CN112522242A - Application of Zika virus serine protease in removal of recombinant protein affinity tag - Google Patents
Application of Zika virus serine protease in removal of recombinant protein affinity tag Download PDFInfo
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- CN112522242A CN112522242A CN202011593865.5A CN202011593865A CN112522242A CN 112522242 A CN112522242 A CN 112522242A CN 202011593865 A CN202011593865 A CN 202011593865A CN 112522242 A CN112522242 A CN 112522242A
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- Prior art keywords
- recombinant protein
- zika virus
- serine protease
- protein
- target protein
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- 108010008281 Recombinant Fusion Proteins Proteins 0.000 title claims abstract description 44
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- 108010022999 Serine Proteases Proteins 0.000 title claims abstract description 28
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 64
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 49
- 239000013604 expression vector Substances 0.000 claims abstract description 20
- 238000000034 method Methods 0.000 claims abstract description 18
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- 238000001976 enzyme digestion Methods 0.000 claims abstract description 7
- 238000010276 construction Methods 0.000 claims abstract description 6
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- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 108010056787 lysyl-arginyl-glutamyl-glutamic acid Proteins 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 108010077112 prolyl-proline Proteins 0.000 description 1
- 238000001814 protein method Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 230000001988 toxicity Effects 0.000 description 1
- 231100000419 toxicity Toxicity 0.000 description 1
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Abstract
The invention discloses an application of Zika virus serine protease in removing a recombinant protein affinity tag. In the method, in the construction process of the recombinant protein expression vector, a gene sequence for expressing asparagine-lysine-arginine-glycine-serine (NKRRGS) is inserted between a gene sequence for expressing an affinity label and a gene sequence for expressing a target protein to construct and obtain the recombinant protein expression vector; and then transferring the expression vector into host cells for recombinant protein expression, purifying the recombinant protein, then adding Zika virus serine protease, mixing, carrying out enzyme digestion reaction, and purifying to obtain the target protein without the affinity tag. The Zika virus serine protease can be widely applied to the preparation, separation and purification processes of soluble protein and membrane protein, and has wide application prospect.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to application of Zika virus serine protease in removal of a recombinant protein affinity tag.
Background
Protein purification is a technique for separating a target protein from other proteins, and plays an important role in biological research. The target protein to be purified can be isolated directly from the organism, but such an operation is extremely inefficient. The development of genetic engineering techniques has facilitated the development of a variety of protein expression systems. The target protein can be efficiently expressed in some host cells by cloning the gene of the target protein and then constructing the gene into an expression vector. Recombinant proteins are widely applied to scientific research and industrial production processes, and most target proteins from different sources can be separated and purified by adopting a recombinant protein method. However, some target proteins are still difficult to isolate and purify by this method, mainly due to their low expression level, low solubility and toxicity to host cells. Therefore, the method adopting the affinity tag can effectively improve the yield of the target protein. Many affinity tags such as glutathione-S-transferase (GST), polyhistidine tag, etc. are widely used in the purification of recombinant proteins, and the addition of such affinity tags greatly increases the efficiency of protein purification, and can improve the yield and purity of the target protein. Because the added affinity tag does not belong to the target protein, the function of the protein can be influenced by the tag, therefore, in the construction process of the recombinant protein, the protease enzyme cutting site is artificially introduced, and the affinity tag in the recombinant protein is removed by a protease enzyme cutting method, so as to achieve the purpose of purifying the target protein.
Disclosure of Invention
The present invention is directed to overcoming the above-mentioned disadvantages of the prior art and providing the use of the serine protease of Zika virus for removing the affinity tag of recombinant proteins.
There are currently a variety of proteases of different origin for the separation of recombinant proteins from affinity tags. However, since the cleavage sites recognized by the protease derived from Zika virus are composed of positively charged amino acids such as lysine and arginine, the protease derived from Zika virus is particularly suitable for cleaving a target protein having no cleavage site in the protein sequence. Furthermore, the protease of Zika virus is localized on the surface of the cell membrane, and the membrane system has little influence on the enzymatic activity thereof, so that such protease is suitable for cleaving the affinity tags at both ends of the target membrane protein. The Zika virus serine protease is made available for removal of affinity tags in such recombinant proteins by adding an asparagine-lysine-arginine-glycine-serine (NKRRGS) sequence between the protein of interest and the affinity tag.
The first purpose of the invention is to provide an application of Zika virus serine protease in removing recombinant protein affinity labels, wherein the amino acid sequence of the Zika virus serine protease is shown in SEQ ID NO.1, the enzyme cutting site of the Zika virus serine protease is amino acid sequence NKRRGS, and the target protein of the recombinant protein does not contain the amino acid sequence NKRRGS.
It is a second object of the present invention to provide a method for removing an affinity tag, comprising the steps of:
in the construction process of the recombinant protein expression vector, inserting a gene sequence for expressing asparagine-lysine-arginine-glycine-serine (NKRRGS) between a gene sequence for expressing an affinity tag and a gene sequence for expressing a target protein to construct and obtain the recombinant protein expression vector; then transferring the expression vector into host cells for recombinant protein expression, purifying the recombinant protein, then adding Zika virus serine protease, mixing, carrying out enzyme digestion reaction, and purifying to obtain the target protein with the affinity tag removed;
the Zika virus serine protease has an amino acid sequence shown in SEQ ID NO.1, and the target protein does not contain an amino acid sequence NKRRGS.
Preferably, the expression vector is pET29b vector, pET vector or pGEX vector.
Preferably, the affinity tag is a glutathione-S-transferase (GST) tag or a polyhistidine tag.
The corresponding expression vector is selected according to the expression host chosen. A commonly used expression host is E.coli (BL21DE 3).
Preferably, the purified recombinant protein is obtained by affinity chromatography, and the purification after the enzyme digestion reaction is to obtain the target protein through molecular sieve column purification.
Compared with the prior art, the invention has the following beneficial effects:
1. the invention shows that the Zika virus serine protease can be used for the excision of an affinity label. The removal of the affinity tag can be carried out using the enzyme as long as the target protein does not contain the cleavage site of the enzyme.
2. The Zika virus serine protease can be widely applied to the preparation, separation and purification processes of soluble protein and membrane protein, and has wide application prospect.
Drawings
FIG. 1 is a schematic diagram of an overall implementation of the process of the invention; a: introducing the enzyme cutting site of Zika virus serine protease into the N or C terminal of a target protein, and B: an affinity purification tag such as polyhistidine or GST affinity purification tag is introduced to the N-or C-terminus of the target protein, and after the target protein is purified, the affinity tag can be removed using Zika virus serine protease.
FIG. 2 is a SDS-PAGE electrophoretic analysis of the target protein 1; m, molecular weight standard; 1, a purified recombinant protein; 2-5, the components eluted from the gel column after enzyme digestion contain purified target protein; and 6, components still remained on the gel after enzyme digestion and buffer washing, wherein the components comprise the recombinant protein which is not cut, GST tag and other hybrid proteins.
Detailed Description
The following examples are further illustrative of the present invention and are not intended to be limiting thereof.
The general implementation of the process of the invention is illustrated in FIG. 1.
In the construction process of the recombinant protein expression vector, a gene sequence for expressing asparagine-lysine-arginine-glycine-serine (NKRRGS) is introduced between a gene sequence for expressing an affinity tag and a gene sequence for expressing a target protein (namely, an expressed NKRRGS enzyme cutting site and the affinity tag can be introduced at the N or C end of the target protein) to construct the expression vector. Then the expression vector is transferred into a host cell for expression, recombinant protein expression and recombinant protein purification are sequentially carried out, Zika virus serine protease (the amino acid sequence of which is shown as SEQ ID NO. 1) is added and mixed with the purified recombinant protein, the affinity tag is cut from the NKRRGS enzyme cutting site, and the purified target protein is eluted.
Example 1
The removal of the glutathione-S-transferase (GST) tag during the purification of the protein of interest 1 (molecular weight 31kDa) was carried out. The method comprises the following steps:
1. construction of target protein 1 expression vector: mixing asparagine-lysine-arginine-glycine-serine
(NKRRGS) sequence gene and target protein 1 gene were synthesized and cloned into bacterial expression vector pGEX system plasmid containing GST. The resulting recombinant expression vector can express recombinant proteins: contains GST label, Zika virus serine protease cleavage site and target protein.
2. Expression of recombinant protein: the recombinant expression vector is transformed into escherichia coli (BL21DE3), cultured on an LB plate containing the corresponding antibiotic, a single colony is selected and inoculated in an LB culture medium, the culture medium is placed at 37 ℃ for overnight (12 hours) shaking culture, and then the culture medium is transferred into 1 liter of LB culture medium containing the corresponding antibiotic for 37 ℃ shaking culture. When the absorbance at 600nm reached 1.0, IPTG was added to 1mM to induce expression of the recombinant protein, and after the cells were further cultured on a shaker at 20 ℃ for 12 hours, the cells were collected by centrifugation at 8000 Xg.
3. And (3) purifying the recombinant protein: each 4g of the bacterial cells were suspended in 40mL of a buffer solution (20mM sodium phosphate, pH7.8,300 mM sodium chloride), disrupted in an ultrasonic disrupter for 15min in an ice bath, centrifuged at 18000 Xg for 20 minutes at 4 ℃ and the supernatant was mixed with 4mL of glutathione-Sepharose 4B, and the gel was washed with 40mL of a buffer solution (20mM sodium phosphate, pH7.8,100mM sodium chloride).
4. Enzyme digestion to remove GST tag: 0.5mg of Zika virus serine protease (amino acid sequence shown in SEQ ID NO. 1) was added to 2mL of a buffer (20mM sodium phosphate, pH7.8,100mM sodium chloride) and mixed with 4mL of glutathione-Sepharose 4B, and the mixture was shaken at 16 ℃ for 12 hours, and then the mixture was transferred to a column, and the eluted solution was collected. The gel was washed 2-5 times with 4mL of buffer (20mM sodium phosphate, pH7.8,100mM sodium chloride). The GST tag and the non-cleaved recombinant protein will remain on the gel, while the target protein is eluted.
5. And (3) detection of a purification effect: the purity of the target protein can be checked by analyzing the collected fractions by SDS-PAGE.
The results are shown in FIG. 2: wherein 1 represents purified recombinant protein (containing GST label, Zika virus serine protease enzyme cutting site and target protein), 2-5 represents components eluted from a gel column after being cut by Zika virus serine protease, contains purified target protein, has the size of about 31kDa and is consistent with the molecular weight of the target protein; 6 denotes the fractions remaining on the gel after cleavage, buffer washing, including the non-cleaved recombinant protein, GST tag and other hetero-proteins. The above results demonstrate that Zika virus serine protease can precisely cleave the NKRRGS site on the protein to cleave the affinity tag.
Sequence listing
<110> institute of bioengineering of academy of sciences of Guangdong province
Application of <120> Zika virus serine protease in removal of recombinant protein affinity tag
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 231
<212> PRT
<213> Zika Virus (Zika Virus)
<400> 1
Gly Ser Gly Ala Leu Trp Asp Val Pro Ala Pro Lys Glu Val Lys Lys
1 5 10 15
Gly Glu Thr Thr Asp Gly Val Tyr Arg Val Met Thr Arg Arg Leu Leu
20 25 30
Gly Ser Thr Gln Val Gly Val Gly Val Met Gln Glu Gly Val Phe His
35 40 45
Thr Met Trp His Val Thr Lys Gly Ala Ala Leu Arg Ser Gly Glu Gly
50 55 60
Arg Leu Asp Pro Tyr Trp Gly Asp Val Lys Gln Asp Leu Val Ser Tyr
65 70 75 80
Cys Gly Pro Trp Lys Leu Asp Ala Ala Trp Asp Gly Leu Ser Glu Val
85 90 95
Gln Leu Leu Ala Val Pro Pro Gly Glu Arg Ala Lys Asn Ile Gln Thr
100 105 110
Leu Pro Gly Ile Phe Lys Thr Lys Asp Gly Asp Ile Gly Ala Val Ala
115 120 125
Leu Asp Tyr Pro Ala Gly Thr Ser Gly Ser Pro Ile Leu Asp Lys Cys
130 135 140
Gly Arg Val Ile Gly Leu Tyr Gly Asn Gly Val Val Ile Lys Asn Gly
145 150 155 160
Ser Tyr Val Ser Ala Ile Thr Gln Gly Lys Arg Glu Glu Glu Thr Pro
165 170 175
Val Glu Met Thr Gly Lys Ser Val Asp Met Tyr Ile Glu Arg Ala Gly
180 185 190
Asp Ile Thr Trp Glu Lys Asp Ala Glu Val Thr Gly Asn Ser Pro Arg
195 200 205
Leu Asp Val Ala Leu Asp Glu Ser Gly Asp Phe Ser Leu Val Glu Glu
210 215 220
Asp Gly Pro Pro Met Arg Glu
225 230
Claims (5)
1. The application of Zika virus serine protease in removing recombinant protein affinity tags is characterized in that the amino acid sequence of the Zika virus serine protease is shown in SEQ ID NO.1, and the target protein of the recombinant protein does not contain the amino acid sequence NKRRGS.
2. A method for removing an affinity tag, comprising the steps of:
in the construction process of the recombinant protein expression vector, inserting a gene sequence for expressing asparagine-lysine-arginine-glycine-serine between a gene sequence for expressing an affinity label and a gene sequence for expressing a target protein to construct and obtain the recombinant protein expression vector; then transferring the expression vector into host cells for recombinant protein expression, purifying the recombinant protein, then adding Zika virus serine protease, mixing, carrying out enzyme digestion reaction, and purifying to obtain the target protein with the affinity tag removed;
the amino acid sequence of the Zika virus serine protease is shown in SEQ ID NO.1, and the target protein does not contain the amino acid sequence NKRRGS.
3. The method of claim 2, wherein the expression vector is pET29b vector, pET vector or pGEX vector.
4. The method of claim 2, wherein the affinity tag is a glutathione-S-transferase tag or a polyhistidine tag.
5. The method of claim 2, wherein the purified recombinant protein is purified by affinity chromatography, and the purification after the enzymatic cleavage is performed by molecular sieve column purification to obtain the target protein.
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2020
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