CN117535329A - Carrier capable of removing tag and construction method and application thereof - Google Patents

Abstract

The invention discloses a carrier capable of removing labels, a construction method and application thereof, and relates to the technical field of genetic engineering. The invention obtains a recombinant plasmid by inserting the base sequence of the enzyme cutting site corresponding to PSP enzyme and the coding gene of the purification tag on the plasmid pET-28 (a), and the protein expressed by the plasmid can be subjected to enzyme cutting on the target protein by using PSP enzyme after primary purification, and the high-purity protein without the tag can be obtained after secondary nickel column purification, thereby providing a new purification strategy for scientific researchers with high requirements on protein purity.

Description

Carrier capable of removing tag and construction method and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a vector capable of removing a label, and a construction method and application thereof.

Background

Proteins are one of the important components of organisms, and research on human vital activities has been mainly focused on the structure and function of proteins. Because the expression quantity of most proteins in vivo is low, in-situ extraction cannot meet the requirement of protein research, in-vitro expression and purification of recombinant proteins are always important research contents of research in the field of biology. The pET28 (a, b, c) -6 XHis tag plasmid is available on the market, and due to 6 histidines carried by the vector and redundant amino acid sequences introduced by molecular cloning, the recombinant expressed protein is 1-2kDa larger than the actual protein, and the increase of the molecular weight is one of reasons for deviating the structure and function research of the recombinant protein from the actual situation to a certain extent. Therefore, in order to make the research on the structure and function of recombinant proteins more careful, how to eliminate the increased amino acids of recombinant proteins is the most critical issue at present.

In view of this, the present invention has been made.

Disclosure of Invention

The invention aims to provide a carrier capable of removing labels, a preparation method and application thereof.

In order to achieve the above object of the present invention, the following technical solutions are specifically adopted:

in a first aspect, the invention provides a vector with removable tag, wherein the vector is a recombinant vector pET28-8His obtained by inserting a base sequence of a protease cleavage site and two base sequences of histidine into a pET28-6His plasmid; wherein the protease is PSP enzyme; the base sequence of two histidines inserted into the recombinant vector is positioned at the N end of pET28-6His plasmid and after 6 XHis tag, a new purification tag 8 XHis tag is formed at the N end.

In a second aspect, the invention provides a host cell comprising a vector comprising a removable tag as described above.

In a third aspect, the invention provides a construction method of a vector with removable tag, which comprises taking SEQ ID NO.1-2 sequence as a primer, taking pET28-6His plasmid as a template, connecting a target fragment after polymerase chain reaction to obtain plasmid, and verifying whether the obtained plasmid is inserted into PSP restriction enzyme site.

In a fourth aspect, the invention provides the use of a tag-removable vector for purification of an exogenous recombinant protein.

The invention has the following beneficial effects:

according to the invention, a recombinant plasmid is obtained by inserting the base sequence of the enzyme cutting site corresponding to PSP enzyme and the coding gene of the His tag into the pET28-6His plasmid, the protein expressed by the plasmid can be subjected to enzyme cutting by using the PSP enzyme after primary purification, and the high-purity protein without the tag can be obtained after secondary nickel column purification, so that a novel purification strategy is provided for scientific researchers with high requirements on protein purity.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.

FIG. 1 is a schematic diagram of a protein-depleted tag of the present invention;

FIG. 2 is a map of the plasmid constructed in example 1;

FIG. 3 is a flow chart showing the preparation of the tag-removable plasmid of example 2;

FIG. 4 is a process diagram of the removal of protein tags in example 3;

FIG. 5 is a comparative analysis of SDS-PAGE of FXR LBD protein of example 3, wherein FIG. 5A is an analysis of protein-removed tags of the present invention, FIG. 5B is an analysis of pET28-6His tags, UI is overnight cultured mycoprotein, I is overnight induced mycoprotein, top is supernatant protein after ultracentrifugation, ppt is precipitated protein after ultracentrifugation, UB is protein not captured by nickel matrix packing, wash is non-specific binding protein eluted by primary purification, elu, elu2, elu3 is target protein after primary purification;

FIG. 6 is a SDS-PAGE analysis before and after cleavage of the tag from AIM1 protein in experimental example 1, wherein UB1, UB12, UB13 are non-tagged target proteins, E1, E2, E3, E4 are tagged proteins;

FIG. 7 is a chart showing molecular sieves before and after cleavage of the tag by AIM1 protein in Experimental example 1.

Detailed Description

In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

The pET series vector has the advantages of the lowest basic expression level in an escherichia coli expression system, truly regulated 'rheostat' control of the expression level, provision of various fusion tags and expression system configuration and the like, and becomes a system with the greatest prokaryotic expression reference.

prescission protease (PSP enzyme) is a fusion protein consisting of human rhinovirus type 14 3C protease and GST, which specifically recognizes sequences including Leu-Phe-Gln/Gly-Pro and cleaves between Gln and Gly residues. The method can specifically separate the exogenous protein expressed by the vector from the GST tag fused with the exogenous protein, thereby obtaining the exogenous target protein with higher purity.

At present, the protein expressed by the pET28 (a, b, c) plasmid carrying the 6 XHis tag is 1-2kD larger than the actual protein in molecular weight, the His tag of the expressed target protein can not be removed by taking pET28-6His as a vector, the purity of the finally obtained protein is not ideal, and the function of the finally obtained protein can be influenced to a certain extent due to the existence of the tag. The inventor researches and discovers that on the basis of pET28-6His plasmid, the base sequence of the enzyme cutting site corresponding to PSP enzyme and the coding gene of His tag are inserted before the multiple cloning site, the target protein can be subjected to enzyme cutting by PSP enzyme after the target protein is initially purified, then nickel column is used for purification, and finally purer target protein is obtained at the part which is not captured, and the principle is shown in figure 1.

Based on the above, the invention provides a vector capable of removing tags, which is a recombinant vector pET28-8His obtained by inserting a base sequence of a protease cleavage site and a base sequence of two histidines into a pET28-6His plasmid; wherein the protease is PSP enzyme; the base sequence of two histidines inserted into the recombinant vector is positioned at the N end of pET28-6His plasmid and after 6 XHis tag, a new purification tag 8 XHis tag is formed at the N end.

In an alternative embodiment, the recombinant vector further comprises a multiple cloning site and a C-terminal 6 XHis tag, and the base sequence of the corresponding cleavage site for PSP enzyme is inserted before the multiple cloning site.

In an alternative embodiment, the base sequence of the cleavage site corresponding to the PSP enzyme is followed by the base sequence of the 8 XHis tag.

In an alternative embodiment, the recombinant vector comprises a coding gene of 8 XHis tag, a base sequence of a cleavage site corresponding to PSPase, a multiple cloning site and a coding gene of 6 XHis tag in sequence.

In an alternative embodiment, the multiple cloning site of the recombinant vector and the gene encoding the 6 XHis tag further comprise a terminator.

The invention provides a host cell comprising a vector comprising the removable tag described above.

The invention provides a construction method of a vector capable of removing a label, which comprises the steps of commercially synthesizing a nucleotide fragment SEQ ID NO.1-2 with a sticky end as a primer, taking a pET28-6His plasmid as a template, generating the sticky end through an enzyme digestion reaction, connecting a synthetic fragment to obtain a plasmid, and verifying whether the obtained plasmid is inserted into a PSP enzyme digestion site;

the forward sequence SEQ ID NO.1 of the nucleotide fragment is as follows:

TCGAGTGCGGCCGCAAGCTTGTCGACGGAGCTCGAATTCGGATC CGCTAGCCATATGGGGCCCCTGGAACAGAACTTCCAGGTGATGATGAT GATGATGGTGGTGGCTGCTGCC；

the reverse sequence SEQ ID NO.2 is:

CACGCCGGCGTTCGAACAGCTGCCTCGAGCTTAAGCCTAGGCGA TCGGTATACCCCGGGGACCTTGTCTTGAAGGTCCACTACTACTACTACT ACCACCACCGACGACGGGTAC。

the invention provides an application of a vector with removable labels in purification of exogenous recombinant proteins.

After the vector is introduced into host cells, the recombinant strain is cultured and exogenous gene expression is induced, and the expression product is collected, separated and purified.

In an alternative embodiment, after the target protein generated by the expression of the vector is purified for the first time, the target protein is digested by PSP enzyme and purified again;

the ratio of the target protein to PSP enzyme is 50-100:1.

In an alternative embodiment, the re-purifying comprises purifying using a Ni-NTA column;

in an alternative embodiment, the repurification comprises purification using His Trap HP 1 ml.

The features and capabilities of the present invention are described in further detail below in connection with the examples.

Example 1

The embodiment provides a vector capable of removing tags, which sequentially comprises a coding gene of 8 XHis tag, a base sequence of a cleavage site corresponding to PSP enzyme, a gene of a first beta-gamma crystalline lens structural domain of a human AIM1 (Absent in melanoma 1) gene and a coding gene of 6 XHis tag, wherein the nucleotide sequence SEQ ID NO.3 of the human AIM1 is as follows:

CCGAAGGTGGTTGTGTACGAGAAACCGTTCTTTGAGGGTAAGTGCGTTGAGCTGGAAACCGGCATGTGCAGCTTTGTGATGGAGGGTGGCGAAACCGAGGAAGCGACCGGTGACGATCACCTGCCGTTCACCAGCGTTGGTAGCATGAAAGTGCTGCGTGGCATCTGGGTGGCGTATGAAAAGCCGGGTTTCACCGGCCACCAGTACCTGCTGGAGGAAGGCGAGTATCGTGATTGGAAAGCGTGGGGTGGCTACAACGGTGAACTGCAAAGCCTGCGCCCGATCCTG。

FIG. 2 is a map of the plasmid provided in this example.

Example 2

The embodiment provides a construction method of a label-removable carrier, which is shown in fig. 3, and comprises the following steps:

(1) Designing a primer: after the base sequence of the enzyme cleavage site corresponding to PSP enzyme is added to 8His labels, the sequence is used as a forward primer before the gene of the first beta-gamma lens structural domain of the human AIM1 gene, and the reverse primer is used after reverse complementation.

(2) Polymerase chain reaction: the original plasmid pET28-6His plasmid is used as a template, the designed forward and reverse primers are added into a PCR reaction system, and the target fragment is obtained after PCR is performed by high-fidelity polymerase.

(3) Ligation reaction: the resulting target fragment was phosphorylated with PNK enzyme and ligated with DNA ligase.

(4) And (3) constructing a carrier: the ligated plasmid is transformed into DH5 alpha, and then the colony PCR technology is combined with DNA sequencing to determine that the enzyme cleavage site of PSP enzyme is successfully inserted, and finally the plasmid pET28-8His containing the enzyme cleavage site is successfully obtained.

Example 3

This example provides the use of the removable tagged vector of example 2 in the purification of exogenous recombinant proteins. The specific process is shown in fig. 4.

Recombinant plasmids of FXR LBD were constructed as in example 2, with the corresponding amino acid sequence SEQ ID NO.4 as follows:

MGSSHHHHHHHHLEVLFQGPHMMELTPDQQTLLHFIMDSYNKQRMPQEITNKILKEEFSAEENFLILTEMATNHVQVLVEFTKKLPGFQTLDHEDQIALLKGSAVEAMFLRSAEIFNKKLPSGHSDLLEERIRNSGISDEYITPMFSFYKSIGELKMTQEEYALLTAIVILSPDRQYIKDREAVEKLQEPLLDVLQKLCKIHQPENPQHFACLLGRLTELRTFNHHHAEMLMSWRVNDHKFTPLLCEIWDVQ。

the recombinant plasmid was transformed into E.coli BL21 (DE 3), cultured overnight at a constant temperature of 37℃to give colonies, and then the colonies were picked up in 100ml of LB medium, and cultured overnight in a shaking table at 37℃with the addition of kanamycin. The cultured bacterial liquid was expanded to 2L of LB medium at a ratio of 1:50, and the culture was continued in a shaker at 37 ℃. When the OD value of the bacterial liquid reaches 0.6-0.8, standing at 4 ℃ for half an hour, cooling, adding 0.2mM IPTG to induce expression after cooling, and initiating overnight at 25 ℃ at the temperature of a shaking table. Coli was lysed in buffer A containing 40mM Tris-HCl 8.0, 250mM NaCl, 10mM imidazole, 4mM beta-ME and 0.1mM PMSF.

First, the method for primary purification of FXR LBD protein: the thalli are crushed under high pressure by using a low-temperature Gao Yapo crushing instrument until the thalli are crushed completely. The crushed bacterial liquid is collected by a round bottom ultracentrifuge tube, ultracentrifuged at 14000rpm for 60 minutes at 4 ℃, and the supernatant is taken after centrifugation. During centrifugation, nickel matrix fillers were pre-equilibrated, after which the fillers were incubated with the centrifuged protein supernatant solution in an empty 50mL centrifuge tube at 4 ℃ for 2 hours. After incubation, the protein supernatant solution containing the packing was transferred to a hollow column with a two-way valve, the flow rate was controlled, elution of non-specifically bound proteins was performed with a large volume of buffer A, then the target proteins were eluted with buffer B containing 40mM Tris-HCl 8.0, 250mM NaCl, 250mM imidazole and a high concentration of 0.1mM PMSF, the eluate was collected and the presence of the proteins was detected using Bradford.

The purity of the protein was checked by SDS-PAGE, and the result is shown in FIG. 5A, and the purity of the protein obtained at this time was: 75%. And then PSP enzyme is used for enzyme digestion of target protein, and purification is carried out again. The His Trap HP 1ml was used for further purification to obtain a protein with a purity > 95%.

Comparative example

The present comparative example relates to the use of another recombinant vector in the purification of foreign recombinant proteins, which differs from the vector of example 1 in that the N-terminal tag is 6 XHis tag, the corresponding amino acid sequence SEQ ID NO.5 is as follows:

MGSSHHHHHHELTPDQQTLLHFIMDSYNKQRMPQEITNKILKEEFSAEENFLILTEMATNHVQVLVEFTKKLPGFQTLDHEDQIALLKGSAVEAMFLRSAEIFNKKLPSGHSDLLEERIRNSGISDEYITPMFSFYKSIGELKMTQEEYALLTAIVILSPDRQYIKDREAVEKLQEPLLDVLQKLCKIHQPENPQHFACLLGRLTELRTFNHHHAEMLMSWRVNDHKFTPLLCEIWDVQ。

the recombinant vector was subjected to the same primary purification as in example 3, and the results are shown in FIG. 5B, and the purity of the protein after primary purification was obtained as follows: 60%.

Comparing the comparative example with example 3, it can be found that the recombinant plasmid constructed according to the present invention has a better purification effect in the purification of foreign recombinant proteins than the recombinant plasmid of the comparative example.

Experimental example 1

This experimental example 1 provides an alignment test of the purified protein obtained in example 3 with the tagged protein.

The protein of example 3 was subjected to further purification, and then the tag was excised, and the purity of the protein was checked by SDS-PAGE, and the results are shown in FIG. 6.

Wherein UB1, UB2, UB3 is a target protein without a tag, E1, E2, E3, E4 is a protein with a tag. The protein purity after tag cleavage is higher than when tagged, and the tagged protein can grow into larger crystals of the individual, thus indicating that the protein properties are more stable after tag cleavage.

In addition, the inventors analyzed and compared proteins before and after cleavage of the tag by molecular sieves, and found that the recombinant proteins were more uniform in conformation after cleavage of the tag, and the results are shown in fig. 7.

The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims (10)

1. A vector capable of removing a tag, which is characterized in that the vector is a recombinant vector pET28-8His obtained by inserting a base sequence of a protease cleavage site and two base sequences of histidine into a pET28-6His plasmid; the protease is PSP enzyme;

the base sequences of two histidines inserted into the recombinant vector are positioned at the N end of the pET28-6His plasmid and then are positioned at the 6 XHis tag, and a new purification tag 8 XHis tag is formed at the N end.

2. The removable tag vector of claim 1, wherein the recombinant vector further comprises a multiple cloning site and a C-terminal 6 xhis tag, and wherein the base sequence of the corresponding cleavage site of the pspase is inserted before the multiple cloning site.

3. The tag-removable vector according to claim 2, wherein the base sequence of the cleavage site corresponding to the pspase follows the base sequence of the 8 xhis tag.

4. The vector of claim 3, wherein the recombinant vector comprises a coding gene of 8 xHis tag, a base sequence of a cleavage site corresponding to PSP enzyme, a multiple cloning site and a coding gene of 6 xHis tag in sequence.

5. The removable tagged vector of claim 1, wherein the multiple cloning site of the recombinant vector and the coding gene for the 6 xhis tag further comprise a terminator.

6. A host cell comprising the vector of any one of claims 1 to 5.

7. The method of constructing a tag-removable vector according to any one of claims 1 to 5, comprising generating a cohesive end by an enzyme digestion reaction using the sequence of SEQ ID No.1 to 2 as a primer and the pET28-6His plasmid as a template, ligating the fragment to obtain a plasmid, and verifying whether the obtained plasmid is inserted into a PSP cleavage site.

8. Use of a tag-removable vector according to any one of claims 1 to 5 for purification of an exogenous recombinant protein.

9. The use of the tag-removable vector according to claim 7, wherein the target protein produced after expression of the vector is purified for the first time, and then digested with PSP enzyme to be purified again;

the mass ratio of the target protein to the PSP enzyme is 50-100:1.

10. The use of a removable label carrier according to claim 8, wherein the repurification comprises purification using a nickel column;

preferably, the repurification comprises purification using His Trap HP 1 ml.