CN110078791B

CN110078791B - Method for realizing protein crosslinking based on amino acid specificity recognition

Info

Publication number: CN110078791B
Application number: CN201910405266.7A
Authority: CN
Inventors: 陈必强; 邵文铉; 张晓楠; 谭天伟
Original assignee: Beijing University of Chemical Technology
Current assignee: Beijing University of Chemical Technology
Priority date: 2019-05-15
Filing date: 2019-05-15
Publication date: 2021-06-22
Anticipated expiration: 2039-05-15
Also published as: CN110078791A

Abstract

The invention relates to a peptide chain tag for realizing protein crosslinking, which has a peptide chain length of 8-15 amino acids, at least one lysine and/or glutamine in the peptide chain, and an amino acid Sequence of at least one of Sequence No. 1-4. The invention also relates to a method for realizing protein crosslinking, which inserts peptide chain genes with two specified amino acids of lysine or glutamine into the N end of the protein and expresses the corresponding protein by escherichia coli. The glutamine transaminase (TGase) has the function of specifically recognizing and crosslinking the primary amine group of lysine and the amide group of glutamine, realizes ordered crosslinking among proteins, can ensure no obvious loss of the activity of the proteins, and has wide application prospect.

Description

Method for realizing protein crosslinking based on amino acid specificity recognition

Technical Field

The invention belongs to the technical field of biology, and relates to a method for realizing protein crosslinking based on amino acid specificity identification, in particular to a peptide chain label for realizing protein crosslinking and a method for realizing protein crosslinking based on the peptide chain label.

Background

Glutamine transaminase (TGase), was first discovered by Clarke et al in guinea pig liver and subsequently in animals, plants, fish, microorganisms. Transglutaminase (TGase) catalyzes the transacylation reaction between a glutamine residue and a primary amine group of a protein or polypeptide. With the maturation and commercial production of microbial transglutaminase (TGase) technology, the TGase is applied to more and more fields, such as food fields (food processing of meat, milk, beans, etc.), biomedical fields (drug carriers, medical scaffolds), material fields, etc. Glutamine transaminase (TGase) has recently attracted much attention in modifying the functionality of proteins and enzymes. Noriho Kamiya demonstrated that the lipase could not be crosslinked except for the crude enzyme solution of Rhizopus lipase by crosslinking the crude enzyme solution of lipase using glutamine transaminase, whereas pure enzyme could not be crosslinked after purifying Rhizopus fat with a nickel column.

Therefore, there is a need to develop a method for achieving protein ordered cross-linking by using transglutaminase, while ensuring no significant loss of protein activity.

Disclosure of Invention

The technical problem to be solved by the invention is to provide a peptide chain label with specific amino acid aiming at the defects of the prior art, and the protein with the peptide chain label, which is obtained by inserting the deoxyribonucleotide sequence of the peptide chain label into a protein gene and expressing the protein gene, can realize protein crosslinking by utilizing glutamine transaminase. The invention also provides a method for realizing protein crosslinking by utilizing glutamine transaminase, which inserts the gene with the peptide chain label of the specific amino acid into a protein gene for expression culture, and the obtained protein with the peptide chain label realizes protein ordered crosslinking by utilizing the glutamine transaminase, and simultaneously can ensure that the activity of the protein has no obvious loss.

To this end, the invention provides in a first aspect a peptide tag for effecting protein cross-linking, which peptide tag has a length of 8 to 15 amino acids, at least one lysine and/or glutamine in the peptide tag, and an amino acid Sequence comprising at least one of Sequence nos. 1 to 4.

According to the invention, the lysine and/or glutamine sites are located close to the N-terminus in the peptide chain.

In some embodiments of the invention, the deoxyribonucleotide Sequence of the peptide chain comprises at least one of Sequence nos. 5-8.

In a second aspect, the present invention provides a method of effecting protein cross-linking, comprising:

step L, inserting the deoxyribonucleotide sequence of the peptide chain label into the 5' end of the protein gene fragment to obtain the protein gene fragment of the deoxyribonucleotide sequence with the peptide chain label;

step M, expressing a protein gene segment of the deoxyribonucleotide sequence with the peptide chain label to obtain a protein with the peptide chain label;

step N, crosslinking the protein with the peptide chain label in the presence of glutamine transaminase (TGase) to obtain crosslinked protein;

wherein the peptide chain tag is according to any one of claims 1 to 3.

According to the invention, in step N, glutamine transaminase is added to a substrate solution containing protein with a peptide chain tag, and the substrate solution are mixed and then subjected to a crosslinking reaction to prepare the crosslinked protein.

In some embodiments of the invention, the transglutaminase is used in an amount of 1wt% to 3wt% based on the volume of the substrate solution.

In the present invention, the substrate solution is formed by dissolving a protein containing a peptide-tagged peptide in a buffer solution.

In some embodiments of the invention, the peptide-tagged protein is present in an amount of 5-10mg/mL based on the total volume of the substrate solution.

In some preferred embodiments of the present invention, the buffer solution is a 0.05mol/L Tris-HCl solution.

In some embodiments of the invention, the temperature of the crosslinking is 20 to 30 ℃.

In some embodiments of the invention, the crosslinking time is 4 to 12 hours.

According to the invention, said step M comprises:

b, connecting the gene segment of the protein of the deoxyribonucleotide sequence with the peptide chain label to a vector to construct a recombinant expression vector;

step C, transferring the recombinant expression vector into a host cell to obtain an expression strain;

and D, performing fermentation culture on the expression strain, performing induction expression by using an inducer, then performing cell breakage on the thalli, re-suspending, centrifuging, collecting supernatant, and performing separation and purification to obtain a pure protein product with the peptide chain label.

In the present invention, the host cell is Escherichia coli, and preferably the host cell is Escherichia coli BL21(DE 3).

In the present invention, the vector is pET32b (+) vector.

In the invention, the inducer is IPTG.

In some embodiments of the invention, the inducer is added in an amount of 0.05% by weight of the volume of the fermentation broth.

According to the invention, in step D, the expression strain is inoculated into a culture medium for fermentation culture, and then an inducer is added for induction expression culture.

According to a preferred embodiment of the present invention, the expression strain is inoculated into a culture medium for fermentation culture, and when the OD600 reaches 0.8, an inducer is added for induction expression culture.

In the invention, the culture medium is an LB culture medium or a TB culture medium.

In some embodiments of the invention, the temperature of the fermentation culture is 37 ℃.

In some embodiments of the invention, the temperature of the induced expression culture is 15-25 ℃.

In some particularly preferred embodiments of the invention, the fermentation culture is carried out in a rocking bed.

In some particularly preferred embodiments of the present invention, the shaker speed in the present invention is 180 rpm.

In some particularly preferred embodiments of the invention, the inducible expression culture is performed in a shaker.

In some particularly preferred embodiments of the present invention, the shaker speed is 180 rpm.

In some particularly preferred embodiments of the invention, the time for the induction of expression culture is 12-20 hours.

According to the present invention, in the step D, the method for separation and purification includes an affinity purification method.

In some preferred embodiments of the invention, the affinity purification is performed using a nickel column.

In some particularly preferred embodiments of the invention, the affinity purification process is performed using 0.05mol/L Tris-HCl solution to remove the contaminating proteins and 0.1mol/L imidazole solution for elution.

The invention uses gene engineering technology, expresses protein with peptide chain label by colon bacillus, and realizes protein crosslinking by utilizing the specificity recognition of glutamine transaminase (TGase) to amino acid in peptide chain. The method has the following advantages:

(1) the peptide chain modification of the protein is realized at the gene level, and the protein does not need to be modified in vitro;

(2) adopts a biocatalyst-glutamine transaminase (TGase) to crosslink protein with a peptide chain label, and has the function of specific recognition;

(3) glutamine transaminase (TGase) does not become a part of a cross-linked product, while chemical cross-linking agents such as glutaraldehyde and genipin become a part of the product, so that the glutamine transaminase (TGase) can be recycled;

(4) the protein molecule is modified at the gene level, so that the protein molecule is provided with a peptide chain, and the peptide chain is crosslinked, and the structure and the performance of the protein molecule are not influenced.

Drawings

The invention is described in further detail below with reference to the attached drawing figures:

FIG. 1 glutamine transaminase cross-links enhanced green fluorescent protein with S-peptide tag.

FIG. 2 Glutamine transaminase cross-links enhanced green fluorescent protein with an F-peptide tag and an M-peptide tag.

FIG. 3 protein electrophoresis of transglutaminase crosslinked F-peptide tagged carbonic anhydrase and M-peptide tagged formate dehydrogenase.

FIG. 4 results of enzyme activities of transglutaminase to crosslink F-peptide-tagged carbonic anhydrase and M-peptide-tagged formate dehydrogenase.

FIG. 5 protein electrophoresis of transglutaminase crosslinked K2 peptide-tagged carbonic anhydrase and M peptide-tagged formate dehydrogenase.

FIG. 6 results of enzyme activities of transglutaminase to crosslink K2 peptide-tagged carbonic anhydrase and M peptide-tagged formate dehydrogenase.

Detailed Description

In order that the invention may be readily understood, a more particular description thereof will be rendered by reference to the appended drawings. However, before the invention is described in detail, it is to be understood that this invention is not limited to particular embodiments described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

Unless otherwise defined, all terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described.

I. Term(s) for

The term "water" as used herein means deionized water, distilled water or ultrapure water unless otherwise specified or limited.

Embodiments of

As mentioned above, except for the fact that the crude lipase solution can be crosslinked by using glutamine transaminase, the remaining lipase can not be crosslinked, especially the pure lipase purified by nickel column can not be crosslinked at all. Meanwhile, the enzyme activity loss is obvious after enzyme crosslinking. In view of this, the present inventors have conducted extensive studies on the use of transglutaminase to achieve protein crosslinking.

The inventor researches and discovers that protein with a peptide chain label, which is obtained by inserting a deoxyribonucleotide sequence of the peptide chain label into a protein gene fragment and expressing the deoxyribonucleotide sequence of the peptide chain label, can realize ordered protein crosslinking by utilizing glutamine transaminase and simultaneously ensure that the activity of the protein is not obviously lost. The present invention has been made based on the above findings.

To this end, the peptide chain tag for achieving protein crosslinking according to the first aspect of the present invention has a peptide chain length of 8 to 15 amino acids, at least one lysine and/or glutamine in the peptide chain, and an amino acid Sequence including at least one of Sequence nos. 1 to 4, as shown in table 1.

TABLE 1

Serial number	Peptide chain tag	Peptide chain tag amino acid sequence
			1	S peptide	N-KETAAAKFERQHMDS-C
2	F peptide	N-DYKDDDDG-C
			3	M peptides	N-EQGLISEEDL-C
4	K2 peptide	N-GKGGGGKGGG-C

The present inventors have found that a protein gene fragment having a peptide-labeled deoxyribonucleotide sequence is obtained by inserting the deoxyribonucleotide sequence of the peptide chain into a protein gene fragment and then bringing the site of lysine and/or glutamine close to the 5' -end; coli is taken as a host cell to express a protein gene segment with a deoxyribonucleotide sequence of a peptide chain label, so that the N end of the protein has the peptide chain label, and the specific recognition crosslinking of the protein with the peptide chain label is realized by utilizing the function that glutamine transaminase (TGase) has specific recognition crosslinking on a primary amine group of lysine and an amide group of glutamine. Thus, the above-mentioned peptide chain tag can be considered as the basis for achieving the ability of the protein to be cross-linked by TGase, whereas "in the peptide chain, the site of lysine and/or glutamine is close to the N-terminus. "then can be considered as a necessary condition for the protein to be cross-linked by TGase.

Accordingly, the deoxyribonucleotide Sequence of the peptide chain includes at least one of Sequence nos. 5 to 8, as shown in table 2.

TABLE 2

According to some embodiments of the invention, the method of achieving protein cross-linking according to the second aspect of the invention comprises:

step N, adding glutamine transaminase (TGase) into a substrate solution containing protein with a peptide chain label to perform a crosslinking reaction to prepare crosslinked protein;

wherein the peptide chain tag is according to the first aspect of the invention.

The present inventors characterized the obtained protein by SDS-PAGE and showed that the molecular weight of the protein became large, confirming that the protein formed a covalent bond, i.e., belonging to the result of isopeptide bond formation of TGase crosslinked protein, thereby confirming that the protein having a peptide chain tag was crosslinked in the presence of transglutaminase (TGase) in this step to obtain a crosslinked protein;

the inventors of the present invention have found through experimental studies that the amount of enzyme used affects the crosslinking rate of protein, and if the amount of enzyme is too small, the crosslinking rate is too slow, but if the amount of enzyme is too large, the cost is too high, and preferably, the amount of transglutaminase used is 1wt% to 3wt% based on the volume of the substrate solution.

In the invention, the substrate solution is formed by dissolving the protein with the peptide chain tag in a buffer solution, and the content of the protein with the peptide chain tag is 5-10mg/mL based on the volume of the substrate solution.

In some preferred embodiments, the buffer solution is a 0.05mol/L Tris-HCl solution.

In the step N, the crosslinking temperature is 20-30 ℃, and the crosslinking time is 4-12 hours.

In the present invention, there is no particular limitation on the method for expressing a protein gene fragment having a peptide-tagged deoxyribonucleotide sequence, and a method conventionally used in the art can be used to express a protein gene fragment having a peptide-tagged deoxyribonucleotide sequence. For example, in some embodiments: the step M comprises the following steps:

step B, connecting the gene fragment of the protein of the deoxyribonucleotide sequence with the peptide chain label to a pET32B (+) vector to construct a recombinant expression vector;

step C, transferring the recombinant expression vector into escherichia coli BL21(DE3) serving as a host cell to obtain an expression strain;

and D, inoculating the expression strain into an LB culture medium or a TB culture medium for fermentation culture, adding an inducer IPTG (isopropyl-beta-D-thiogalactoside) for induction expression culture when OD600 reaches 0.8-1.0, then breaking the thalli, resuspending, centrifuging, collecting supernatant, separating and purifying to obtain a pure protein product with a peptide chain label, wherein the addition amount of the inducer is 0.05 wt% of the volume of the fermentation liquid.

In the step D, fermentation culture is carried out in a shaking table, wherein the rotation speed of the shaking table is 180 rpm; the temperature of the fermentation culture was 37 ℃.

In the step D, performing inducible expression culture in a shaking table, wherein the rotation speed of the shaking table is 180 rpm; the temperature of the induced expression culture is 15-25 ℃, and the time of the induced expression culture is 12-20 hours.

In the present invention, the method for separation and purification in step D is not particularly limited, and for example, the protein in the product may be separated and purified by affinity purification.

In some preferred embodiments of the invention, the affinity purification is performed using a nickel column, and during the affinity purification, the hetero-proteins are removed using 0.05mol/L Tris-HCl solution and eluted using 0.1mol/L imidazole solution. The protein thus obtained has high separation and purification efficiency.

In some particularly preferred embodiments of the present invention, the specific method for cross-linking the peptide chain tagged enhancer protein by transglutaminase is as follows:

(1) construction of recombinant expression vectors

Connecting the gene fragment of the protein with the deoxyribonucleotide sequence with a peptide chain label to a pET32b (+) vector to construct a recombinant expression vector;

(2) construction of recombinant strains

Transferring the recombinant expression vector into escherichia coli to obtain an expression strain;

(3) expression of protein with peptide chain tag

Fermenting and culturing the expression strain, and performing induced expression by taking IPTG as an inducer. And (3) the thalli are resuspended after cell breaking, centrifuged, and the supernatant is collected for affinity purification to obtain the protein with the peptide chain label.

In the step (3), the fermentation culture method comprises the following steps: the expression strain is cultured in LB culture medium at 37 deg.C and 180rpm by shaking table until OD is reached₆₀₀When reaching 0.8, IPTG is added, the temperature is set to 15-25 ℃, shaking culture is carried out at 180rpm, and continuous culture is carried out for 12-20 hours. The amount of IPTG added was 0.05% of the volume of the fermentation broth.

Further, the target protein in the supernatant is subjected to affinity purification by adopting a nickel column to obtain pure protein; in the nickel column affinity purification, the impurity protein is removed by adopting 0.05mol/L Tris-HCl solution, and elution is carried out by using 0.1mol/L imidazole solution.

Furthermore, for the specific recognition crosslinking of a protein with a peptide-tag, the transglutaminase is used in an amount of 1 to 3% relative to the total volume of the substrate solution containing the peptide-tag-containing protein, the crosslinking temperature is 20 to 30 ℃ and the crosslinking time is 4 to 12 hours.

Particularly preferably, the substrate solution is formed by dissolving the protein with the peptide chain tag in a buffer solution, and the content of the protein with the peptide chain tag is 5-10mg/mL based on the volume of the substrate solution.

More specifically, the buffer solution is 0.05mol/L Tris-HCl solution.

The invention relates to a method for realizing protein crosslinking by using a genetic engineering method and an enzymatic crosslinking method, namely expressing a protein with a peptide chain label through escherichia coli and then utilizing the specific recognition of glutamine transaminase (TGase) on amino acid in a peptide chain.

The invention firstly provides a peptide chain label with specific amino acid, the amino acid sequence of the peptide chain label is shown in table 1, and the deoxyribonucleotide sequence of the peptide chain label is shown in table 2. Secondly, the invention provides a method for realizing protein crosslinking based on amino acid specificity recognition. Expressing the protein gene segment of the deoxyribonucleotide sequence with the specific amino acid peptide chain label by taking escherichia coli as a host cell to obtain the protein with the specific amino acid peptide chain label; performing affinity purification on the target protein by using a nickel column; the specific recognition crosslinking of the protein with the peptide chain label is realized by utilizing the function that glutamine transaminase (TGase) has specific recognition crosslinking on a primary amine group of lysine and an amide group of glutamine. The invention relates to a method for realizing protein crosslinking by utilizing the specific recognition of amino acid by glutamine transaminase (TGase), which provides a foundation for the aspects of protein modification, enzyme molecule performance modification and the like.

III, examples

The present invention will be specifically described below with reference to specific examples. The experimental methods described below are, unless otherwise specified, all routine laboratory procedures. The experimental materials described below, unless otherwise specified, are commercially available.

Example 1: enhanced green fluorescent protein with S peptide label crosslinked by glutamine transaminase

(1) Construction of recombinant expression vectors

Connecting the gene fragment of the enhanced green fluorescent protein of the deoxyribonucleotide sequence with an S peptide label to a pET32b (+) vector to construct a recombinant expression vector;

(2) construction of recombinant strains

(3) expression of protein with peptide chain tag

Fermenting and culturing the expression strain, and performing induced expression by taking IPTG as an inducer. The expression strain is cultured in LB culture medium at 37 deg.C and 180rpm by shaking table until OD is reached₆₀₀When the temperature reached 0.8, IPTG was added and the temperature was set at 25 ℃ and shaking-cultured at 180rpm for 12 hours. The amount of IPTG added was 0.05% of the volume of the fermentation broth.

And (3) the thalli are resuspended after cell breaking, centrifuged, and the supernatant is collected for affinity purification, so as to obtain the enhanced green fluorescent protein with the S peptide tag. In the nickel column affinity purification, the impurity protein is removed by adopting 0.05mol/L Tris-HCl solution, and elution is carried out by using 0.1mol/L imidazole solution.

And (3) carrying out specific recognition crosslinking on the purified enhanced green fluorescent protein with the S peptide tag by using glutamine transaminase. The amount of transglutaminase used was 1wt% relative to the total volume of the substrate solution containing the S-peptide-tagged enhanced green fluorescent protein, the crosslinking temperature was 20 ℃, and the crosslinking time was 12 hours.

The results in fig. 1 demonstrate that, lane 1 is glutamine transaminase, lane 2 is enhanced green fluorescent protein, and

lanes

3 and 4 are cross-linked products, and according to the newly generated bands in

lanes

3 and 4, dimers and tetramers of enhanced green fluorescent protein are formed, which indicates that the enhanced green fluorescent protein can be successfully cross-linked by inserting S-peptide tag. The results of color comparison of the pre-reaction sample, the 5-hour reaction sample, the 10-hour reaction sample, and the 24-hour reaction sample show that the fluorescence of the enhanced green fluorescent protein begins to disappear after 10 hours of crosslinking, indicating that the crosslinking is excessive. When the strong green fluorescent protein is combined with 1(A) and 1(B), the strong green fluorescent protein forms a dimer and a tetramer and can still keep fluorescence, and the structure of the protein per se is not influenced.

Example 2: glutamine transaminase cross-linked enhanced green fluorescent protein with F peptide and M peptide tags

(1) Construction of recombinant expression vectors

Respectively connecting the gene segments of the enhanced green fluorescent protein with the deoxyribonucleotide sequence labeled by the F peptide and the deoxyribonucleotide sequence labeled by the M peptide to a pET32b (+) vector to construct a recombinant expression vector;

(2) construction of recombinant strains

(3) expression of protein with peptide chain tag

And (3) the thalli are resuspended after cell breaking, centrifuged, and the supernatant is collected for affinity purification to respectively obtain the enhanced green fluorescent protein with the F peptide label and the M peptide label. In the nickel column affinity purification, the impurity protein is removed by adopting 0.05mol/L Tris-HCl solution, and elution is carried out by using 0.1mol/L imidazole solution.

And (3) carrying out specific recognition crosslinking on the purified enhanced green fluorescent protein with the F peptide and the M peptide by using glutamine transaminase. The amount of transglutaminase used was 3wt% relative to the total volume of the substrate solution containing the enhanced green fluorescent protein labeled with F-peptide, the crosslinking temperature was 30 ℃ and the crosslinking time was 12 hours.

In FIG. 2(A),

lanes

1, 2 and 3 are the purified enhanced green fluorescent protein with F peptide tag, and

lanes

4, 5 and 6 are the purified enhanced green fluorescent protein with M peptide tag.

In FIG. 2(B),

lanes

1, 2 and 3 show the results of transglutaminase-crosslinking the F peptide-labeled and M peptide-labeled enhanced green fluorescent proteins for 6 hours, and

lanes

4, 5 and 6 show the results of transglutaminase-crosslinking the F peptide-labeled and M peptide-labeled enhanced green fluorescent proteins for 12 hours.

From the above results, it is known that the formed high molecular weight protein is a dimer, i.e., the enhanced green fluorescent protein with the F peptide tag and the M peptide tag forms a "one-to-one" cross-linking result.

The samples before reaction, the samples after 4 hours of reaction, the samples after 8 hours of reaction and the samples after 12 hours of reaction were compared. The results show that: when the strong green fluorescent protein forms a dimer, the enhanced green fluorescent protein band with the F peptide tag and the M peptide tag can still keep fluorescence, and the structure of the protein is not influenced at the moment.

Example 3: glutamine transaminase cross-linking F-peptide-tagged carbonic anhydrase and M-peptide-tagged formate dehydrogenase

(1) Construction of recombinant expression vectors

Respectively connecting gene fragments of carbonic anhydrase with the deoxynucleotide sequence of the F peptide tag and the formate dehydrogenase with the deoxynucleotide sequence of the M peptide tag to a pET32b (+) vector to construct a recombinant expression vector;

(2) construction of recombinant strains

(3) expression of protein with peptide chain tag

Fermenting and culturing the expression strain, and performing induced expression by taking IPTG as an inducer. Placing the carbonic anhydrase expression strain in LB culture medium, shaking-culturing at 37 deg.C and 180rpm until OD is reached₆₀₀When the temperature reached 0.8, IPTG was added and the temperature was set at 25 ℃ and shaking-cultured at 180rpm for 20 hours. The amount of IPTG added was 0.05% of the volume of the fermentation broth. Culturing the formate dehydrogenase expression strain in LB culture medium at 37 deg.C and 180rpm with shaking table until OD is reached₆₀₀When the temperature reached 0.8, IPTG was added thereto, the temperature was set at 20 ℃ and shaking culture was carried out at 180rpm, and continuous culture was carried out for 16 hours. The amount of IPTG added was 0.05% of the volume of the fermentation broth.

And (3) the thalli are resuspended after cell breaking, centrifuged, and the supernatant is collected for affinity purification to respectively obtain carbonic anhydrase with F peptide tags and formate dehydrogenase with M peptide tags. In the nickel column affinity purification, the impurity protein is removed by adopting 0.05mol/L Tris-HCl solution, and elution is carried out by using 0.1mol/L imidazole solution.

The purified carbonic anhydrase with F peptide tag and M peptide tag formate dehydrogenase are specifically identified and cross-linked by using glutamine transaminase. The transglutaminase was used in an amount of 2 wt% relative to the total volume of the substrate solution containing the M-peptide-tagged formate dehydrogenase, at a crosslinking temperature of 25 ℃ and for a crosslinking time of 4 hours.

The results in FIG. 3 demonstrate that, lane 1 is glutamine transaminase, lane 2 is a mixture of F-peptide-tagged carbonic anhydrase and M-peptide-tagged formate dehydrogenase, lane 3 is the product of 0 hour cross-linking, and

lanes

4 and 5 are the products of 2 and 4 hours cross-linking, respectively. The new band produced in

lanes

3 and 4 has a molecular weight of about 120 kDa. According to the results shown in the bands, a "one-to-one" cross-link should be formed for Carbonic Anhydrase (CA) and Formate Dehydrogenase (FDH).

The results in FIG. 4 show that the enzyme activity of the cross-linked carbonic anhydrase and formate dehydrogenase is not significantly reduced from the enzyme activity of the mixed double enzymes. The result shows that the cross-linking process has little loss of enzyme activity.

Example 4: glutamine transaminase cross-linking K2 peptide-tagged carbonic anhydrase and M peptide-tagged formate dehydrogenase

(1) Construction of recombinant expression vectors

Respectively connecting gene fragments of carbonic anhydrase with K2 peptide-labeled deoxyribonucleotide sequence and formate dehydrogenase with M peptide-labeled deoxyribonucleotide sequence to pET32b (+) vector to construct recombinant expression vector;

(2) construction of recombinant strains

(3) expression of protein with peptide chain tag

Fermenting and culturing the expression strain, and performing induced expression by taking IPTG as an inducer. Placing the carbonic anhydrase expression strain in LB culture medium, shaking-culturing at 37 deg.C and 180rpm until OD is reached₆₀₀When reaching 0.8, IPTG is added, the temperature is set to 25 ℃, shaking culture is carried out at 180rpm, and continuous culture is carried outAnd culturing for 20 hours. The amount of IPTG added was 0.05% of the volume of the fermentation broth. Culturing the formate dehydrogenase expression strain in LB culture medium at 37 deg.C and 180rpm with shaking table until OD is reached₆₀₀When the temperature reached 0.8, IPTG was added thereto, the temperature was set at 20 ℃ and shaking culture was carried out at 180rpm, and continuous culture was carried out for 16 hours. The amount of IPTG added was 0.05% of the volume of the fermentation broth.

And (3) the thalli are resuspended after cell breaking, centrifuged, and the supernatant is collected for affinity purification to respectively obtain carbonic anhydrase with K2 peptide tags and formate dehydrogenase with M peptide tags. In the nickel column affinity purification, the impurity protein is removed by adopting 0.05mol/L Tris-HCl solution, and elution is carried out by using 0.1mol/L imidazole solution.

The specific recognition cross-linking of purified K2 peptide-tagged carbonic anhydrase and M peptide-tagged formate dehydrogenase was performed using glutamine transaminase. The transglutaminase was used in an amount of 2 wt% relative to the total volume of the substrate solution containing the M-peptide-tagged formate dehydrogenase, at a crosslinking temperature of 25 ℃ and for a crosslinking time of 12 hours.

The results in FIG. 5 demonstrate that lane 1 is glutamine transaminase, lane 2 is the result of crosslinking for 10 minutes, lane 3 is the product of crosslinking for 0.5 hours, and

lanes

4, 5, 6, 7, 8 are the results of crosslinking for 1 hour, 4 hours, 6 hours, 8 hours, and 12 hours, respectively. Lane 2 shows that a band with a molecular weight of about 120KDa was produced, which should form a "one-to-one" cross-link for Carbonic Anhydrase (CA) and Formate Dehydrogenase (FDH). The new band in lane 5, which has a molecular weight of about 200 kDa. According to the results shown in the bands, a "one-to-two" cross-link should be formed for Carbonic Anhydrase (CA) and Formate Dehydrogenase (FDH).

The results in FIG. 6 show that the enzyme activity of the cross-linked carbonic anhydrase and formate dehydrogenase is not significantly reduced from that of the mixed double enzymes. The result shows that the cross-linking process has little loss of enzyme activity.

It should be noted that the above-mentioned embodiments are only for explaining the present invention, and do not constitute any limitation to the present invention. The present invention has been described with reference to exemplary embodiments, but the words which have been used herein are words of description and illustration, rather than words of limitation. The invention can be modified, as prescribed, within the scope of the claims and without departing from the scope and spirit of the invention. Although the invention has been described herein with reference to particular means, materials and embodiments, the invention is not intended to be limited to the particulars disclosed herein, but rather extends to all other methods and applications having the same functionality.

Sequence listing

<110> Beijing university of chemical industry

<120> method for realizing protein crosslinking based on amino acid specificity recognition

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 15

<212> PRT

<213> (S peptide)

<400> 1

Lys Glu Thr Ala Ala Ala Lys Phe Glu Arg Gln His Met Asp Ser

1 5 10 15

<210> 2

<211> 8

<212> PRT

<213> (F peptide)

<400> 2

Asp Tyr Lys Asp Asp Asp Asp Gly

1 5

<210> 3

<211> 10

<212> PRT

<213> (M peptide)

<400> 3

Glu Gln Gly Leu Ile Ser Glu Glu Asp Leu

1 5 10

<210> 4

<211> 10

<212> PRT

<213> (K2 peptide)

<400> 4

Gly Lys Gly Gly Gly Gly Lys Gly Gly Gly

1 5 10

<210> 5

<211> 45

<212> DNA

<213> (S peptide)

<400> 5

aaagaaaccg ctgctgctaa attcgaacgc cagcacatgg acagc 45

<210> 6

<211> 24

<212> DNA

<213> (F peptide)

<400> 6

gattacaagg atgacgacga tggt 24

<210> 7

<211> 30

<212> DNA

<213> (M peptide)

<400> 7

gagcagggtc tcatctctga agaggatctg 30

<210> 8

<211> 30

<212> DNA

<213> (K2 peptide)

<400> 8

ggtaaaggtg gtggtggtaa aggtggtggt 30

Claims

1. A peptide chain tag for effecting protein crosslinking, which is composed of a peptide chain having an amino acid Sequence shown in Sequence No.2 and a peptide chain having an amino acid Sequence shown in Sequence No.3, or a peptide chain having an amino acid Sequence shown in Sequence No.3 and a peptide chain having an amino acid Sequence shown in Sequence No. 4; in the peptide chain, the site of lysine and/or glutamine is near the N-terminal; the protein with the peptide chain label can realize ordered protein crosslinking by utilizing glutamine transaminase (TGase).

2. A nucleic acid encoding the peptide tag according to claim 1, which consists of a peptide chain having a deoxyribonucleotide Sequence such as shown in Sequence No.6 and a deoxyribonucleotide Sequence such as shown in Sequence No.7, or a peptide chain having a deoxyribonucleotide Sequence such as shown in Sequence No.7 and a deoxyribonucleotide Sequence such as shown in Sequence No. 8.

3. A method of effecting protein cross-linking, comprising:

wherein the peptide chain tag is the peptide chain tag of claim 1 or the peptide chain tag encoded by the nucleic acid of claim 2.

4. The method according to claim 3, wherein, in step N, transglutaminase is added to a substrate solution containing a peptide-tagged protein to perform a crosslinking reaction to produce a crosslinked protein; the usage amount of the glutamine transaminase is 1-3 wt% based on the volume of the substrate solution; and/or the substrate solution is formed by dissolving the protein with the peptide chain tag in a buffer solution, and the content of the protein with the peptide chain tag is 5-10mg/mL based on the volume of the substrate solution; the buffer solution is a Tris-HCl solution of 0.05 mol/L; in step N, the temperature of the crosslinking is 20-30 ℃; and/or the crosslinking time is 4 to 12 hours.

5. The method of claim 3, wherein the step M comprises:

6. The method of claim 4, wherein the step M comprises:

7. The method of claim 5, wherein the host cell is E.coli; and/or, the vector is a pET32b (+) vector; and/or, the inducer is IPTG.

8. The method of claim 7, wherein the host cell is Escherichia coli BL21(DE 3); and/or the addition amount of the IPTG is 0.05 percent of the volume of the fermentation liquor.

9. The method of claim 6, wherein the host cell is E.coli; and/or, the vector is a pET32b (+) vector; and/or, the inducer is IPTG.

10. The method of claim 9, wherein the host cell is escherichia coli BL21(DE 3); and/or the addition amount of the IPTG is 0.05 percent of the volume of the fermentation liquor.

11. The method according to any one of claims 5 to 10, wherein in step D, the expression strain is inoculated into a culture medium for fermentation culture, and then an inducer is added for inducible expression culture; the culture medium is an LB culture medium or a TB culture medium; and/or the temperature of the fermentation culture is 37 ℃.

12. The method of claim 11, wherein the fermentation culture is carried out in a shaker; the shaker speed was 180 rpm.

13. The method of claim 11, wherein the expression strain is inoculated into a culture medium for fermentation culture, and when the OD600 reaches 0.8-1.0, an inducer is added for induction expression culture; the temperature of the induced expression culture is 15-25 ℃, and the time of the induced expression culture is 12-20 hours.

14. The method of claim 13, wherein the inducible expression culture is performed in a shaker; the shaker speed was 180 rpm.

15. The method according to any one of claims 5 to 10, wherein in step D, the method for separation and purification comprises affinity purification; and (4) carrying out affinity purification by adopting a nickel column.

16. The method of claim 15, wherein the affinity purification process comprises removing the contaminating proteins with a 0.05mol/L Tris-HCl solution and eluting with a 0.1mol/L imidazole solution.

17. The method according to claim 11, wherein in step D, the separation and purification method comprises an affinity purification method; and (4) carrying out affinity purification by adopting a nickel column.

18. The method of claim 17, wherein the affinity purification process comprises removing the contaminating proteins with a 0.05mol/L Tris-HCl solution and eluting with a 0.1mol/L imidazole solution.

19. The method according to any one of claims 12 to 14, wherein in step D, the method for separation and purification comprises affinity purification; and (4) carrying out affinity purification by adopting a nickel column.

20. The method of claim 19, wherein the affinity purification process comprises removing the contaminating proteins with a 0.05mol/L Tris-HCl solution and eluting with a 0.1mol/L imidazole solution.