polynucleotide)" refers to a molecule composed of more than one nucleotide, which can be transcribed and/or translated into a polypeptide or protein with physiological activity.

The "encode/encoding" used herein refers to the process by which the polynucleotide is transcribed and/or translated to produce a polypeptide, or to further form a protein. The "an isolated polynucleotide that encodes a fragment of a glucan binding functional block" refers to the polynucleotide that is transcribed and/or translated to produce a glucan binding functional block Fragment. The coding can be performed in vivo or in vitro. The encoding can be performed in homologous cells or heterologous cells.

The term "isolated" in this article refers to being separated from the original environment, not specifically in a free state. For example, an isolated polynucleotide means that it has been separated from its original gene body and is in a free state or is further genetically engineered to be built into a plasmid.

"Purification/purify" as used herein refers to enriching a target molecule, which can be achieved by increasing the content of the target molecule in an environment, or increasing the content of the target molecule in an environment containing the target molecule. The content of non-target molecules is reduced, and the result of increasing the concentration of the target molecules in the environment is achieved.

The first aspect of this disclosure:

The first aspect of this disclosure is about an affinity tag. In a specific embodiment, the amino acid sequence of the affinity tag is SEQ ID NO: 02; the restriction condition is that the amino acid sequence is not SEQ ID NO: 01. In another specific embodiment, the amino acid sequence of the affinity tag is SEQ ID NO: 10; the restriction condition is that the amino acid sequence is not SEQ ID NO: 01.

The aforementioned "the amino acid sequence of the affinity tag is SEQ ID NO" or similar descriptions mean that the amino acid sequence of the affinity tag includes the indicated sequence, but is not limited to the indicated sequence. For example, the aforementioned "the amino acid sequence of the affinity tag is SEQ ID NO: 02" means that the amino acid sequence of the affinity tag includes SEQ ID NO: 02 (in a specific embodiment, the main It is composed of SEQ ID NO: 02), but those with ordinary knowledge in the field should modify the referenced sequence based on the general knowledge in the field according to their needs, so that the modified sequence includes sequences other than SEQ ID NO: 02 . The present disclosure does not exclude those with ordinary knowledge in the art from extending one to several amino acids at the N-terminus or C-terminus of the affinity tag of the present disclosure. The present disclosure does not exclude those with ordinary knowledge in the field from extending other protein sequences at the N-terminus or C-terminus of the affinity tag of the present disclosure based on specific usage requirements. For example, the sequence of the fusion partner can be added to the N-terminus or C-terminus of SEQ ID NO: 02 (to form a recombinant fusion protein) to increase the solubility of the recombinant protein. Examples of the fusion partner include DsbC of Escherichia coli, MsyB of Escherichia coli, FklB of Escherichia coli and Saccharomyces cerevisiae ( Saccharomyces cerevisiae ) small ubiquitin-like modifier (SUMO). In addition, other affinity tags such as His tag, Strep tag and T7 tag can also be combined with the N-terminal or C-terminal of SEQ ID NO: 02. In this way, the antibodies corresponding to the affinity tags can be used to detect the performance of the recombinant protein (for example, applied to the Western blot method). Unless the modified sequence is equivalent to SEQ ID NO: 01, it should still belong to the scope of this disclosure.

In a specific embodiment, the affinity tag has an affinity to form a bond with dextran. The affinity can be judged by fluorescence microscope observation, dissociation constant measurement, or purification effect. Among them, fluorescence microscope observation is to pass the DBD with fluorescent substance through a resin composed of dextran, and then determine the affinity by the fluorescence intensity. The dissociation constant is determined by fixing the DBD in a micro-porous plate, then adding different concentrations of biotin-dextran solution to the reaction and washing, and then adding the extravidin-alkaline phosphatase ) And perform color reaction; plot the absorbance value corresponding to the concentration of biotin-dextran solution, and calculate the dissociation constant with the one-site saturation ligand-binding equation in the SigmaPlot software. The purification effect is to pass the DBD-bearing protein through a resin composed of dextran and extract it, and then determine the affinity of the purified protein by electrophoresis or concentration measurement.

The second aspect of this disclosure:

The second aspect of the present disclosure relates to an isolated polynucleotide which encodes an affinity tag; wherein the amino acid sequence of the affinity tag is SEQ ID NO: 02; the restriction is that the amine of the affinity tag The base acid sequence is not SEQ ID NO: 01.

In a specific embodiment, the isolated polynucleotide is SEQ ID NO: 14; the limitation is that it is not SEQ ID NO: 13. In another specific embodiment, the isolated polynucleotide is SEQ ID NO: 22; its limitation is that it is not SEQ ID NO: 13.

The aforementioned "the isolated polynucleotide is SEQ ID NO" or similar descriptions mean that the sequence of the isolated polynucleotide includes the indicated sequence, but is not limited to the indicated sequence. For example, if the isolated polynucleotide is SEQ ID NO: 14 it means that the sequence of the isolated polynucleotide includes SEQ ID NO: 14, but those with ordinary knowledge in the field can consider their needs based on Common knowledge in the field modifies the indicated sequence so that the modified sequence includes sequences other than SEQ ID NO: 14. The modified sequence, unless it is equivalent to SEQ ID NO: 13, should still belong to the scope of this disclosure.

In a specific embodiment, the isolated polynucleotide is built into a performance cassette of a performance vector. In a feasible embodiment, the expression vector can be replicated in Escherichia coli, lactic acid bacteria, Bacillus subtilis, or a combination thereof. In a feasible aspect, the expression vector can perform protein expression in a prokaryotic cell expression system; wherein the prokaryotic cell includes E. coli, lactic acid bacteria, Bacillus subtilis, or a combination thereof. In another feasible aspect, the expression vector can be expressed in eukaryotic cells; wherein the eukaryotic cells include yeast, Chinese hamster ovary (CHO) cells, mouse myeloma NS0 cells, baby hamsters Kidney (baby hamster kidney, BHK) cells, mouse myeloma SP2/0 cells, human embryonic kidney (human embryonic kidney) cells HEK 293 cell line, HEK 293 EBNA cell line, human retinal cell PER.C6 ^® cell line and Africa Green monkey kidney cell COS-7 cell line, or a combination thereof.

The third aspect of this disclosure:

The third aspect of the present disclosure relates to a recombinant protein, which contains an affinity tag as described above. In a specific embodiment, the recombinant protein includes a target protein and the affinity tag calibrated to the target protein. The "target protein" mentioned herein refers to a protein to be produced for experimental or commercial implementation purposes. In an experimental embodiment, the target protein is a green fluorescent protein. In a feasible aspect, the affinity tag is calibrated to the N-terminus of the target protein. In another feasible embodiment, the affinity tag is calibrated to the C-terminus of the target protein.

In a feasible embodiment, the recombinant protein is produced by a prokaryotic cell expression system; wherein the nucleotides encoding the target protein and the nucleotides encoding the affinity tag are constructed by genetic engineering technology to form an expression The expression cassette of the carrier is used for production in the prokaryotic expression system. In a feasible embodiment, the prokaryotic cell comprises Escherichia coli, lactic acid bacteria, Bacillus subtilis, or a combination thereof. In another feasible aspect, the expression vector can be expressed in eukaryotic cells; wherein the eukaryotic cells include yeast, CHO cells, NS0 cells, BHK cells, SP2/0 cells, HEK 293 cells, HEK 293 EBNA cells, PER.C6 ^® cells and COS-7 cells, or combinations thereof.

Feasibly, the recombinant protein can be used in the preparation of subunit vaccines; wherein the target protein is the antigen of the subunit vaccine, and the target protein is purified through the disclosed affinity tag, thereby improving the production efficiency of the subunit vaccine.

The fourth aspect of the present invention:

A method for protein purification, comprising: (a) calibrating a target protein with the affinity tag of the present disclosure; wherein the calibrated target protein is stored in a fluid; (b) mixing the fluid and a resin to form a mixture , And make the mixture pass through a tube column; wherein the resin contains dextran; (c) make a washing liquid flow through the tube column to obtain the target protein from the resin; wherein the washing liquid contains dextran.

In a feasible implementation aspect, the calibration is achieved through genetic engineering technology. Specifically, the nucleotide encoding the target protein and the nucleotide encoding the affinity tag are constructed in a performance cassette of an expression vector by genetic engineering technology, and performed in prokaryotic or eukaryotic cells which performed.

The fluid generally refers to the environment containing the target protein, for example, a buffer solution or prokaryotic cell culture fluid or eukaryotic cell culture fluid used to express the target protein. Although the fluid is mostly in a liquid form, it is not limited to a liquid form.

In a possible implementation aspect, the mixing in step (b) refers to bringing the fluid into contact with the resin by any physical means (such as stirring, shaking, or shaking). Although not intending to be limited by any theory, in this step (b), the affinity tag calibrated to the target protein will bind to the dextran in the resin, thereby binding the target protein to the resin. In a specific embodiment, the column in step (b) is a polypropylene column (for example, a product sold by QIAGEN), which can hold the resin and the target protein bound to the resin.

In a specific embodiment, although not intending to be limited by any theory, in this step (c), the washing liquid contains glucan, so that when the column is washed with the washing liquid, the washing liquid The dextran competes with the dextran in the resin for binding with the target protein, so that the target protein is separated from the resin and washed out with the washing liquid. In a feasible aspect, the concentration of glucan in the washing liquid is 0.1 to 0.5% (w/v). In a feasible implementation aspect, a person with ordinary knowledge in the field can adjust the concentration of glucan in the resin and the washing liquid according to their needs and process conditions.

In a feasible aspect, the pH of the washing liquid is 6-8. More preferably, the pH value can be adjusted according to actual operating conditions. In a specific embodiment, the resin is a resin containing dextran such as Sephacryl ^TM S-300 HR, Sephacryl ^TM S-500 HR, Superdex G75, or Superdex G100.

The following paragraphs will list specific embodiments of the present disclosure, and these embodiments are intended to exemplarily illustrate the features of the present disclosure but not to limit the scope thereof. Each specific embodiment and feature described in this disclosure should be understood as interchangeable and combinable with each specific embodiment contained therein.

Example 1 : The construction of the expression vector that expresses the target protein and the affinity tag of this disclosure.

The purpose of this embodiment is to construct different dextran binding domains (DBD) by recombinant DNA technology to study the most suitable affinity tags. In this example, the Leuconostoc mesenteroides strain ATIT-08 isolated from kimchi was used as the source of the full-length DNA of the glucan binding functional block. Escherichia coli ( E. coli ) ECOS ^TM 9-5 (Yisheng, Taiwan) is used as the host cell for DNA selection. E. coli BL21 (DE3) (Thermo, USA) is used as the host cell for protein expression. These Escherichia coli are cultivated on Luria-Bertani (LB) medium, and 30 μg/mL kanamycin or 1.5% (w/v) agar is added as needed. DeMan-Rogosa-Sharpe (MRS) medium (Merck, USA) was used to culture Leuconostoc. Both the culture medium and agar-agar were purchased from American BD Company.

The Leuconostoc strain ATIT-08 was inoculated into 5 mL of MRS liquid medium and cultured at 30°C for 16 hours. Use DNA purification kit (Tissue & Cell Genomic DNA Purification kit; GMbiolab, Taiwan) to extract chromosomes from strains. The DBDKPNIF/DBDSALIR primer combination (SEQ ID NO: 25 and SEQ ID NO: 26) was used to increase the DBD DNA. The 50 μL PCR reaction mixture contains 1x GDP-HiFi PCR buffer, 200 μM dNTPs, 1 μM amplification primers, 200 ng Leuconostoc genomic DNA and 1 U GDP-HiFi DNA polymerase (Genedirex, USA) . PCR reaction conditions are 96°C for 2 minutes (1 step); 94°C for 15 seconds, 55°C for 30 seconds, 68°C for 30 seconds (35 cycles); 68°C for 5 minutes (1 Steps). After the PCR reaction is completed, after confirming that the estimated size of the DNA fragment is obtained, the PCR product is recovered using the PCR Clean Up Kit (GMbiolab, Taiwan).

Then use CloneJET PCR Cloning Kit (Thermo, USA) for gene selection, and transform the ligation mixture into E. coli ECOS ^TM 9-5. Then use colony polymerase chain reaction (SEQ ID NO: 25/SEQ ID NO: 26) to select transformed strains that may contain insert DNA. After electrophoresis confirms that the recombinant plastids in the transformed strain have extrapolated DNA, the plastids in the transformed strain are extracted and DNA sequencing is performed. The plastid with the correct DNA sequence is named pJET-LCDBD.

DBD Fusion expression vector pET -DBD The construction.

Because the DBD DNA contains the Bam HI cut site commonly used in gene selection, mutation primers are designed for the restriction site and the overlap extension polymerase chain reaction is used for site-directed mutation. The operation flow of point mutation is summarized as follows.

Using the plastid pJET-LCDBD as a template, use the DBDKPNIF/DBDBAMHIM2 (SEQ ID NO: 25/SEQ ID NO: 28) and DBDBAMHIM1/DBDSALIR (SEQ ID NO: 27 and SEQ ID NO: 26) primer sets for DNA fragment amplification. . The 50 μL PCR reaction mixture contains 1 times GDP-HiFi PCR buffer B, 200 μM dNTPs, 1 μM amplification primers, 100 ng pJET-LCDBD and 1 U GDP-HiFi DNA polymerase.

PCR reaction conditions are 96°C for 2 minutes (1 step); 94°C for 15 seconds, 55°C for 30 seconds, 68°C for 30 seconds (35 cycles); 68°C for 5 minutes ( 1 step). After the PCR reaction is completed, after confirming that the DNA fragment of the estimated size is obtained, the PCR product is recovered using Gel-M ^TM gel extraction system kit (GMbiolab, Taiwan).

Next, using the two recovered PCR products as templates, DBDKPNIF/DBDSALIR primer combination (SEQ ID NO: 25/SEQ ID NO: 26) was used to amplify the DBD DNA fragment. PCR reaction conditions are 96°C for 2 minutes (1 step); 94°C for 15 seconds, 55°C for 30 seconds, 68°C for 1 minute (35 cycles); 68°C for 5 minutes ( 1 step). After this step, DBD DNA of site-directed mutation can be obtained, the sequence of which is SEQ ID NO: 13 and encodes SEQ ID NO: 01.

After the PCR product was cleaved with Kpn I and Sal I, the DNA fragment was inserted into the pET29a (+) cleaved with the same restriction enzyme by T4 DNA ligase. Transform the bonded product into E. coli ECOS ^TM 9-5. Then, the colony polymerase chain reaction (SEQ ID NO: 25 and SEQ ID NO: 26) is used to select the transformed strains that may carry extrapolated DNA. After electrophoresis confirms that the recombinant plastids in the transformed strain have extrapolated DNA, the plastids in the transformed strain are extracted and DNA sequencing is performed. The plastid with the correct DNA sequence is named pET-DBD.

Green fluorescent protein expression vector pET -GFP With green fluorescent protein -DBD Fusion expression vector pET -GFP-DBD The construction.

The green fluorescent protein is used as the target protein in the research of this disclosure. The GFPNDEIF/GFPKPNIR primer combination (SEQ ID NO: 29/SEQ ID NO: 30) was used to increase the enhanced green fluorescent protein (GFP) gene. The 50 μL PCR reaction mixture contains 1 times GDP-HiFi PCR buffer, 200 μM dNTPs, 1 μM amplification primers, 100 ng pBRLP-8-P23-GFPT and 1 U GDP-HiFi DNA polymerase. PCR reaction conditions are 96°C for 2 minutes (1 step); 94°C for 15 seconds, 55°C for 30 seconds, 68°C for 30 seconds (35 cycles); 68°C for 5 minutes (1 Steps). After the PCR reaction is completed, after confirming that a DNA fragment of the estimated size is obtained, the PCR product is recovered using the PCR Clean Up Kit.

After the PCR product is cut with Nde I and Kpn I, the DNA fragments are respectively inserted into the plastid pET29a (+) or pET-DBD cut with the same restriction enzyme using T4 DNA ligase. Transform the bonded product into E. coli ECOS ^TM 9-5. Then through the colony polymerase chain reaction (SEQ ID NO: 29/SEQ ID NO: 30), select the transformed strains that may have extrapolated DNA. After electrophoresis confirms that the recombinant plastids in the transformed strain have extrapolated DNA, the plastids in the transformed strain are extracted and DNA sequencing is performed. The pET29a (+) with the GFP gene was named pET-GFP. The pET-DBD with the GFP gene was named pET-GFP-DBD.

DBD The deletion mutation.

1. DBD-D1:

The DBD-D1KPNIF/DBDSALIR primer combination (SEQ ID NO: 31/SEQ ID NO: 26) was used to increase the DBD-D1 DNA. The 50 μL PCR reaction mixture contains 1 times GDP-HiFi PCR buffer, 200 μM dNTPs, 1 μM amplification primers, 100 ng pET-GFP-DBD and 1 U GDP-HiFi DNA polymerase. PCR reaction conditions are 96°C for 2 minutes (1 step); 94°C for 15 seconds, 55°C for 30 seconds, 68°C for 30 seconds (35 cycles); 68°C for 5 minutes (1 Steps). After the PCR reaction is completed, after confirming that a DNA fragment of an estimated size is obtained, the PCR product is recovered using Gel-M ^TM gel extraction system kit. The PCR product is the disclosed affinity tag DBD-D1, and its sequence is SEQ ID NO: 14, and it encodes SEQ ID NO: 02.

2. DBD-D2, DBD-D3, DBD-D4, DBD-D5, DBD-D7, and DBD-D8:

Use the primer sets shown in Table 1 below to amplify DNA fragments. The 50 μL PCR reaction mixture contains 1x GDP-HiFi PCR buffer B, 200 μM dNTPs, 1 μM amplification primers, and 100 ng pET- GFP-DBD and 1 U GDP-HiFi DNA polymerase. PCR reaction conditions are 96°C for 2 minutes (1 step); 94°C for 15 seconds, 55°C for 30 seconds, 68°C for 30 seconds (35 cycles); 68°C for 5 minutes ( 1 step). After the PCR reaction is completed, after confirming that a DNA fragment of an estimated size is obtained, the PCR product is recovered using Gel-M ^TM gel extraction system kit. Two PCR products were prepared for each affinity label experiment sample.

Table 1:

Then use the two recovered PCR products as templates, and use the DBDKPNIF/DBDSALIR (SEQ ID NO: 25/SEQ ID NO: 26) primer combination for gene amplification. PCR reaction conditions are 96°C for 2 minutes (1 step); 94°C for 15 seconds, 55°C for 30 seconds, 68°C for 1 minute (35 cycles); 68°C for 5 minutes ( 1 step). After this step, the corresponding DBD-D2, DBD-D3, DBD-D4, DBD-D5, DBD-D7, and DBD-D8 fragments can be obtained respectively. Use PCR Clean Up Kit to recover PCR products.

The sequence of the affinity tag DBD-D2 of the present disclosure is SEQ ID NO: 15, and encodes SEQ ID NO: 03.

The sequence of the affinity tag DBD-D3 of the present disclosure is SEQ ID NO: 16, and encodes SEQ ID NO: 04.

The sequence of the affinity tag DBD-D4 of the present disclosure is SEQ ID NO: 17, and encodes SEQ ID NO: 05.

The sequence of the affinity tag DBD-D5 of the present disclosure is SEQ ID NO: 18 and encodes SEQ ID NO: 06.

The sequence of the affinity tag DBD-D7 of the present disclosure is SEQ ID NO: 20 and encodes SEQ ID NO: 08.

The sequence of the affinity tag DBD-D8 of the present disclosure is SEQ ID NO: 21 and encodes SEQ ID NO: 09.

3. DBD-D6:

DBDKPNIF/DBD-D6SALIR (SEQ ID NO: 25/SEQ ID NO: 40) primer combination was used to increase DBD-D6 DNA. The 50 μL PCR reaction mixture contains 1 times GDP-HiFi PCR buffer, 200 μM dNTPs, 1 μM amplification primers, 100 ng pET-GFP-DBD and 1 U GDP-HiFi DNA polymerase. PCR reaction conditions are 96°C for 2 minutes (1 step); 94°C for 15 seconds, 55°C for 30 seconds, 68°C for 30 seconds (35 cycles); 68°C for 5 minutes (1 Steps). After the PCR reaction is completed, after confirming that a DNA fragment of an estimated size is obtained, the PCR product is recovered using Gel-M ^TM gel extraction system kit. The PCR product is the disclosed affinity tag DBD-D6, and its sequence is SEQ ID NO: 19, and it encodes SEQ ID NO: 07.

4. DBD-D9:

The DBD-D9KPNIF/DBDSALIR (SEQ ID NO: 44/SEQ ID NO: 26) primer combination was used to increase the DBD-D9 DNA. The 50 μL PCR reaction mixture contains 1 times GDP-HiFi PCR buffer, 200 μM dNTPs, 1 μM amplification primers, 100 ng pET-GFP-DBD and 1 U GDP-HiFi DNA polymerase. PCR reaction conditions are 96°C for 2 minutes (1 step); 94°C for 15 seconds, 55°C for 30 seconds, 68°C for 30 seconds (35 cycles); 68°C for 5 minutes (1 Steps). After the PCR reaction is completed, after confirming that a DNA fragment of an estimated size is obtained, the PCR product is recovered using Gel-M ^TM gel extraction system kit. The PCR product is the disclosed affinity tag DBD-D9, and its sequence is SEQ ID NO: 22, and it encodes SEQ ID NO: 10.

5. DBD-D10:

The DBD-D1KPNIF/DBD-D8SALIR (SEQ ID NO: 31/SEQ ID NO: 45) primer combination was used to increase the DBD-D10 DNA. The 50 μL PCR reaction mixture contains 1 times GDP-HiFi PCR buffer, 200 μM dNTPs, 1 μM amplification primers, 100 ng pET-GFP-DBD and 1 U GDP-HiFi DNA polymerase. PCR reaction conditions are 96°C for 2 minutes (1 step); 94°C for 15 seconds, 55°C for 30 seconds, 68°C for 30 seconds (35 cycles); 68°C for 5 minutes (1 Steps). After the PCR reaction is completed, after confirming that a DNA fragment of an estimated size is obtained, the PCR product is recovered using Gel-M ^TM gel extraction system kit. The PCR product is the disclosed affinity tag DBD-D10, and its sequence is SEQ ID NO: 23, and it encodes SEQ ID NO: 11.

6. DBD-D11:

The DBD-D1KPNIF/DBD-D11SALIR (SEQ ID NO: 31/SEQ ID NO: 46) primer combination was used to increase the DBD-D11 DNA. The 50 μL PCR reaction mixture contains 1 times GDP-HiFi PCR buffer, 200 μM dNTPs, 1 μM amplification primers, 100 ng pET-GFP-DBD and 1 U GDP-HiFi DNA polymerase. PCR reaction conditions are 96°C for 2 minutes (1 step) 94°C for 15 seconds, 55°C for 30 seconds, 68°C for 30 seconds (35 cycles); 68°C for 5 minutes (1 step) step). After the PCR reaction is completed, after confirming that a DNA fragment of an estimated size is obtained, the PCR product is recovered using Gel-M ^TM gel extraction system kit. The PCR product is the disclosed affinity tag DBD-D11, and its sequence is SEQ ID NO: 24, and it encodes SEQ ID NO: 12.

Example 2 : Purify the target protein through the affinity tag disclosed in this disclosure.

Green fluorescent protein - Deletion mutation DBD The construction of a fusion expression carrier.

After cutting the cut mutation DBD DNA with Kpn I and Sal I, use T4 DNA ligase to insert the DNA fragment into the pET-GFP cut with the same restriction enzyme. Transform the bonded product into E. coli ECOSTM 9-5. Then through the colony polymerase chain reaction (T7 promoter/T7 terminator (SEQ ID NO: 47/SEQ ID NO: 48)), select the transformed strains that may carry extrapolated DNA. After electrophoresis confirms that the recombinant plastids in the transformed strain have extrapolated DNA, the plastids in the transformed strain are extracted and DNA sequencing is performed. The plasmids with the correct DNA sequence are named pET-GFP-DBD-D1~pET-GFP-DBD-D11.

Transformation of fusion protein expression vector and induction expression of recombinant fusion protein.

The fusion protein expression vector pET-GFP-DBD and pET-GFP-DBD-D1~ pET-GFP-DBD-D11 were transformed into E. coli BL21 (DE3). A single colony was selected and inoculated into 5 mL LB medium containing kanamycin (final concentration 30 μg/mL), and cultured at 25°C and 180 rpm. After overnight incubation, add 100 μL of the pre-culture broth to 10 mL of LB medium containing kanamycin (final concentration of 30 μg/mL), and shake culture to OD ₆₀₀ at 37°C and 180 rpm About 0.4 to 0.6, add 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and induce expression of recombinant protein at 25°C. After 4 hours of induction, take 2 mL of the bacterial solution and centrifuge (20,630×g, 5 minutes, 4°C), collect the bacterial part, and divide the soluble protein and insoluble protein (ie, soluble or not). Use protein electrophoresis to observe the solubility of the recombinant fusion protein. Use ImageQuantTL software (GE, USA) to analyze the percentage of recombinant protein in the film to the total protein, which is the performance percentage.

The results of protein electrophoresis showed (Figure 1) that the fusion proteins prepared in the aforementioned Example 1 can all behave normally, although the expression levels of GFP-DBD-D7, GFP-DBD-D9 and GFP-DBD-D10 are relatively low. In other words, the affinity tag disclosed in the present disclosure does not affect the performance of the target protein. It can be further observed from the experimental results in FIG. 1 that the affinity tag disclosed in the present disclosure will affect the solubility of the fusion protein. Specifically, GFP-DBD, GFP-DBD-D1 and GFP-DBD-D8 are mainly soluble proteins; GFP-DBD-D2 and GFP-DBD-D5 are mainly insoluble proteins (Table 2).

Table 2

Reorganization GFP-DBD or GFP-DBD Purification of deletion mutant fusion protein.

Add 100 μL of Sephacryl ^TM S-300 HR resin (GE Healthcare, Sweden) to the soluble protein fraction obtained by induction expression at 25°C. After mixing for 30 minutes, put the mixture into a spin column. The separation tube was further placed in a collection tube, and after centrifugation (100×g, 1 minute), the effluent was removed. Wash the resin with 1.2 mL fragmentation buffer (50 mM sodium phosphate, 0.05 g/L CaCl ₂ , 0.3 M NaCl, 1% triton X-100, pH 7.2) (used as a washing buffer), and then 200 μL dissociation buffer Solution (elution buffer; 50 mM sodium phosphate, 0.5% dextran, pH 7.2) to extract the recombinant fusion protein on the resin (100 μL/tube). Use protein electrophoresis to observe the purification of the recombinant fusion protein.

The results of protein electrophoresis showed (Figure 2) that among the affinity tags constructed in Example 1, only the fusion proteins of DBD, DBD-D1 and DBD-9 can bind to Sephacryl ^TM S-300 HR resin. The binding force of DBD-D1 to resin is better than that of DBD-9, showing similar affinity to DBD. This result shows that the present disclosure successfully tested a DBD fragment that can retain the affinity with dextran, which means that there is no need to express the full length of DBD, and a specific fragment can be used as an affinity tag, which is more efficient in terms of protein expression. In addition, the experimental results of this disclosure also show that not any DBD fragment can be used as an affinity tag, which is not easily foreseen from the sequence of DBD before the research of this disclosure.

Example 3 ：Compare the affinity label and conventional use in this disclosure His-tag .

GFP-His tag The performance and purification.

E. coli BL21 (DE3) (pET-GFP) was inoculated in 50 mL LB medium containing kanamycin (final concentration 30 μg/mL), and then cultured with shaking at 37°C and 180 rpm to OD ₆₀₀ is about 0.4~0.6. Then add 0.1 mM IPTG and perform the induction expression of recombinant protein at 25°C. After 4 hours of induction, the cells were collected by centrifugation (7,354×g, 30 minutes, 4°C), and the cells were suspended in 40 mL of lysis buffer; 20 mM sodium phosphate, 500 mM NaCl, 1% triton X-100, pH 7.4). Utilize the ultrasonic breaker to break the bacteria. Centrifuge the broken bacteria solution (39,191×g, 30 minutes, 4°C) and collect the supernatant. Add 2 mL of Ni Sepharose ^TM excel resin (GE Healthcare, Sweden) to the supernatant, and shake at 160 rpm for 30 minutes at 4°C to thoroughly mix the supernatant with the resin. Pour the supernatant containing resin into polypropylene columns (QIAGEN, Germany), and discard the effluent. Use 60 mL of washing buffer (20 mM sodium phosphate, 500 mM NaCl, 30 mM imidazole, pH 7.4) to remove non-specifically bound proteins on the resin. Finally, 25 mL of elution buffer (20 mM sodium phosphate, 500 mM NaCl, 250 mM imidazole, pH 7.4) was used to extract the recombinant protein on the resin. Use protein electrophoresis to observe the purification of recombinant protein.

GFP-DBD versus GFP-DBD-D1 The performance and purification.

Inoculate E. coli BL21 (DE3) (pET-GFP-DBD) or E. coli BL21 (DE3) (pET-GFP-DBD-D1) in 50 cells containing kanamycin (final concentration 30 μg/mL) In mL LB medium, shake culture at 37°C and 180 rpm until OD ₆₀₀ is about 0.4~0.6. Then add 0.1 mM IPTG and perform the induction expression of recombinant protein at 25°C. After 4 hours of induction, centrifugation (7,354×g, 30 minutes, 4°C) to collect the bacterial cells, and suspend the bacterial cells in 40 mL of disrupting buffer (50 mM sodium phosphate, 0.05 g/L CaCl ₂ , 0.3 M NaCl, 1% triton X-100, pH 7.2). Utilize the ultrasonic breaker to break the bacteria. Centrifuge the broken bacteria solution (39,191 × g, 30 minutes, 4°C) and collect the supernatant. Add 2 mL of Sephacryl ^TM S-300 HR resin to the supernatant, place it at 4°C and shake at 160 rpm for 30 minutes to mix the supernatant with the resin. Pour the resin-containing supernatant into a polypropylene column (QIAGEN, Germany) and discard the effluent. Wash off non-specifically bound proteins on the resin with 60 mL of fragmentation buffer (used as a washing buffer). Finally, 25 mL of elution buffer (50 mM sodium phosphate, 0.5% dextran, pH 7.2) was used to extract the recombinant protein on the resin. Use protein electrophoresis to observe the purification of recombinant protein.

The results of protein electrophoresis showed that all recombinant GFP with affinity tags can be purified (Figure 3). The disclosed affinity tags can indeed be applied to protein purification processes. In addition, the DBD-D1 disclosed in the present disclosure can achieve the highest yield, which is better than the His tag commonly used in the field, and is also better than the full length of DBD. Accordingly, the affinity tag disclosed in the present disclosure is not only more efficient in terms of protein expression, but also more excellent from the viewpoint of purification efficiency.

table 3

List of introductions:

no

Figure 1 is a protein electrophoresis diagram showing the induction performance of recombinant fusion protein (Example 2 of the present disclosure).

Figure 2 is a protein electrophoresis diagram showing the purification result of recombinant GFP-DBD or GFP-DBD deletion mutant fusion protein (Example 2 of the present disclosure).

Figure 3 is a protein electrophoresis diagram showing the purification results of GFP-His-tag, GFP-DBD, and GFP-DBD-D1 (Example 3 of the present disclosure).

no

<110> Agricultural Science and Technology Research Institute

<120> Glucan affinity label and its application

<140> TW 107138199

<141> 2018-10-29

<160> 48

<170> PatentIn version 3.5

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Claims (15)

An affinity tag whose amino acid sequence is SEQ ID NO: 02. An affinity tag whose amino acid sequence is SEQ ID NO: 10. An isolated polynucleotide encoding an affinity tag; wherein the amino acid sequence of the affinity tag is SEQ ID NO: 02. Such as the isolated polynucleotide of claim 3, which is SEQ ID NO: 14. An isolated polynucleotide encoding an affinity tag; wherein the amino acid sequence of the affinity tag is SEQ ID NO: 10. Such as the isolated polynucleotide of claim 5, which is SEQ ID NO:22. A recombinant protein, which is a fusion protein comprising a target protein and an affinity tag as described in claim 1 or 2, wherein the target protein is not SEQ ID NO: 01 or a fragment thereof. Such as the recombinant protein of claim 7, wherein the affinity tag is calibrated to the N-terminus of the target protein. Such as the recombinant protein of claim 7, wherein the affinity tag is calibrated to the C-terminus of the target protein. A method for protein purification, comprising: (a) labeling a target protein with an affinity tag as described in item 1 or 2 of the claim; wherein the calibrated target protein is stored in a fluid; (b) mixing The fluid and a resin form a mixture, and the mixture is passed through a tube column; wherein the resin contains dextran; and (c) a washing solution is passed through the tube column to obtain the target protein from the resin; Wherein the washing liquid contains dextran. Such as the method of claim 10, wherein the calibration is achieved through genetic engineering technology. Such as the method of claim 10, wherein the resin is Sephacryl ^™ S-300 HR, Sephacryl ^™ S-500HR, Superdex G75, or Superdex G100. The method according to claim 10, wherein the concentration of the glucan in the washing liquid is 0.1 to 0.5% (w/v). Such as the method of claim 10, wherein the pH of the washing liquid is 6 to 8. Such as the method of claim 10, wherein the pipe string is a polypropylene pipe string.

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