CN112094879B - Enzyme-catalyzed C-terminal selective amidation and hydrazidation modification method for polypeptide - Google Patents

Abstract

The invention discloses an enzyme-catalyzed C-terminal selective amidation and hydrazidation modification method of polypeptide. The invention provides a method for carrying out amidation and/or hydrazide modification on a polypeptide C-terminal amino acid, which is to carry out amidation and/or hydrazide modification on the polypeptide C-terminal amino acid by adopting a specific protein; the specific protein is a protein shown as SEQ ID No. 1. The method can realize amidation and/or hydrazide modification of the C-terminal amino acid of the polypeptide, is a water-phase reaction, has mild conditions, selectively modifies the C-terminal, does not affect other modification groups of the side chain, does not need protection of the side chain, has wide substrate spectrum and no limitation of the amino acid sequence of the substrate polypeptide, and can modify any amino acid except proline at the terminal of the modified polypeptide.

Description

Enzyme-catalyzed C-terminal selective amidation and hydrazidation modification method for polypeptide

Technical Field

The invention relates to the field of polypeptide chemistry, in particular to an enzyme-catalyzed C-terminal selective amidation and hydrazidation modification method of a polypeptide.

Background

Terminal functionalization of polypeptides has a very important influence on their biochemical properties. The specific modification of the C end can prolong the in vivo metabolism half-life of the polypeptide, reduce the immunogenicity or reduce the toxic and side effects. In the biochemical property study of polypeptides, researchers often need to add different functional groups (e.g., fluorescent groups, synthetic macromolecules, or immunological labels) to the polypeptide structure. In addition, efficient and accurate methods for protecting, deprotecting and selectively activating the C-terminal of the polypeptide are also urgently needed technologies in industrial polypeptide synthesis production. Therefore, the selective C-terminal modification method of the polypeptide is a hot spot field of the research of the current bioactive macromolecules and is an urgent problem to be solved in the industrial production of the polypeptide.

Due to the complexity of the polypeptide structure, the chemical modification steps are multiple, the yield is low, and the difficulty is high. The enzymatic modification has high position and stereoselectivity, almost no need of additional side chain protection and deprotection, and no racemization side reaction, thereby having great advantages.

Disclosure of Invention

The invention aims to provide an enzyme-catalyzed C-terminal selective amidation and/or hydrazidation modification method for a polypeptide.

In a first aspect, the invention claims a method for amidation and/or hydrazidation modification of the C-terminal amino acid of a polypeptide.

The method for amidating and/or hydrazidating the C-terminal amino acid of the polypeptide, which is claimed by the invention, is to perform amidating and/or hydrazidating modification on the C-terminal amino acid of the polypeptide to be modified by using a specific protein as a catalyst.

Wherein the specific protein is the protein with an amino acid sequence of SEQ ID No. 1.

Further, the method may comprise the steps of: the C-terminal amidated polypeptide to be modified is catalyzed by the specific protein to react with nucleophilic organic amine with a modifying group, so that amidation and/or hydrazidation modification of the C-terminal amino acid of the polypeptide to be modified is realized.

The nucleophilic organic amine with a modifying group may specifically be any one of the following:

in the method, the modified polypeptide C-terminal amino acid is any one of 19 kinds of amino acids constituting the biological protein other than proline (i.e., glycine, alanine, valine, leucine, isoleucine, phenylalanine, tryptophan, serine, tyrosine, cysteine, methionine, asparagine, glutamine, threonine, aspartic acid, glutamic acid, lysine, arginine, or histidine).

Furthermore, the modified polypeptide C-terminal amino acid is the C-terminal 1 st amino acid.

Further, in the method, the side chain protection of the modified polypeptide is not needed in the amidation and/or hydrazide modification of the C-terminal amino acid of the polypeptide to be modified.

Further, in the method, the solvent for carrying out the reaction is water.

Further, in the method, the reaction may be carried out at a temperature of 10 to 70 ℃.

Further, in the method, the temperature at which the reaction is carried out may be 25 ℃.

Further, in the method, the reaction is carried out at a final concentration of 1 to 20mM of the polypeptide to be modified having C-terminal amidation in the reaction system; the final concentration of the nucleophilic organic amine with the modification group in the reaction system is 0.5-2M, and the final concentration of the specific protein in the reaction system is 0.7-700 mug/mL.

Further, in the method, the catalytic reaction is carried out at a final concentration of 5mM of the C-terminally amidated polypeptide to be modified in the reaction system; the final concentration of the nucleophilic organic amine with the modification group in the reaction system is 0.5-2M, and the final concentration of the specific protein in the reaction system is 0.7-700 mu g/mL.

Further, in the method, the reaction is carried out for 3-174 min.

In a particular embodiment of the invention, the N-terminus of the C-terminally amidated polypeptide to be modified is acetylated. It is to be noted that the modified polypeptide substrate in the claimed method does not have to be protected by N-terminal acetylation.

In a particular embodiment of the invention, the C-terminally amidated polypeptide is in the form of: Ac-DFSKX-NH₂(ii) a Wherein Ac represents acetylation; D. f, S, K is an uppercase alphabet representation of amino acids; x is any one of 19 kinds of amino acids constituting the organism protein other than proline. The nucleophilic organic amine with a modifying group is N₂H₄。

In a second aspect, the present invention claims the use of a specific protein as described in the first aspect above or a gene encoding said specific protein or an expression cassette, recombinant vector or recombinant bacterium or transgenic cell line comprising said encoding gene for amidation and/or hydrazidation modification of the C-terminal amino acid of a polypeptide.

In a third aspect, the invention also claims a kit capable of performing amidation and/or hydrazidation modification on the C-terminal amino acid of the polypeptide.

The invention claims a kit capable of amidating and/or hydrazidating a C-terminal amino acid of a polypeptide, comprising a nucleophilic organic amine with a modifying group as described in the first aspect and any one of the following:

(a1) a specific protein as described in the previous first aspect;

(a2) a gene encoding the specific protein;

(a3) expression cassette, recombinant vector or recombinant bacterium or transgenic cell line containing coding gene

Further, the kit may further comprise a readable carrier carrying the method of the first aspect as hereinbefore described. The readable carrier may be a paper or an optical disc or the like, which is carrying the method.

In the second and third aspects, the specific meaning of the amidation and/or hydrazide modification of the C-terminal amino acid of the polypeptide is the same as that described in the first aspect.

Experiments prove that amidation and/or hydrazide modification of the C-terminal amino acid of the polypeptide can be realized by using the method provided by the invention. The invention has the advantages that: (1) water phase reaction under mild condition; (2) c-terminal is selectively modified, and other modifying groups on a side chain are not influenced; (3) the side chain does not need protection; (4) the substrate spectrum is wide, the limitation of the amino acid sequence of the substrate polypeptide is avoided, and the tail end of the modified polypeptide can be modified by any amino acid except proline.

Drawings

FIG. 1 is a plasmid map of pET21a-PAM 12B.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 enzyme-catalyzed C-terminal Selective amidation and hydrazide modification of Polypeptides

In this example, the 1 st amino acid at the C-terminus of the polypeptide was selectively amidated and hydrazide-modified with a polypeptide-terminal amidating enzyme (PAM12B) shown in SEQ ID No. 1.

The reaction process comprises the following steps: catalyzing polypeptide to be modified with C-terminal amidated by PAM12B shown in SEQ ID No.1 and nucleophilic organic amine (NH) with modified group₂R) reaction, thereby realizing amidation and hydrazidation modification of C-terminal amino acid of the polypeptide to be modified.

Wherein the nucleophilic organic amine with the modification group has a general formula of NH₂R can be any one of the following specifically:

preparation of polypeptide terminal amidation enzyme (PAM12B)

Replacing a small fragment between NdeI and XhoI enzyme cutting sites of pET21a (+) plasmid by using a double-stranded DNA molecule shown in SEQ ID No.2 (a coding gene of polypeptide terminal amidating enzyme PAM12B shown in SEQ ID No. 1) to obtain a recombinant expression plasmid pET21a-PAM12B (a plasmid map is shown in figure 1);

pET21a (+) plasmid: novagen, catalog No.: 69740-3 CN.

Coli BL21(DE 3): tiangen Biochemical technology (Beijing) Ltd., Cat No.: CB 105-02.

1. The recombinant expression vector pET21a-PAM12B constructed above is transformed into Escherichia coli BL21(DE3) to obtain a recombinant strain. Recombinant bacteria expression can obtain the recombinant bacteria with His fused at the C terminal₆Labeled PAM 12B.

2. Inoculating the recombinant bacterium obtained in the step 1 into LB liquid culture medium containing 50 mug/mL ampicillin, and culturing at 37 ℃ and 220rpm until the bacterium liquid OD_600nm30ppm of IPTG was added to the culture system at 0.6. degreeCuntil, and the mixture was induced at 30 ℃ and 220rpm for 16 hours.

3. After completion of step 2, the culture system was centrifuged at 8000rpm, and the pellet was collected.

4. After the step 3 is completed, the cells are resuspended in 20mM (pH 7.5) phosphate buffer and then sonicated, wherein the sonication power is 250W and the sonication time is 20min (sonication for 5s, and cessation of 10 s).

5. After completion of step 4, the sonicated product was centrifuged at 12000 Xg for 1h at 4 ℃ and the supernatant collected.

6. Purifying the supernatant obtained in the step 5 by using a nickel column (GE Healthcare Bio-science AB, the product number is 17-5248-01), loading the supernatant into the nickel column, eluting by using a mobile phase A and a mobile phase B in sequence, and collecting the eluent of the mobile phase B; desalting the eluate with desalting column (GE Healthcare Bio-science AB, cat # 17-1408-01), and eluting with mobile phase C to obtain purified protein solution.

Mobile phase A: 20mM (pH 7.5) phosphate buffer (containing 20mM imidazole and 0.5M NaCl); mobile phase B: 20mM (pH 7.5) phosphate buffer (containing 200mM imidazole and 0.5M NaCl); mobile phase C: 20mM (pH 7.5) phosphate buffer.

7. And (4) concentrating the purified protein solution obtained in the step (6) by using a protein concentration tube, and then quantifying the protein concentration by using a BCA method, wherein the protein concentration of PAM12B is 7.0 mg/ml.

Secondly, PAM12B carries out selective amidation and/or hydrazidation modification on C-terminal of polypeptide

Because the substrate binding pocket of PAM12B mainly recognizes the 1-and 2-amino acids at the C-terminus of the polypeptide, and further selectively catalyzes amidation modification of the 1-amino acid at the C-terminus of the polypeptide after binding to the amino acid, there are two polypeptides to be modified whose C-terminus is amidated, one of which is Ac-DFSKX and the other is Ac-DFSXL. Wherein Ac represents acetylation; D. f, S, K, L is an capitalized letter representation of amino acids; x may be specifically any of 20 amino acids constituting the biological protein. Two types of test polypeptides were obtained by artificial synthesis.

The nucleophilic organic amine with a modifying group to be tested is in particular N₂H₄。

The reaction system and reaction conditions are shown in table 1. The side chain of the polypeptide is not protected in the reaction process.

TABLE 1 reaction System and reaction conditions

Solvent(s)	H2O
		Substrate concentration (mM)	1-20
Reaction volume (μ l)	50-100
		Hydrazine concentration (M)	0.5-2
Enzyme amount (μ g)	0.1-10
		Temperature (. degree.C.)	10-70

After the reaction is finished, the yield of the hydrazide modification of the 1 st amino acid at the C terminal of the polypeptide is determined and detected by an HPLC method.

1、Ac-DFSKX-NH₂+N₂H₄→Ac-DFSKX-N₂H₃+NH₃

The experimental protocol and the measurement results are shown in table 2.

TABLE 2 modification of the C-terminal amino acid X at position 1 of the polypeptide by hydrazidation

Note: the reaction system is 100 mul; the reaction temperature is 25 ℃; capitalized letter representation of amino acids in the Ac-DFSKX column; x may specifically be any of 20 amino acids constituting the biological protein; p₅NH₂Represents Ac-DFSKX-NH 2; -means that the method is not amenable to modification when the amino acid at position 1 of the C-terminus of the polypeptide substrate is P; when the 1-amino acid at the C-terminal end of the polypeptide substrate is N or Q, the method generates byproducts when the method is modified.

2、Ac-DFSXL-NH₂+N₂H₄→Ac-DFSXL-N₂H₃+NH₃

The experimental protocol and the measurement results are shown in table 3.

TABLE 3 modification of the C-terminal 1 st amino acid L of the polypeptide by hydrazidation

Note: the reaction system is 100 mul; the reaction temperature is 25 ℃; capitalized letters in the Ac-DFSXL column are capitalized letter representations of amino acids; x may specifically be any of 20 amino acids constituting the biological protein; p₅NH₂Represents Ac-DFSXL-NH 2.

In both of the above experimental protocols, it was found that the amino acids at the other positions of the modified polypeptide were not modified by hydrazide formation, except for the amino acid at position 1 at the C-terminus (except for the cases where the C-terminus is P, N and Q).

And (4) conclusion: the method is characterized in that a substrate of the modified enzyme is screened, except proline, high-efficiency hydrazide labeling can be realized for polypeptides at different C terminals, and the nucleophilicity of hydrazine groups is stronger than that of ammonia and water, so that the hydrazide rate is higher than that of hydrolysis reaction in a water phase, a dynamic equilibrium reaction tends to generate hydrazide products, and a series of catalytic labeling of hydrazide derivatives can be realized based on the same principle.

<110> institute for microbiology of Chinese academy of sciences

<120> enzyme-catalyzed C-terminal selective amidation and hydrazidation modification method of polypeptide

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Pro Asp Asp Ala Tyr Leu Val Arg Arg Leu Arg Asp Ala Gly Ala Val

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Asn Ser Ile Ser Gly Trp Ser Ala Arg Gly Gly Gln Thr Arg Asn Pro

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Tyr Arg Pro Ser His Ser Pro Cys Gly Ser Ser Ser Gly Ser Ala Val

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Gly Cys Ile Val Cys Pro Ala Ala Ile Asn Gly Val Val Gly Leu Lys

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Pro Thr Val Gly Leu Val Ser Arg Asp Gly Ile Ile Pro Ile Ser Phe

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Ala Val Leu Thr Ala Ile Ala Gly Arg Asp Pro Ala Asp Pro Ala Thr

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Ala Thr Met Pro Gly Arg Ala Val Tyr Asp Tyr Thr Ala Arg Leu Asp

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ccagatgcgc tgaaagaggc cgcagagcgt gatcgtgaac gtcgtgacgg tcgtctgcgt 240

ggtccgctgc acggtatccc actgctgttg aaagacaaca tcaacgccgc accgatggcg 300

acgtctgcgg gtagcctggc actgcaagac ttccgtccgg atgatgcgta tctggttcgt 360

cgcctgcgtg atgcgggtgc agtggttctg ggtaagacga atttgtctga atgggggaac 420

ttccgtagca ataactccat ctccggttgg agcgcgcgtg gtggccagac ccgtaatccg 480

taccgcccca gccacagccc gtgtggcagc agcagcggta gcgccgttgc agttgcggca 540

aacctggcga gcgtcgcgat tggcaccgaa accgacggct gcattgtctg cccggcagcg 600

atcaatggtg tcgtaggtct gaaacctacc gtgggtctgg tcagccgcga tggtatcatt 660

ccgattagct tcagccaaga tacgcctggt ccgatggcgc gtagcgttgc cgacgctgcg 720

gctgtgctga cggcgattgc aggtcgcgat cctgcggacc cggcgaccgc gacgatgccg 780

ggtcgcgcgg tttacgacta tacggcccgt ctggacccgc agggcctgcg tggcaagcgc 840

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cgttatttca acacgcatcg tgcaccgttg cgctcgttgg cggacctgat tgcattcaac 1080

caagctcaca gcaagcagga gctggccttg tttggtcaag aactgctggt cgaggcggat 1140

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Claims (8)

1. A method for carrying out hydrazide modification on C-terminal amino acid of polypeptide adopts specific protein as a catalyst to carry out hydrazide modification on the C-terminal amino acid of the polypeptide to be modified;

the specific protein is the protein with the amino acid sequence of SEQ ID No. 1;

the reaction solvent for the hydrazide modification is water.

2. The method of claim 1, wherein: the method comprises the following steps: catalyzing the polypeptide to be modified with amidated C terminal by the specific protein to react with nucleophilic organic amine with a modifying group, thereby realizing hydrazide modification of C terminal amino acid of the polypeptide to be modified;

the nucleophilic organic amine with a modification group is any one of the following:

。

3. the method of claim 1, wherein: in the method, the modified polypeptide C-terminal amino acid is any one of other 19 amino acids which form organism proteins besides proline;

in the method, the modified polypeptide C-terminal amino acid is the 1 st amino acid at the C terminal.

4. The method of claim 3, wherein: in the method, side chain protection is not required for the modified polypeptide.

5. The method of claim 1, wherein: in the method, the reaction is carried out at a temperature of 10 to 70 ℃.

6. The method of claim 2, wherein: in the method, the reaction is carried out at a final concentration of 1 to 20mM of the polypeptide to be modified having C-terminal amidation in the reaction system; the final concentration of the nucleophilic organic amine with the modification group in the reaction system is 0.5-2M, and the final concentration of the specific protein in the reaction system is 0.7-700 mu g/mL.

7. The method of claim 1, wherein: in the method, the reaction is carried out for 3-174 min.

8. The application of a specific protein or a coding gene of the specific protein or an expression cassette, a recombinant vector or a recombinant bacterium containing the coding gene or a transgenic cell line in the hydrazide modification of the C-terminal amino acid of the polypeptide;

the specific protein is the protein with the amino acid sequence of SEQ ID No. 1;

the reaction solvent for the hydrazide modification is water.

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