Unnatural amino acid, application thereof, recombinant protein containing unnatural amino acid and recombinant protein conjugate

文档序号：182664 发布日期：2021-11-02 浏览：44次中文

阅读说明：本技术 一种非天然氨基酸及其应用、包含其的重组蛋白以及重组蛋白偶联物 (Unnatural amino acid, application thereof, recombinant protein containing unnatural amino acid and recombinant protein conjugate ) 是由杨金纬叶诚浩夏钢霍鹏超丁文陈龙飞焦琳黄浩衡新宫丽颖祝静静于 2021-07-29 设计创作，主要内容包括：本发明提供了一种非天然氨基酸,为具有如式(Ⅰ)所示结构的化合物或其对映异构体。本发明还提供了所述非天然氨基酸的应用。进一步地,本发明还提供了一种包含所述非天然氨基酸的重组蛋白以及由所述重组蛋白制得的蛋白偶联物。本发明提供的非天然氨基酸制备简便,安全性好,插入蛋白时不易失活,与偶联部分的结合率高,所得偶联物的稳定性也更高,本发明提供的非天然氨基酸可应用至众多领域,尤其是在制备重组蛋白或重组蛋白偶联物中,(The invention provides an unnatural amino acid which is a compound with a structure shown as a formula (I) or a compound thereofEnantiomers. The invention also provides the application of the unnatural amino acid. Further, the present invention also provides a recombinant protein comprising the unnatural amino acid and a protein conjugate prepared from the recombinant protein. The unnatural amino acid provided by the invention has simple preparation, good safety, difficult inactivation when protein is inserted, high combination rate with a coupling part, and higher stability of the obtained conjugate, can be applied to a plurality of fields, particularly in the preparation of recombinant protein or recombinant protein conjugate,)

1. An unnatural amino acid which is characterized in that the unnatural amino acid is a compound having a structure shown as a formula (I) or an enantiomer thereof,

wherein X and Z each independently represent a substituted or unsubstituted C0-C20 straight or branched alkylene group, one or more-CH groups therein₂Optionally substituted by one or more of-O-, -S-, -NH-, -C (O) -, -S (O) -, Y represents-C (O) -, -S (O) -, or-CH₂-, and A represents a substituted or unsubstituted C6-C20 aryl group;

when X, Z and A each independently represent a substituted group, the substituent is selected from one or more of hydroxyl, mercapto, halogen, nitro, cyano, alkyl, alkenyl, alkynyl, alkoxy, acyl, amido, carboxyl, ester, amino, sulfonyl, sulfinyl, cycloalkyl, heterocyclyl, aryl, heteroaryl.

2. The unnatural amino acid according to claim 1, wherein said substituents are selected from the group consisting of one or more of hydroxy, mercapto, halogen, nitro, cyano, C1-C6 alkyl, C1-C6 alkoxy, acyl, amido, carboxy, ester, amino, sulfonyl, sulfinyl, C3-C8 cycloalkyl, C3-C8 heterocyclyl, C6-C20 aryl, C4-C10 heteroaryl;

preferably, X and Z each independently represent a C0-C10 linear or branched alkylene group, preferably a C0-C6 linear alkylene group, of which one or more-CH groups₂-may optionally be replaced by one or more of-O-, -S-, -NH-; more preferably, X and Z are not both C0 alkylene; and/or

The A represents a substituted or unsubstituted C6-C10 aryl group, and more preferably, the A represents a substituted or unsubstituted phenyl or naphthyl group.

3. The unnatural amino acid according to claim 1 or 2, wherein said unnatural amino acid is a compound having the structure represented by formula (I-1),

wherein X, Z and A are each independently as defined in claim 1 or 2;

preferably, the unnatural amino acid is a compound having a structure as shown in formula (I-2),

wherein X is as defined in claim 1 or 2;

R₁and R₂Each independently represents hydrogen, hydroxyl, sulfydryl, halogen, nitro, cyano, C1-C6 alkyl, C1-C6 alkoxy, acyl, amido, carboxyl, ester group, amino, sulfonyl, sulfinyl, C3-C8 naphthenic base, C3-C8 heterocyclic group, C6-C20 aryl or C4-C10 heteroaryl.

4. The unnatural amino acid as claimed in claim 3, wherein the unnatural amino acid is a compound having a structure represented by formula (I-3), formula (I-4), formula (I-5) or formula (I-6),

wherein X' represents a linear alkylene group having 0 to 6 carbon atoms, preferably having 0 to 4 carbon atoms, wherein one or more-CH groups₂-is optionally substituted with-O-and/or-NH-;

the R is₁And R₂Each independently of the other asAs defined in claim 3.

5. The unnatural amino acid according to any of claims 1 to 4, wherein said unnatural amino acid is a compound having one of the following structures:

6. use of the unnatural amino acid of any one of claims 1 to 5 in the preparation of a recombinant protein or recombinant protein conjugate; preferably, the recombinant protein is recombinant human growth hormone; alternatively, preferably, the recombinant protein conjugate is a recombinant human growth hormone-polyethylene glycol conjugate.

7. A recombinant protein characterized in that at least one position in the amino acid sequence of the recombinant protein is the unnatural amino acid as claimed in any one of claims 1 to 5.

8. A recombinant protein conjugate comprising the end carbonyl group of the unnatural amino acid of claim 7 and an "NH" group₂The coupling moiety of the-O- "terminal group forms an oxime bond.

9. The recombinant protein conjugate according to claim 8, wherein the recombinant protein is recombinant human growth hormone, and the recombinant protein conjugate contains "NH₂The coupling moiety of the-O- "terminal group is a compound containing" NH "-" terminal group₂-O- "terminal polyethylene glycol, preferably polyethylene glycol with molecular weight of 10-100 KD; more preferably, the amino acid sequence of the recombinant human growth hormone is shown as SEQ ID NO. 1; further preferably, the non-natural amino acid according to any of claims 1 to 5 is comprised at position 107 in the amino acid sequence SEQ ID No. 1.

10. Use of the recombinant protein conjugate of claim 9 in the manufacture of a medicament for the treatment of growth and development disorders caused by insufficient endogenous growth hormone secretion, growth and development disorders caused by turner's syndrome, or adult growth hormone deficiency.

Technical Field

The invention relates to the field of biological pharmacy, in particular to an unnatural amino acid, a recombinant protein containing the unnatural amino acid and a conjugate formed by the recombinant protein.

Background

By introducing the unnatural amino acid containing special groups into the protein, various scientific researches and product development and applications can be realized, for example, the photosensitive unnatural amino acid is introduced into the protein, or the unnatural amino acid is specially marked, so that the interaction between the proteins can be conveniently researched; for another example, the enzyme is directionally transformed by using unnatural amino acids to improve the enzyme activity and the stability of the enzyme, or to facilitate the efficient immobilization of the enzyme; for another example, a safe live bacterial or live viral vaccine can be prepared by utilizing the feature that unnatural amino acid insertion cannot be achieved in a conventional host. An important application of introducing unnatural amino acids into proteins by using codon expansion technology is to perform site-directed modification on proteins, change the characteristics of the proteins such as function, stability and half-life period, and can be used for developing innovative biological drugs. At present, a series of achievements exist, such as PEG fixed-point coupling recombination human growth hormone preparation long-acting recombinant protein, micromolecule toxin fixed-point coupling monoclonal antibody development of antibody coupling drugs and the like. It follows that unnatural amino acids have a very important role and a very wide range of applications.

The prior art (e.g. CN 102838671B, CN 106146663a, j.am. chem.soc.2009,131,8720, etc.) discloses an unnatural amino acid Lys-azido, the structural formula of which is shown below:

lys-azido terminal azide structure (-N)₃) Capable of reacting with alkyne-containing structures (e.g. BCN, i.e.) The conjugate (for example, Chinese patent CN 103153927B) is obtained by chemically linking modified carrier drugs (such as PEG and the like) and has high specific selectivity. However, the coupling method and the chemical modification method need to introduce an alkyne structure with higher cost, and an acceptable drug antibody coupling ratio can be obtained only when the equivalent is larger, so that the corresponding production cost is increased, and the process is more complicatedThe process conditions are harsh.

Therefore, it is necessary to develop an unnatural amino acid with a novel structure, easy preparation, and low cost, so as to expand the variety and applications of the amino acid.

Disclosure of Invention

To overcome the above-mentioned disadvantages of the prior art, it is an object of the present invention to provide an unnatural amino acid.

Another object of the present invention is to provide the use of said unnatural amino acid.

It is also an object of the present invention to provide a recombinant protein and a recombinant protein conjugate.

The unnatural amino acid provided by the invention is a compound with a structure shown in a formula (I) or an enantiomer thereof,

when X, Z and A each independently represent a substituted group, the substituent may be selected from one or more of hydroxyl, thiol, halogen, nitro, cyano, alkyl, alkenyl, alkynyl, alkoxy, acyl, amide, carboxyl, ester, amino, sulfonyl, sulfinyl, cycloalkyl, heterocyclyl, aryl, heteroaryl.

As shown in example 11, the present inventors found that the azide structure (-N) at the Lys-azido terminal is expensive and complicated₃) Is easily reduced to amino structure (-NH) when inserted into protein₂) Thereby losing the activity of coupling and thus reducing the yield in the process preparation.

The non-natural amino acid provided by the invention introduces carbonyl as an active reaction group at the tail end, so that the structure is novel, the preparation is simple and convenient, the coupling condition is mild, the production cost is low, and the loss of the reaction activity caused by the structural change is not easy to occur when the non-natural amino acid is inserted into a protein sequence; the unnatural amino acid provided by the invention also comprises an aryl connected with a terminal carbonyl group, the stability of the obtained conjugate can be further enhanced by introducing the aryl, and the conjugate is not easy to decompose even under the condition of lower pH. In addition, the unnatural amino acid provided by the invention also comprises an alkylene group with a certain chain length, so that the compound has better flexibility and is easier to form various conjugates.

Among the unnatural amino acids provided by the present invention, "C0-Cn" includes C0-C1, C0-C2, and … … C0-Cn, and when C0 is represented, this means that the group is not present, and C atoms at both ends are directly bonded. For example, the "C0-C6" group refers to a moiety having 0-6 carbon atoms, i.e., the group is absent, contains 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms; the "C6-C10" group refers to 6-10 carbon atoms in the moiety, i.e., 6, 7, 8, 9, or 10 carbon atoms.

In the non-natural amino acids provided herein, "aryl" refers to a carbocyclic aromatic system containing one or two rings, wherein the rings may be joined together in a fused fashion. "aryl" includes monocyclic or bicyclic aryl groups such as phenyl, naphthyl, tetrahydronaphthyl aromatic groups. Preferably aryl is C6 to C10 aryl, more preferably aryl is phenyl and naphthyl, most preferably phenyl.

In some preferred embodiments according to the present invention, the substituent may be selected from one or more of hydroxyl, thiol, halogen, nitro, cyano, C1-C6 alkyl, C1-C6 alkoxy, acyl, amide, carboxyl, ester, amino, sulfonyl, sulfinyl, C3-C8 cycloalkyl, C3-C8 heterocyclic, C6-C20 aryl, and C4-C10 heteroaryl.

In some preferred embodiments according to the invention, X and Z may each independently represent a C0-C10 linear or branched alkylene group, one or more of which are-CH₂-may optionally be replaced by one or more of-O-, -S-, -NH-; in some more preferred embodiments according to the invention, X and Z may each independently represent a C0-C6 linear alkylene group, one or more-CH therein₂-may optionally be replaced by one or more of-O-, -S-, -NH-; in some more preferred embodiments according to the invention, said X and Z are not both C0 alkylene, i.e. the X and Z groups cannot be both absent.

In some preferred embodiments according to the present invention, the a may represent a substituted or unsubstituted C6 to C10 aryl group, and more preferably, the a may represent a substituted or unsubstituted phenyl or naphthyl group.

In some preferred embodiments according to the present invention, the unnatural amino acid can be a compound having a structure as shown in formula (I-1),

wherein X, Z and A are each independently as defined in any one of the preceding claims.

In some preferred embodiments according to the present invention, the unnatural amino acid can be a compound having a structure as shown in formula (I-2),

wherein X is as defined in any of the above claims;

R₁and R₂Each independently represents hydrogen, hydroxyl, sulfydryl, halogen, nitro, cyano, C1-C6 alkyl, C1-C6 alkoxy, acyl, amido, carboxyl, ester group, amino, sulfonylA sulfinyl group, a C3-C8 cycloalkyl group, a C3-C8 heterocyclic group, a C6-C20 aryl group or a C4-C10 heteroaryl group.

In some more preferred embodiments according to the present invention, the unnatural amino acid is a compound having a structure represented by formula (I-3), formula (I-4), formula (I-5), or formula (I-6),

wherein X' represents a linear alkylene group having 0 to 6 carbon atoms, more preferably a linear alkylene group having 0 to 4 carbon atoms, of which one or more-CH groups₂-is optionally substituted with-O-and/or-NH-;

the R is₁And R₂Each independently as defined in any of the above claims.

The unnatural amino acids provided herein include optically pure enantiomers and racemates.

In some more preferred embodiments according to the invention, the unnatural amino acid provided herein is a compound having one of the following structures:

the invention also provides the application of the unnatural amino acid in any technical scheme in preparation of recombinant protein or recombinant protein conjugate.

In the application of the present invention, the recombinant protein may be obtained by inserting the unnatural amino acid described in any of the above embodiments into any kind of protein commonly used in the art at any position and in any number. The recombinant protein conjugate may be any recombinant protein obtained after conjugation with a conjugation moiety common in the art, wherein the conjugation moiety may include, but is not limited to, one or more of a polymer (e.g., polyethylene glycol of any molecular weight), a protein, a polypeptide, or a small molecule drug.

In some preferred embodiments according to the present invention, the recombinant protein may be a recombinant human growth hormone, and the recombinant protein conjugate may be a recombinant human growth hormone-polyethylene glycol conjugate.

The invention also provides a recombinant protein, wherein at least one site in the amino acid sequence of the recombinant protein is the unnatural amino acid described in any one of the technical schemes.

Further, the recombinant protein can have a structure shown in a formula (II),

in formula (II), D represents a residue of an unnatural amino acid except for the aminocarboxylic acid moiety as described in any of the above embodiments, and P₁、P₂Respectively represents the connection part of the amino group and the carboxyl group of the unnatural amino acid in the amino acid sequence.

The recombinant protein provided by the invention can be prepared by using a preparation method common in the field, and comprises the steps of cloning and expressing the recombinant protein containing the unnatural amino acid by using a gene codon expansion technology.

The recombinant protein provided by the invention can be obtained by inserting the unnatural amino acid described in any one of the above technical schemes into any kind of protein commonly used in the field at any position and in any number, such as recombinant human growth hormone.

The invention also provides a recombinant protein conjugate, wherein the recombinant protein is characterized in that the terminal carbonyl of the unnatural amino acid in the recombinant protein according to any one of the technical schemes is combined with the conjugate containing' NH₂The coupling moiety of the-O- "terminal group forms an oxime bond.

Further, the recombinant protein conjugate can have a structure shown in a formula (III),

in formula (III), D' represents the residue of the recombinant protein according to any one of the preceding claims from which the terminal carbonyl moiety of the unnatural amino acid has been removed, and D "represents the coupling moiety from which" NH "has been removed₂-O- "terminal residue.

In the recombinant protein conjugates provided by the invention, the conjugation moiety may comprise one or more of a polymer (e.g., polyethylene glycol of any molecular weight), a protein, a polypeptide, or a small molecule drug. The different types of coupling moieties may be coupled to the recombinant protein alone or may be linked to the recombinant protein after the different types of coupling moieties have been linked.

In some preferred embodiments according to the present invention, the recombinant protein may be a recombinant human growth hormone, said protein containing "NH₂The coupling moiety of the-O- "terminal group may be a linker containing" NH "-" group₂-O- "terminal polyethylene glycol.

In some more preferred embodiments according to the invention, the compound contains "NH₂The polyethylene glycol of the-O- "terminal group has the following structural formula:

wherein it contains "NH₂The molecular weight of the-O- "terminal polyethylene glycol can be 10-100 KD, including but not limited to about 10KD, 20KD, 30KD, 40KD, 50KD, 60KD, 70KD, 80KD, 90KD, 100KD or any combination of molecular weight regions. Preferably, containing "NH₂The molecular weight of the polyethylene glycol with the end group of-O- "can be 20-50 KD.

In some more preferred embodiments according to the present invention, the amino acid sequence of the recombinant human growth hormone is as set forth in SEQ ID NO 1; further preferably, the non-natural amino acid corresponds to position 107 in the amino acid sequence SEQ ID NO 1 as defined in any one of the above claims.

When the recombinant protein is recombinant human growth hormone, the invention also provides the application of the recombinant protein conjugate in any one of the technical schemes in preparing a medicament for treating growth and development disorder caused by endogenous growth hormone hyposecretion, growth and development disorder caused by Turner's syndrome or adult growth hormone deficiency.

The technical scheme provided by the invention has the following advantages:

(1) the terminal carbonyl group and the aryl group connected with the terminal carbonyl group are introduced into the structure of the unnatural amino acid, so that compared with the existing terminal azido unnatural amino acid (such as Lys-azido), the unnatural amino acid is simpler and more convenient to prepare, has better safety, is not easy to inactivate when being inserted into protein, has higher binding rate with a coupling part, and has higher stability of the obtained conjugate.

(2) The unnatural amino acid provided by the invention can have the property of amino acid as an amino acid derivative, thereby expanding the potential variety of the amino acid and enabling the unnatural amino acid to be applied to a plurality of fields as the amino acid derivative, particularly in the preparation of recombinant proteins or recombinant protein conjugates.

(3) The unnatural amino acid provided by the invention can be successfully identified and inserted into protein in a prokaryotic expression system and a eukaryotic expression system, so that the protein containing the unnatural amino acid at a specific site is generated, and thus, recombinant proteins with different physicochemical properties and biochemical activities can be formed, and the variety and potential application range of the protein are expanded. In addition, the unnatural amino acid has higher expression efficiency in protein and better practicability.

(4) The recombinant protein provided by the invention contains the unnatural amino acid, and the contained terminal carbonyl active group can conveniently form a protein conjugate (or conjugate), such as a polyethylene glycol conjugate, a polyethylene glycol-active drug conjugate and the like. These protein conjugates may have improved biological activity (e.g., anti-tumor activity) due to their engineered properties.

(5) The invention also provides a novel protein conjugate platform, which can realize the combination of multiple proteins and multiple conjugate parts through novel unnatural amino acids contained in the proteins and the linked conjugate parts.

Drawings

FIG. 1 is a map of recombinant human growth hormone expression plasmid pET21-rhGH107 in example 8.

FIG. 2 is an SDS-PAGE of the fermentation products obtained by adding different types of unnatural amino acids according to example 8, wherein the lanes are shown below: lane 1: protein molecular weight Marker; lane 2: wild-type recombinant human growth hormone; lane 3: feeding a recombinant human growth hormone expression product of NBOK; lane 4: feeding a recombinant human growth hormone expression product of NPAK; lane 5: feeding a recombinant human growth hormone expression product of NBPK; lane 6: feeding a recombinant human growth hormone expression product of NBGK; lane 7: feeding a recombinant human growth hormone expression product of NPOK; lane 8: feeding a recombinant human growth hormone expression product of NPOK-2; lane 9: feeding a recombinant human growth hormone expression product of NBGK-2; lane 10: recombinant human growth hormone expression product without any unnatural amino acids added.

FIG. 3 is an SDS-PAGE of the coupling product of the recombinant human growth hormone containing unnatural amino acids and PEG in example 8, wherein the lanes are shown as follows: lane 1: a molecular weight Marker; lane 2: wild-type recombinant human growth hormone; lane 3: coupling product of recombinant human growth hormone containing NBOK and PEG; lane 4: coupling product of NPAK-containing recombinant human growth hormone and PEG; lane 5: coupling product of recombinant human growth hormone containing NBPK and PEG; lane 6: coupling product of recombinant human growth hormone containing NBGK and PEG; lane 7: coupling product of NPOK-containing recombinant human growth hormone and PEG; lane 8: the NPOK-2-containing recombinant human growth hormone is coupled with PEG.

FIG. 4 is an SDS-PAGE of purified PEG-conjugated recombinant human growth hormone of example 8, wherein the lanes are shown below: lane 1: a molecular weight Marker; lane 2: recombinant human growth hormone standards; lane 3: coupling product of NPOK-containing recombinant human growth hormone and PEG; lane 4: NBOK-containing recombinant human growth hormone is coupled with PEG.

FIGS. 5A-5G is the cell activity profile of example 8, wherein FIG. 5A is the cell activity profile of the middle hospital rhGH standard; FIG. 5B isA plot of cell activity of; FIG. 5C isA plot of cell activity of; FIG. 5D is a graph of the cellular activity of rhGH (NPOK); FIG. 5E is a graph showing the cell activity of PEG-rhGH (NPOK); FIG. 5F is a graph showing the cellular activity of rhGH (NBOK); FIG. 5G is a graph showing the cell activity of PEG-rhGH (NBOK).

FIG. 6 is a fluorescence micrograph of the transient expression of the inserted unnatural amino acid in CHO cells of example 9.

FIG. 7 is a map of expression plasmid pCDNA3.1-Trastuzumab-UAG142 in example 10.

FIGS. 8A-8C are HIC-HPLC chromatograms of example 10 after conjugation of NBPK-containing trastuzumab, NBOK-containing trastuzumab, and NPOK-2-containing trastuzumab with the toxin, respectively.

FIG. 9 is a graph showing the effect of NBOK-containing trastuzumab and DM 1-modified NBOK-containing trastuzumab on BT-474 cells in example 10.

FIGS. 10A and 10B are the mass spectra of rhGH mutated at position 140 to Lys-azido and rhGH mutated at position 140 to NBOK in example 11, respectively.

FIGS. 11A and 11B are SDS-PAGE electrophoresis of the coupling of rhGH mutated at position 140 to Lys-azido and rhGH mutated at position 140 to NBOK, respectively, with PEG of 30KD in example 11. Wherein each lane of FIG. 11A is shown as follows: lane 1: a molecular weight Marker; lane 2: wild-type recombinant human growth hormone; lane 3: rhGH-Lys-azido-140; lane 4: rhGH-Lys-azido-140: 30K BCN-PEG is a product of coupling reaction for 72 hours at a molar ratio of 1:15 (the same applies below); lane 5: rhGH-Lys-azido-140: the 30K BCN-PEG is a product of 1:25 coupling reaction for 72 h; FIG. 11B shows the following lanes: lane 1: a molecular weight Marker; lane 2: rhGH-NBOK-140; lane 3: rhGH-NBOK-140: the 30K BCN-PEG is a product of 1:15 coupling reaction for 6 hours; lane 4: rhGH-NBOK-140: the 30K BCN-PEG is a product of 1:15 coupling reaction for 9 h; lane 5: rhGH-NBOK-140: the 30K BCN-PEG is a product of 1:15 coupling reaction for 12 hours; lane 6: rhGH-NBOK-140: the 30K BCN-PEG is a product of 1:15 coupling reaction for 24 hours; lane 7: rhGH-NBOK-140: the 30K BCN-PEG is the product of 1:15 coupling reaction for 48 h.

Detailed Description

The technical solution of the present invention is further described in detail with reference to the following specific examples.

The reagents or raw materials used in the examples of the present invention are commercially available products unless otherwise specified.

Example 1 preparation of unnatural amino acid NBOK

The structural formula of NBOK is shown below:

the reaction process is shown in the following chart:

the preparation process comprises the following steps:

a) in a reaction flask, p-methylacetophenone (4.0mL,30.0mmol) was added, solvent DCE (50.0mL) was added, NBS (6.41g,36.0mmol) and BPO (0.05g,0.3mmol) were added, and the mixture was refluxed at 80 ℃ for 24 hours, then the vessel was cooled in ice water to precipitate a solid, which was removed by filtration, and then saturated Na was used₂CO₃Washed 3 times, extracted 3 times with DCM, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give the crude product 1-1(5.46g, 85% yield) which was used in the next step without purification.

b) In a reaction flask, the product 1-1(2.73g,12.80mmol) was added, the solvent dioxane (40mL) and water (40mL) was added, calcium carbonate (7.68g,76.8mmol) was added, the mixture was refluxed at 105 ℃ for 24 hours, cooled to room temperature, filtered to remove solids, extracted 3 times with DCM, the organic phases were combined, concentrated under reduced pressure, column chromatographed (eluent: PE: EA ═ 3:1) purification gave the product 1-2(1.80g, 94% yield).

c) In a two-necked reaction flask, p-nitrophenyl chloroformate (2.90g, 14.4mmol) was added, solvent DCM (10.0mL) was added, the temperature was reduced to 0 ℃, product 1-2(1.80g,12.0mmol) and pyridine (1.2mL, 14.4mmol) were added, after stirring at room temperature for 18 hours, saturated sodium carbonate solution (10mL) was added to the reaction solution, extraction was performed with DCM (50mL) 3 times, the organic phases were combined, washed with water 2 times, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, purified by column chromatography (eluent: PE: EA ═ 5:1) purification afforded the product 1-3(3.14g, 83% yield).

d) In a reaction flask, product 1-3(1.26g,4.0mmol) and Fmoc-Lys-OH hydrochloride (1.40g,3.33mmol) were added, solvent dioxane (15mL) and water (5mL) were added, triethylamine (1.2mL,8.3mmol) was added, the mixture was reacted at room temperature for 24 hours, and appropriate 1M HCl solution was added, extracted with DCM, and concentrated under reduced pressure to give crude product 1-4, which was used directly in the next step.

e) In a reaction flask, the product 1-4(1.10g,0.19mmol) was dissolved in DCM (10mL), diethylamine (5.0mL) was added, reacted at room temperature for 6 hours, the product precipitated, filtered and slurried with DCM 3 times to obtain the target product NBOK (1-5,817mg, two-step yield 63%).

¹H-NMR (400MHz, heavy water) δ 8.04(d, J ═ 8.4Hz,2H),7.55(d, J ═ 8.0Hz,2H),5.21(s,2H),3.74(t, J ═ 6.0Hz,1H),3.17(t, J ═ 6.4Hz,2H),2.70(s,3H), 1.95-1.83 (m,2H), 1.62-1.52 (m,2H), 1.47-1.35 (m,2H).

EXAMPLE 2 preparation of the unnatural amino acid NPAK

The structural formula of NPAK is shown below:

the reaction process is shown in the following chart:

the preparation process comprises the following steps:

a) into a reaction flask, p-chloroacetophenone (1.00g,6.47mmol) was added, and diethyl malonate (6.84g,47.70mmol) and KHCO were added under a nitrogen atmosphere₃(0.97g,9.70mmol) and K₂CO₃(1.34g,9.70mmol) followed by the addition of Pd (dba)₂(0.019g,0.030mmol) and P (t-Bu)₃HBF₄(0.021g,0.071mmol), replacing nitrogen after adding, heating to 160 deg.C, and reacting for 40 h. After completion of the TLC detection reaction, water (30mL) was added to the reaction solution, extraction was performed 3 times with EA, the organic phases were combined, washed 2 times with water, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure at 0-5 ℃ to give a colorless transparent liquid, which was purified by column chromatography (eluent: PE: EA: 10:1) to give 2-1(0.80g, yield 60%).

b) LiOH (0.30g,11.64mmol) and water (5.0mL) were added to a reaction flask, ethanol (10mL) was added, product 2-1(0.80g,3.88mmol) was added, after stirring at room temperature for 2 hours, the completion of the reaction was checked by TLC, 2M HCl solution was added to the reaction solution to adjust pH to 1-2, extraction was performed with EA 3 times, the organic phases were combined, washed with water 2 times, dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain product 2-2(0.5g, yield 72%).

c) To a reaction flask, product 2-2(0.20g,1.12mmol) was added followed by N-hydroxysuccinimide (NHS,0.19g,1.68mmol), DIPEA (0.07g, 0.56mmol), and DCM (2.0 mL). And cooling to 0-5 ℃, adding a solution of DCC (0.23g,1.12mmol) and DCM (2.0mL), and reacting for 2 hours under the condition of heat preservation. Warmed to room temperature and stirred overnight. After TLC detection reaction was complete, filtration was carried out, washing was carried out with DCM, the mother liquor was concentrated under reduced pressure, and column chromatography was carried out (eluent: PE: EA: 5:1) to obtain 2-3(0.19g, yield 62%).

d) To a reaction flask, the product 2-3(0.10g,0.36mmol) was added, followed by triethylamine (0.04g,0.36mmol), Fmoc-Lys-OH hydrochloride (0.13g,0.36mmol), dioxane (2.0mL) and water (2.0mL), and the reaction was stirred at room temperature for 18 h. After completion of TLC detection, the reaction was concentrated under reduced pressure, extracted 3 times with EA, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by column chromatography (eluent: DCM: MeOH: 15:1) to give 2-4(0.03g, yield 61%) as an oily liquid product.

e) In a reaction flask, product 2-4(0.08g,0.15mmol), DCM (1.0mL) and piperidine (0.04g, 0.47mmol) were added and stirred at room temperature for 3 h. After TLC detection reaction is completed, concentration is carried out under reduced pressure, the mixture is beaten by petroleum ether (5mL) for 1h, filtration is carried out, a filter cake is beaten by petroleum ether (5mL) for 1h, filtration is carried out, beating is carried out on the filter cake for 4 times by ethanol to remove residual piperidine, and finally off-white solid 2-5(0.02g, yield 43%) is obtained.

¹H-NMR (400MHz, heavy water) δ 7.85(d, J ═ 8.2Hz,2H),7.33(d, J ═ 8.2Hz,2H),3.94(t, J ═ 6.3Hz,1H),3.56(s,2H),3.12(t, J ═ 6.8Hz,2H),2.54(s,3H), 1.80-1.70 (m,2H), 1.54-1.45 (m,2H),1.40-1.224(m,2H).

EXAMPLE 3 preparation of unnatural amino acid NBPK

The structural formula of NBPK is shown below:

the reaction process is shown in the following chart:

the preparation process comprises the following steps:

a) in a reaction flask, p-methylacetophenone (4.0mL,30.0mmol) was added, solvent DCE (50.0mL) was added, NBS (6.41g,36.0mmol) and BPO (0.05g,0.3mmol) were added, and the mixture was refluxed at 80 ℃ for 24 hours. After TLC detection reaction is completed, the container is placed into ice water for cooling, solid is separated out, solid is removed by filtration, and saturated Na is used₂CO₃Washed 3 times, extracted 3 times with DCM, the organic phases were combined, dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give crude product 3-1(5.46g, 85% yield) which was used in the next step without purification.

b) In a reaction flask, NaH (0.58g,14.64mmol, 60%) was added, dried solvent THF (20mL) was added, and ethylene glycol (6.7mL,122.0mmol) was slowly added under ice-cooling, and stirred at room temperature for 1 hour. Then the product 3-1(2.60g,12.2mmol) was added and heated at 70 ℃ under reflux for 48h until the reaction was complete. Slowly cooled in ice bathSaturated NH is dropped₄After quenching NaH with Cl, washing with water, extraction with EtOAc 3 times, combining the organic phases, drying with anhydrous sodium sulfate, filtration, concentration under reduced pressure, and purification by column chromatography (eluent: PE: EA ═ 2:1) yielded the product 3-2(1.39g, yield 59%).

c) In a reaction flask, product 3-2(1.39g,7.2mmol) was added, solvent DCM (10mL) was added, p-nitrophenyl chloroformate (1.74g,8.64mmol) and pyridine (0.7mL,8.64mmol) were added under cooling in an ice bath, and the mixture was stirred at room temperature for 18 hours. After completion of the TLC detection reaction, water was added and the reaction mixture was washed with EtOAc and extracted 3 times, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and purified by column chromatography (eluent: PE: EA ═ 3:1) to give the product 3-3(2.27g, yield 88%).

d) In a reaction flask, the product 3-3(2.27g,6.32mmol) was added, the solvent dioxane (16mL) and water (4mL) was added, Fmoc-Lys-OH hydrochloride (2.13g,5.27mmol) was added, triethylamine (1.85mL,13.2mmol) was added and the reaction was stirred at room temperature for 18 hours to completion. Adding appropriate amount of 1M HCl to adjust pH to about 2, extracting with ethyl acetate, mixing organic phases, adding anhydrous sodium sulfate, drying, filtering, and concentrating under reduced pressure to obtain crude product 3-4, which is directly used in the next step.

e) In a reaction flask, the product 3-4 of the previous step was dissolved in DCM (10mL), and diethylamine (5mL) was added and reacted at room temperature for 6 hours. After TLC detection of completion of the reaction, concentration under reduced pressure and column chromatography (eluent: DCM: MeOH: H)₂O40: 10:1) to give the product as a white solid (3-5,0.95g, 49% yield over two steps).

¹H-NMR (400MHz, heavy water) δ 8.04(d, J ═ 8.0Hz,2H),7.56(d, J ═ 8.0Hz,2H),4.72(s,2H),4.25(s,2H),3.81(s,2H),3.74(t, J ═ 6.0Hz,1H), 3.16-3.08 (m,2H),2.70(s,3H), 1.97-1.79 (m,2H), 1.60-1.48 (m,2H), 1.48-1.35 (m,2H).

Example 4 preparation of the unnatural amino acid NPOK

The structural formula of NPOK is shown below:

the reaction process is shown in the following chart:

the preparation process comprises the following steps:

a) in a reaction flask, triphosgene (BTC,2.18g,7.35mmol) was added, solvent THF (10.0mL) was added, p-hydroxyacetophenone (2.0g,14.7mmol) and pyridine (1.5mL,17.64mmol) were added under cooling in an ice bath, and the mixture was reacted at room temperature for 24 hours. After completion of the TLC check reaction, the appropriate amount of water was added, extracted 3 times with EtOAc and the organic phases were combined. Dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure to give crude 4-1(1.20g) which was used directly in the next step.

b) In a reaction flask, Boc-lysine (1.1g,5.0mmol) was added, solvent DCM (10.0mL) was added, and product 4-1(1.20g) and triethylamine (2mL,15mmol) were added. Stirring at room temperature for 24 hr, detecting by TLC, adding appropriate amount of 1M HCl to adjust pH to weak acidity, extracting with DCM for 3 times, and mixing organic phases. Dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to column chromatography (eluent: DCM: MeOH ═ 5:1) to give the product 4-2(1.70g, yield 84%).

c) In a reaction flask, the product 4-2(1.70g,4.2mmol) was added, solvent DCM (5mL) was added, trifluoroacetic acid (5mL) was added, and the mixture was reacted at room temperature for 1 hour. After TLC detection, the reaction mixture is directly concentrated under reduced pressure and subjected to column chromatography (eluent: DCM: MeOH: H)₂O40: 10:1) gave 4-3(1.19g, 92% yield).

¹H-NMR (400MHz, heavy water) δ 8.09(d, J ═ 8.6Hz,2H),7.31(d, J ═ 8.6Hz,2H),3.78(t, J ═ 6.0Hz,1H),3.27(t, J ═ 6.8Hz,2H),2.70(s,3H), 1.98-1.87 (m,2H), 1.72-1.60 (m,2H), 1.55-1.43 (m,2H).

EXAMPLE 5 preparation of the unnatural amino acid NBGK

The structural formula of NBGK is shown below:

the reaction process is shown in the following chart:

the preparation process comprises the following steps:

a) in a reaction flask, p-methylacetophenone (8.0mL,60.0mmol) was added, solvent DCE (80.0mL) was added, NBS (12.82g,72.0mmol) and BPO (145mg,0.6mmol) were added, and the mixture was refluxed at 90 ℃ for 24 hours. After TLC detection reaction is completed, the container is placed into ice water for cooling, solid is separated out, solid is removed by filtration, and saturated Na is used₂CO₃Wash 3 times, extract 3 times with DCM and combine the organic phases. After drying over anhydrous sodium sulfate, filtration and concentration under reduced pressure, the crude product 5-1(11.12g, yield 87%) was obtained and used in the next step without further purification.

b) In a reaction flask, mixingMS (14g) and LiOH (1.45g,34.54mmol) were dissolved in DMF (70mL), stirred at room temperature for 20min, glycine methyl ester hydrochloride (2.0g,15.7mmol) was added, and after stirring for 45min, product 5-1(4.0g,18.8mmol) was added and stirred at room temperature for 18 h. After TLC monitoring the reaction was complete, the solid was removed by filtration, the filter cake was washed with EA and the filtrate was washed twice with water. Dried over anhydrous sodium sulfate, concentrated under reduced pressure to give crude product 5-2, which was used directly in the next step.

c) In a reaction flask, the product 5-2 in the previous step is dissolved in dioxane (20mL), 1M NaOH is slowly added dropwise, and after 2 hours of reaction, the hydrolysis reaction is monitored by TLC to be completed to obtain the product 5-3. 20mL of saturated NaHCO was added₃After that, Fmoc-OSu dissolved in dioxane (10mL) was slowly added, stirred at room temperature overnight, TLC monitored the completion of the reaction, made weakly acidic with 1M HCl, extracted with EA, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and subjected to column chromatography (eluent: DCM: MeOH 10:1) to obtain 5-4(3.60g, yield 89%).

d) In a reaction flask, product 5-3(3.60g,8.0mmol), NBS (1.10g,9.6mmol), EDCI (1.85g,9.6mmol) and DCM (50mL) as a solvent were added and the mixture was reacted at room temperature for 18 hours. After completion of the reaction monitored by TLC, it was washed 3 times with water, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to give the product 5-5(3.20g, yield 75%).

e) In a reaction flask, the product 5-5(3.20g,6.0mmol) was added, the solvent dioxane (40mL) and water (10mL) was added, Fmoc-Lys-OH hydrochloride (3.0g,7.2mmol) was added, triethylamine (2.0mL,15.0mmol) was added, and the reaction was stirred at room temperature for 18 hours until completion. The pH was adjusted to about 2 by the addition of an appropriate amount of 1M HCl, extracted with EA, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and subjected to column chromatography (eluent: DCM: MeOH: AcOH ═ 20:1:0.5) to give the product 5-5(3.50g, yield 75%).

f) In a reaction flask, the product 5-5 was dissolved in DCM (20mL), diethylamine (20mL) was added and reacted at room temperature for 6 hours. TLC after monitoring the reaction was complete, concentrated under reduced pressure and column chromatographed (eluent: DCM: MeOH: H)₂O30: 10:1) gave the final product as a white powder 5-7(0.55g, 37% yield).

¹H-NMR (400MHz, heavy water) δ 7.98(d, J ═ 8.2Hz,2H),7.50(d, J ═ 8.2Hz,2H),3.84(s,2H),3.71(s,1H),3.31(s,2H),3.17(t, J ═ 6.9Hz,2H),2.67(s,3H), 1.97-1.73 (m,2H), 1.58-1.45 (m,2H),1.44-1.27(m,2H).

EXAMPLE 6 preparation of the unnatural amino acid NPOK-2

The structural formula of NPOK-2 is shown as follows:

the reaction process is shown in the following chart:

the preparation process comprises the following steps:

a) in a reaction flask, p-acetylphenol (2.05g,15.0mmol) and bromoacetic acid (2.50g,18.0mmol) were added, and an aqueous solution (6mL) of NaOH (1.20g,30mmol) was further added. The mixture was refluxed at 100 ℃ for 24 hours until the reaction was complete. The reaction was cooled to room temperature, acidified with 1M hydrochloric acid, precipitated solid and filtered to give the crude white product 6-1(3.32g, 113% yield) which was used in the next step without further purification.

b) In a reaction flask, crude product 6-1 from the previous step (3.32g,17.0mmol) was dissolved in DCM (50mL) and NHS (2.35g,20.4mmol) and EDCI (3.90g,20.4mmol) were added. The mixture was stirred at ambient temperature for 18 hours until the reaction was complete. Extract with DCM, combine the organic phases, dry over anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Further purification by column chromatography (eluent: DCM: MeOH: AcOH ═ 20:1:0.5) afforded the product 6-2(1.67g, yield 38%).

c) In a reaction flask, add product 6-2(1.67g,5.7mmol), add solvent dioxane (20mL) and water (50mL), add Fmoc-Lys-OH hydrochloride (1.9g,4.8mmol), add triethylamine (1.7mL,12.0mmol), stir at room temperature for 18 hours until reaction is complete, add the appropriate amount of 1M HCl to adjust pH to about 2, extract with EA, combine the organic phases, add anhydrous sodium sulfate to dry, filter, concentrate under reduced pressure. The resulting crude 6-3 was dissolved directly in DCM (10mL) and diethylamine (5mL) was added. The mixture was stirred at ambient temperature for 18 hours until the reaction was complete. Concentrating under reduced pressure, and performing column chromatography (eluent: DCM: MeOH: H)₂O ═ 40:10:1) purification afforded the final product 6-4(709mg, two step yield 39%).

¹H-NMR (400MHz, heavy water) δ 7.91(d, J ═ 8.8Hz,2H),6.98(d, J ═ 8.8Hz,2H),4.60(s,2H),3.59(t, J ═ 6.4Hz,1H),3.19(t, J ═ 6.8Hz,2H),2.52(s,3H),1.87 to 1.65(m,2H),1.56 to 1.40(m,2H),1.35 to 1.15(m,2H).

EXAMPLE 7 preparation of the unnatural amino acid NBGK-2

The structural formula of NBGK-2 is shown below:

the reaction process is shown in the following chart:

the preparation process comprises the following steps:

a) to a reaction flask, bromoacetic acid (2.10g,15.0mmol) and an aqueous solution (10mL) of NaOH (0.80g,20mmol) were added and stirred for 10 minutes. Paracetanilide (1.40g,10.0mmol) was then added and the mixture refluxed at 100 ℃ for 18 hours until the reaction was complete. The reaction was cooled to room temperature, filtered and washed with water to give 7-1(1.30g, 67% yield) as a white crude product which was used in the next step without further purification.

b) In a reaction flask, add product 7-1(1.30g,6.7mmol) and NaHCO₃(1.70g,20.1mmol) in water (20 mL). Fmoc-OSu (2.80g,8.1mmol) and DMF (20mL) were added. The mixture was stirred at 60 ℃ for 18 hours until the reaction was complete. Cooled to room temperature and extracted with EA. The water phase is kept, the pH value is adjusted to about 2 by using 1M hydrochloric acid, and then EA is used for extraction to obtain an organic phase. Adding anhydrous sodium sulfate, drying, filtering, and concentrating under reduced pressure. The product 7-2 was obtained and used in the next step without further purification.

c) In a reaction flask, the product of the previous step, 7-2 (about 6.7mmol), NHS (0.90g,8.0mmol) and EDCI (1.50g,8.0mmol) were dissolved in DMF (50 mL). The reaction mixture was stirred at room temperature for 24 hours until the reaction was complete. Water was added and extracted with DCM to obtain the organic phase. Adding anhydrous sodium sulfate, drying, filtering, and concentrating under reduced pressure. The product 7-3 was obtained and used in the next step without further purification.

d) In a reaction flask, the product of the previous step 7-3 (about 6.7mmol), Fmoc-Lys-OH hydrochloride (2.30g,5.6mmol), and triethylamine (2.0mL,14.0mmol) were added. The reaction mixture was stirred at room temperature for 3 hours until the reaction was complete. The pH was then adjusted to about 2 with 1M hydrochloric acid and extracted with EA. Adding anhydrous sodium sulfate, drying, filtering, and concentrating under reduced pressure. The product 7-4 was obtained and used in the next step without further purification.

e) In the reaction flask, the product 7-4 of the previous step was added. The solvents DCM (20mL) and diethylamine (10mL) were added. The reaction mixture was stirred at room temperature for 12 hours until the reaction was complete. Concentrating under reduced pressure, adding acetonitrile (50mL) to dissolve again, concentrating under reduced pressure, and repeating for 3 times to remove excessive diethylamine. DCM was added and slurried twice to give the final product 7-5(1.65g, 51% overall yield).

¹H-NMR (400MHz, heavy water) δ 7.71(d, J ═ 8.8Hz,2H),6.51(d, J ═ 8.8Hz,2H),3.80(s,2H),3.53(t, J ═ 6.8Hz,1H),3.09(t, J ═ 6.8Hz,2H),2.39(s,3H), 1.73-1.60 (m,2H), 1.42-1.33 (m,2H), 1.25-1.14 (m,2H).

Example 8

The NPOK, NPAK, NBOK, NBPK, NBGK, NPOK-2 and NBGK-2 prepared in examples 1 to 7 were used to prepare recombinant human growth hormones in prokaryotic expression systems, and PEG site-directed conjugates were prepared.

(1) Acquisition of helper plasmids

The helper plasmid pSupAR-MbPylRS is purchased from a plasmid preservation tissue Addgene (product number #91705), can express tRNA and tRNA synthetase for specifically recognizing pyrrolysine derived unnatural amino acids in escherichia coli, and is extracted after shake flask culture in LB culture medium added with 37.5mg/L chloramphenicol.

(2) Construction of recombinant human growth hormone expression plasmid containing stop codon in reading frame

The mRNA sequence of the coding gene of homo sapiens growth hormone (amino acid sequence is shown as SEQ ID NO:1) is obtained from the national center for biological information database, a purification label consisting of 6 histidines is added at the C end of the translated protein, meanwhile, the codon of the 107 th amino acid of SEQ ID NO:1 is changed into amber codon (TAG), and then the complete DNA sequence is synthesized by a whole gene synthesis mode to obtain the gene sequence of the recombinant human growth hormone (SEQ ID NO: 2).

The amino acid sequence of homo sapiens growth hormone (SEQ ID NO:1) is as follows:

FPTIPLSRLFDNAMLRAHRLHQLAFDTYQEFEEAYIPKEQKYSFLQNPQTSLCFSESIPTPSNREETQQKSNLELLRISLLLIQSWLEPVQFLRSVFANSLVYGASDSNVYDLLKDLEEGIQTLMGRLEDGSPRTGQIFKQTYSKFDTNSHNDDALLKNYGLLYCFRKDMDKVETFLRIVQCRSVEGSCGF

the gene sequence of the recombinant human growth hormone (SEQ ID NO:2) is as follows:

ATGTTTCCTACTATACCACTATCTCGTCTATTCGATAACGCTATGCTTCGGGCCCATCGTCTTCATCAGCTGGCCTTTGACACCTACCAGGAGTTTGAAGAAGCCTATATCCCAAAGGAACAGAAGTATTCATTCCTGCAGAACCCCCAGACCTCCCTCTGTTTCTCAGAGTCTATTCCGACACCCTCCAACAGGGAGGAAACACAACAGAAATCCAACCTAGAGCTGCTCCGCATCTCCCTGCTGCTCATCCAGTCGTGGCTGGAGCCCGTGCAGTTCCTCAGGAGTGTCTTCGCCAACAGCCTGGTGTACGGCGCCTCTTAGAGCAACGTCTATGACCTCCTAAAGGACCTAGAGGAAGGCATCCAAACGCTGATGGGGAGGCTGGAAGATGGCAGCCCCCGGACTGGGCAGATCTTCAAGCAGACCTACAGCAAGTTCGACACAAACTCACACAACGATGACGCACTACTCAAGAACTACGGGCTGCTCTACTGCTTCAGGAAGGACATGGACAAGGTCGAGACATTCCTGCGCATCGTGCAGTGCCGCTCTGTGGAGGGCAGCTGTGGCTTCCTCGAGCACCACCACCACCACCACTAA

the gene sequence (SEQ ID NO:2) of the recombinant human growth hormone was cloned between Nde I and Xho I sites of pET21a (Novagen, cat #69740-3) by one-step subcloning to obtain expression plasmid pET21-rhGH107, which was confirmed to be identical to the expected sequence by sequencing. pET21-rhGH107 may be used to express recombinant human growth hormone in which the codon for amino acid 107 has been replaced with an amber codon and which contains a6 histidine purification tag at the C-terminus of the protein. The map of the recombinant human growth hormone expression plasmid pET21-rhGH107 is shown in figure 1.

(3) Obtaining of expression Strain of interest

The auxiliary plasmid pSupAR-MbPylRS and the expression plasmid pET21-rhGH107 are co-transformed into competent cells of Escherichia coli origamB (DE3) (Novagen, cat #70911-3), and LB culture medium containing 100mg/L ampicillin and 37.5mg/L chloramphenicol is used for screening to obtain a double-resistant strain, namely the recombinant human growth hormone expression strain.

(4) Recombinant protein expression with insertion of unnatural amino acids

The recombinant human growth hormone-expressing strain after screening was inoculated into 8 parts of 2 XYT medium (yeast extract 16g/L, tryptone 10g/L, NaCl 5g/L, containing 100mg/L ampicillin and 37.5mg/L chloramphenicol), cultured at 37 ℃ until OD600 of the bacterial solution became 2.0. + -. 0.2, IPTG (final concentration of 1mM) and arabinose (final concentration of 0.2) were added to 8 parts of bacterial solution, respectively, NBOK (final concentration of 1mM) was added to 1 part of bacterial solution, NPAK (final concentration of 1mM) was added to 2 parts of bacterial solution, NBPK (final concentration of 1mM) was added to 3 parts of bacterial solution, NBGK (final concentration of 1mM) was added to 4 parts of bacterial solution, NPOK-2 (final concentration of 1mM) was added to 5 parts of bacterial solution, NPOK-2 (final concentration of 1mM) was added to 6 parts of bacterial solution, NBGK-2 (final concentration of 1mM) was added to 7 parts of bacterial solution, the 8 th cell suspension was used as a negative control without addition of unnatural amino acids. Culturing at 37 deg.C for 5-6 hr, centrifuging at 10000rpm for 1min, re-suspending with PBS to OD600 of 10, and performing SDS-PAGE electrophoresis on each bacterial suspension, wherein the SDS-PAGE electrophoresis chart of each bacterial strain is shown in FIG. 2. The results in FIG. 2 show that the expression strain can express the target protein under the condition that 7 unnatural amino acids are respectively added.

(5) Purification of recombinant proteins incorporating unnatural amino acids

And (2) centrifuging each bacterium solution subjected to induced expression at 10000rpm for 5min, taking a precipitate, adding NTA Buffer (20mM Tris-HCl pH7.9, 0.5M NaCl, 10% glycerol) with the growth volume of 1/20 cells and PMSF with the final concentration of 1mM, homogenizing and breaking the cells by ultrasonic waves, centrifuging at 10000rpm for 20min, taking a supernatant, adsorbing the target protein with the His label by using a Ni-NTA chromatographic column, and eluting by using an eluent NTA Buffer (20mM Tris-HCl pH7.9, 0.5M NaCl, 10% glycerol, 250mM imidazole) to obtain a target protein (namely, the recombinant protein with the inserted unnatural amino acid) sample with the purity of about 90%. Each sample was placed in a 10K nitrocellulose dialysis bag, dialyzed overnight in PBS buffer (200 ml dialysate per 1ml protein solution) at pH7.0, and the solution was changed 1 time, and the dialyzed protein solution was collected and the protein concentration was examined by SDS-PAGE.

(6) Coupling reaction of recombinant protein inserted with unnatural amino acid and PEG

As shown in the above synthetic scheme (wherein R₁To R₂In the N-terminal to C-terminal direction of the amino acid sequence).

Taking the example of coupling the recombinant protein inserted with unnatural amino acid by 30KD aminoxy PEG oximation reaction, the coupling reaction is performed as follows: the target protein obtained above was adjusted to 0.5mg/ml with PBS buffer of ph7.0 before the coupling reaction, according to 1:15 (molar ratio, recombinant protein: aminoxy PEG) was added to 30KD aminoxy PEG solid (from Beijing Kekeji science and technology Co., Ltd.), sufficiently shaken and dissolved to obtain a clear and transparent solution, and then the reaction solution was sealed and shaken in a constant temperature shaker (25 ℃ C., 100rpm) for reaction. After 48h the coupling was analysed using SDS-PAGE, see FIG. 3. The results in FIG. 3 show that all 6 target proteins achieved 30KD PEG coupling, further indicating that the 7 unnatural amino acids were inserted into the target protein. The product of coupling of recombinant protein with PEG inserted with unnatural amino acids is abbreviated "PEG-rhGH".

(7) Purification of PEG-rhGH

The coupled PEG-rhGH was separated and purified by Butyl HP hydrophobic chromatography (loading buffer: 50mM NaH)₂PO₄·2H₂O，0.8M(NH₄)₂SO₄pH8.5; elution buffer: 20mM Tris-HCl, pH8.5, 40 column volume gradient elution) to obtain pure electrophoresis grade (>95%) pure PEG-rhGH. The electrophoresis chart is shown in FIG. 4 by taking NPOK-containing and NBOK-containing PEG-rhGH as examples.

Other PEG-rhGH containing unnatural amino acids of the invention also achieved electrophoretic purity after purification.

(8) Activity assay for PEG-rhGH

The specific process is as follows: HEK293-GHR cells (cell line from Chinese patent: 2020112649827) were cultured in DMEM (Gibco, cat # 11995040) medium containing 0.1mg/mL hygromycin B (Biotech, Shanghai, Ltd., cat # A600230-0001), 0.75mg/mL G418 (Biotech, Shanghai, Ltd., cat # A600958-0005), 10% fetal bovine serum (Gibco, cat # 10099141) at 37 ℃ in 5% carbon dioxide to a sufficient amount, and 9X 10G 11995040 was inoculated into each well of a black 96-well cell culture plate (Corning, cat # 3904)⁴HEK293-GHR cells were starved for 16h at 37 ℃ in 5% carbon dioxide at 90. mu.L/well. rhGH Standard (purchased from China institute for food and drug assay, abbreviated as "Zhongzhong institute"), NPOK-containing rhGH (i.e., rhGH (NPOK)), PEG-rhGH (i.e., PEG-rhGH (NPOK)) of PEG-modified NPOK, NBOK-containing PEG-rhGH (i.e., rhGH (NBOK)), PEG-rhGH (i.e., PEG-rhGH (NBOK)) of PEG-modified NBOK, and commercially available rhGH drugs(Changchun Jinsai pharmaceutical industry, Ltd.), and PEG-rhGH medicine sold on the market(Changchun Jinsai pharmaceutical industry, Ltd.) samples were diluted in gradient at a total of 9 concentrations and cultured at 37 ℃ for 4 hours with 5% carbon dioxide. Add 50. mu.L of ONE-Glo per well^TM(Promega, cat. No. E6120) detection reagent, and a microplate reader reads a chemiluminescence value. EC50 was calculated by fitting a curve to the sample concentration and the microplate reader readings. The results are shown in FIGS. 5A-5G and Table 1. The results show that the cell activity at each site before conjugation is equivalent to that of the rhGH standard of the institute. The activity of the coupled cells is reduced to about 25-50% of that of the standard product, and the change rule of the biological activity of the coupled cells is consistent with that of the PEG-rhGH on the market.

The cell activity of other PEG-rhGH containing the unnatural amino acid is also reduced to 25-50% of that of the standard product after coupling, and the change rule of the biological activity is consistent with that of the PEG-rhGH on the market.

TABLE 1

Example 9

Recombinant GFP proteins were prepared in eukaryotic expression systems using the unnatural amino acids NPOK, NPAK, NBOK, NBPK, NBGK, NPOK-2 and NBGK-2 prepared in examples 1-7.

(1) Acquisition of helper plasmids

The helper plasmid pCMV-MbPylRS was purchased from the collection of plasmids addgene (cat #91706) encoding aminoacyltRNA synthetase that specifically recognizes pyrrolysine-derived unnatural amino acids and the corresponding tRNA (recognizing amber codon UAG) in mammalian cells.

(2) Construction of green fluorescent protein expression vector containing amber codon in gene reading frame

The expression vector expressing the wild type green fluorescent protein (the gene coding sequence of which is shown as SEQ ID NO:3) containing the purification label is subjected to point mutation to obtain an expression plasmid pEGFP40, wherein the 40 th amino acid codon of the reading frame is mutated into an amber codon, and the complete sequence of the expression plasmid is shown as SEQ ID NO: 4.

The gene sequence of wild-type green fluorescent protein containing the purification tag (SEQ ID NO:3) is as follows:

ATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAGGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGAAGTACGAAGATTACAAGGATGACGACGATAAGTAA

the gene sequence of expression plasmid pEGFP40 (SEQ ID NO:4) is as follows:

GTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGACGCCATCCACGCTGTTTTGACCTCCATAGAAGACACCGGGACCGATCCAGCCTCCGGACTCTAGAGGATCCCCGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAGGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGAAGTACGAAGATTACAAGGATGACGACGATAAGTAAGAATTCCGGAAGGGTTCGATCCCTACCGGTTAGTAATGAGTTTAAACGGGGGAGGCTAACTGAAACACGGAAGGAGACAATACCGGAAGGAACCCGCGCTATGACGGCAATAAAAAGACAGAATAAAACGCACGGGTGTTGGGTCGTTTGTTCATAAACGCGGGGTTCGGTCCCAGGGCTGGCACTCTGTCGATACCCCACCGAGACCCCATTGGGGCCAATACGCCCGCGTTTCTTCCTTTTCCCCACCCCACCCCCCAAGTTCGGGTGAAGGCCCAGGGCTCGCAGCCAACGTCGGGGCGGCAGGCCCTGCCATAGCAGATCTGCGCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGCATCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGGGGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCAATGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCGACGGATCGGGAGATCTCCCGATCCCCTATGGTCGACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGG

(3) recombinant GFP protein expression with insertion of unnatural amino acids

Chinese hamster ovary cells CHO-K1 (cat # CCL-61-ATC) were purchased from American model culture Collection (ATCC) and cultured adherently using RPMI1640 medium containing 10% fetal bovine serum. The helper plasmid pCMV-MbPylRS and the green fluorescent protein expression plasmid pEGFP40 obtained in the above-mentioned step were extracted, and the plasmid was transfected with lipo2000 (Invitrogen, cat #12566014), transient transfection was performed according to the instructions (cells were inoculated into 24-well plates at 50000 cells/well for 8 wells in total, and transfection was performed 24h after inoculation culture, 500ng plasmid per well) and simultaneously with the transfection, NPOK (final concentration of 1mM) was added to the 1 st well, NPAK (final concentration of 1mM) was added to the 2 nd well, NBOK (final concentration of 1mM) was added to the 3 rd well, NBPK (final concentration of 1mM) was added to the 4 th well, NBGK (final concentration of 1mM) was added to the 5 th well, NPOK-2 (final concentration of 1mM) was added to the 6 th well, NBGK-2 (final concentration of 1mM) was added to the 7 th well, and no unnatural amino acid was added to the 8 th well, as a negative control. And (3) observing the cells under a fluorescence microscope after standing and culturing for 48 hours in a carbon dioxide incubator, wherein the results show that obvious green fluorescence exists in the 1 st to 7 th holes (see figure 6), and the results prove that the unnatural amino acid can be inserted into the green fluorescent protein to obtain the complete green fluorescent protein without influencing the function of the fluorescent protein.

Example 10

NPOK, NPAK, NBOK, NBPK, NBGK, NPOK-2, NBGK-2 prepared in examples 1 to 7 were used to express anti-HER 2 monoclonal antibodies containing unnatural amino acids in eukaryotic expression systems, and conjugates with toxins were prepared.

(1) Acquisition of helper plasmids

(2) Construction of anti-HER 2 antibody (trastuzumab) expression vector containing amber codon inside gene reading frame

Heavy chain and light chain DNAs (corresponding amino acid sequences are SEQ ID NO:5 and SEQ ID NO:6 respectively) for coding Trastuzumab are synthesized in a whole-gene synthesis mode, and are subcloned to a eukaryotic expression vector pCDNA3.1+, and then point mutation is carried out on the obtained expression vector to obtain an expression plasmid pCDNA3.1-Trastuzumab-UAG142 with the 142 th amino acid codon of a heavy chain reading frame mutated into an amber codon, wherein the map of the expression plasmid is shown in figure 7, and the complete sequence of the expression plasmid is shown in SEQ ID NO: 7.

The amino acid sequence of the heavy chain of trastuzumab (SEQ ID NO:5) is as follows:

EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVARIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSRWGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK

the light chain amino acid sequence of trastuzumab (SEQ ID NO:6) is as follows:

DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

the gene sequence of expression plasmid pCDNA3.1-Trastuzumab-UAG142 (SEQ ID NO:7) is as follows:

GACGGATCGGGAGATCTCCCGATCCCCTATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATCTGCTCCCTGCTTGTGTGTTGGAGGTCGCTGAGTAGTGCGCGAGCAAAATTTAAGCTACAACAAGGCAAGGCTTGACCGACAATTGCATGAAGAATCTGCTTAGGGTTAGGCGTTTTGCGCTGCTTCGCGATGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTCTCTGGCTAACTAGAGAACCCACTGCTTACTGGCTTATCGAAATTAATACGACTCACTATAGGGAGACCCAAGCTGGCTAGCGTTTAAACTTAAGCTTATGTACCGGATGCAGCTGCTGAGCTGTATCGCCCTGTCTCTGGCCCTCGTGACCAACAGCGAAGTGCAGCTGGTGGAAAGCGGCGGAGGACTGGTGCAGCCTGGCGGATCTCTGAGACTGAGCTGTGCCGCCAGCGGCTTCAACATCAAGGACACCTACATCCACTGGGTGCGCCAGGCCCCTGGCAAGGGACTGGAATGGGTGGCCAGAATCTACCCCACCAACGGCTACACCAGATACGCCGACAGCGTGAAGGGCCGGTTCACCATCAGCGCCGACACCAGCAAGAACACCGCCTACCTGCAGATGAACAGCCTGCGGGCCGAGGACACCGCCGTGTACTACTGTAGTAGATGGGGAGGCGACGGCTTCTACGCCATGGACTATTGGGGCCAGGGCACCCTCGTGACAGTGTCTAGTGCGTAGACCAAGGGGCCCTCGGTCTTCCCCCTGGCACCCTCCTCCAAGAGCACCTCTGGGGGCACAGCGGCCCTGGGCTGCCTGGTCAAGGACTACTTCCCCGAACCGGTGACGGTGTCGTGGAACTCAGGCGCCCTGACCAGCGGCGTGCACACCTTCCCGGCTGTCCTACAGTCCTCAGGACTCTACTCCCTCAGCAGCGTGGTGACCGTGCCCTCCAGCAGCTTGGGCACCCAGACCTACATCTGCAACGTGAATCACAAGCCCAGCAACACCAAGGTGGACAAGAAAGTTGAGCCCAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCCGGCAAGTGATAAGGCCGGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAACCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTACACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCCCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACGATGATAATATGGCCACAACCATGTACCGCATGCAACTCCTGTCTTGCATTGCACTAAGTCTTGCACTTGTCACAAACAGTGATATCCAGATGACCCAGAGCCCCAGCAGCCTGTCTGCCAGCGTGGGCGACAGAGTGACCATCACCTGTAGAGCCAGCCAGGACGTGAACACCGCCGTGGCCTGGTATCAGCAGAAGCCTGGCAAGGCCCCCAAGCTGCTGATCTACAGCGCCAGCTTCCTGTACAGCGGCGTGCCCAGCAGATTCAGCGGCAGCAGATCCGGCACCGACTTCACCCTGACCATCAGCTCCCTGCAGCCCGAGGACTTCGCCACCTACTACTGCCAGCAGCACTACACCACCCCCCCCACATTTGGCCAGGGCACCAAGGTGGAAATCAAGCGTACGGTGGCTGCACCATCTGTCTTCATCTTCCCGCCATCTGATGAGCAGTTGAAATCTGGAACTGCCTCTGTTGTGTGCCTGCTGAATAACTTCTACCCCAGAGAAGCCAAAGTGCAGTGGAAGGTGGACAACGCCCTGCAGAGCGGAAACAGCCAGGAAAGCGTGACAGAGCAGGATTCCAAGGATTCCACATACAGCCTGAGCAGCACACTGACACTGTCCAAGGCCGACTACGAGAAGCACAAGGTGTACGCCTGCGAAGTGACACACCAGGGACTGTCCTCCCCTGTGACAAAGAGCTTCAACAGAGGAGAATGCTGATGATCTAGAGGGCCCGTTTAAACCCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCCTTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCTGGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGACAATAGCAGGCATGCTGGGGATGCGGTGGGCTCTATGGCTTCTGAGGCGGAAAGAACCAGCTGGGGCTCTAGGGGGTATCCCCACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTAATTCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCTGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCCCGGGAGCTTGTATATCCATTTTCGGATCTGATCAAGAGACAGGATGAGGATCGTTTCGCATGATTGAACAAGATGGATTGCACGCAGGTTCTCCGGCCGCTTGGGTGGAGAGGCTATTCGGCTATGACTGGGCACAACAGACAATCGGCTGCTCTGATGCCGCCGTGTTCCGGCTGTCAGCGCAGGGGCGCCCGGTTCTTTTTGTCAAGACCGACCTGTCCGGTGCCCTGAATGAACTGCAGGACGAGGCAGCGCGGCTATCGTGGCTGGCCACGACGGGCGTTCCTTGCGCAGCTGTGCTCGACGTTGTCACTGAAGCGGGAAGGGACTGGCTGCTATTGGGCGAAGTGCCGGGGCAGGATCTCCTGTCATCTCACCTTGCTCCTGCCGAGAAAGTATCCATCATGGCTGATGCAATGCGGCGGCTGCATACGCTTGATCCGGCTACCTGCCCATTCGACCACCAAGCGAAACATCGCATCGAGCGAGCACGTACTCGGATGGAAGCCGGTCTTGTCGATCAGGATGATCTGGACGAAGAGCATCAGGGGCTCGCGCCAGCCGAACTGTTCGCCAGGCTCAAGGCGCGCATGCCCGACGGCGAGGATCTCGTCGTGACCCATGGCGATGCCTGCTTGCCGAATATCATGGTGGAAAATGGCCGCTTTTCTGGATTCATCGACTGTGGCCGGCTGGGTGTGGCGGACCGCTATCAGGACATAGCGTTGGCTACCCGTGATATTGCTGAAGAGCTTGGCGGCGAATGGGCTGACCGCTTCCTCGTGCTTTACGGTATCGCCGCTCCCGATTCGCAGCGCATCGCCTTCTATCGCCTTCTTGACGAGTTCTTCTGAGCGGGACTCTGGGGTTCGAAATGACCGACCAAGCGACGCCCAACCTGCCATCACGAGATTTCGATTCCACCGCCGCCTTCTATGAAAGGTTGGGCTTCGGAATCGTTTTCCGGGACGCCGGCTGGATGATCCTCCAGCGCGGGGATCTCATGCTGGAGTTCTTCGCCCACCCCAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGTATACCGTCGACCTCTAGCTAGAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTC

(3) insertion of unnatural amino acids

Suspension acclimated HEK293 cells at 0.3X 10⁵Density of/mL inoculation to Wayne293^TMMedium (Quacell Biotechnology, cat # A21501) was prepared in a 1L shake flask, filled with 240mL of liquid, at 120rpm, 5% CO₂Shake culturing under 80% humidity condition until cell density reaches about 1 × 10⁶Performing transfection at the time of/mL, extracting the helper plasmids pCMV-MbPylRS and the expression plasmids pCDNA3.1-Trastuzumab-UAG142 described in the steps (1) and (2), removing endotoxin for later use, using 120 mu g of each expression plasmid and helper plasmid in each shake flask transfection, adding the plasmids into a centrifuge tube, diluting to 7.2mL by using 1 XPBS buffer solution, adding 720 mu g of PEI transfection reagent (Polyethyleneimine) into another centrifuge tube, adding 1 XPBS buffer solution to dilute to 7.2mL, standing for 5 minutes after mixing, standing for 10 minutes after mixing the liquid in the two centrifuge tubes lightly, slowly dripping the PEI transfection reagent (Polyethyleneimine) into 240mL of the shake flasks in total of 14.4mL to 1L, and shaking lightly during dripping, 120rpm and 5% CO₂Shake culturing for 4 hr under 80% humidity, adding 1mM unnatural amino acid, and further culturing at 120rpm and 5% CO₂And shake-culturing for 5 days under the condition of 80% humidity, and harvesting cell culture supernatant for purifying the antibody.

(4) Purification of

Purifying the cell culture supernatant by using HiTrap Protein A and a 1ml pre-packed column, wherein the elution buffer solution is 100mmol/L glycine, 200mmol/L acetate and pH3.5, and obtaining the purified trastuzumab inserted with the unnatural amino acid.

(5) Coupling of

The synthetic route is shown below (taking NBOK as an example):

reacting toxin (DM1-PEG-ONH2) containing aminoxy terminal group with purified trastuzumab (wherein R is₁To R₂In the direction from the N end to the C end of the amino acid sequence) to obtain the monoclonal antibody-toxin conjugate.

The preparation process of DM1-PEG-ONH2 is as follows:

the coupling procedure was specifically performed as follows: mixing the purified trastuzumab inserted with the unnatural amino acid with DM1-PEG-ONH2 at a molar ratio of 1:15, adjusting the pH to 4.0 by using 10M acetic acid, shaking the mixture on a shaking table (25 ℃, 200rpm), sampling for 48h, and detecting the reaction condition of the trastuzumab by using HPLC (HIC-HPLC) based on a hydrophobic chromatography principle. The results showed that 93.0% of the trastuzumab containing NBPK was modified by the toxin, 93.0% of the trastuzumab containing NBOK was modified by the toxin, and 87.4% of the trastuzumab containing NPOK-2 was modified by the toxin, as shown in fig. 8A-8C. Trastuzumab coupling ratio is 100% -the ratio of the remaining uncoupled raw materials.

And (3) exchanging the coupling monoclonal antibody-toxin conjugate through a 50kDa ultrafiltration centrifugal tube to remove unreacted toxin raw materials, wherein the exchange buffer is 20mM histidine buffer (pH6.5).

HIC-HPLC analysis conditions were as follows:

mobile phase A (2M ammonium sulfate, 75mM K)₂HPO₄，pH 7.2±0.2)；

Mobile phase B (75mM K)₂HPO₄25% isopropanol, pH 7.2. + -. 0.2).

(6) Activity assay

The sample was examined for inhibition of cell proliferation using BT-474 cell line (ATCC, cat. No. HTB-20).

The specific process is as follows: BT-474 cells were cultured in a sufficient amount of ATCC hybrid-Care Medium (ATCC, Cat. No. 46-X) containing 10% fetal bovine serum (Gibco, Cat. No. 10099141) at 37 ℃ under 5% carbon dioxide, and seeded into 96-well cell culture plates (Corning, Cat. No. 3599) at 1.5X 10 cells per well⁴BT-474 cells (including vehicle control wells), medium without cells was added to the blank control wells at 80 μ L/well. NBOK-containing trastuzumab and DM 1-modified NBOK-containing trastuzumab were diluted from a gradient of 17 μ g/mL to 0.13 μ g/mL for 8 concentrations, 2 duplicate wells per dilution. The diluted samples were transferred to plates inoculated with BT-474 cells at 10. mu.L/well, 10. mu.L of medium was added to the vehicle control wells and the blank control wells, and incubated at 37 ℃ in 5% carbon dioxide. On day 5, 10. mu.L of CCK-8 (Biyun, cat # C0043) detection reagent was added to each well, and the mixture was developed at 37 ℃ for 6 hours under 5% carbon dioxide, and the optical density value was read by an enzyme-linked microplate reader at a wavelength of 450 nm. The inhibition rate (Growth inhibition) of the test sample was calculated by the following formula: inhibition (%) × (OD compound-OD blank)/(OD vehicle control-OD blank) × 100%. Inhibition was calculated for different concentrations of compound in Excel, followed by a graph of inhibition using GraphPad Prism7 software and calculation of IC 50. The results are shown in FIG. 9. The result shows that the IC50 of the NBOK trastuzumab-containing modified DM1 is about 3 times lower than that of NBOK trastuzumab-containing modified DM1, and the tumor killing effect is obviously improved.

(7) Stability Studies of DM1 modified Trastuzumab with unnatural amino acids

And (3) exchanging the liquid of the coupled monoclonal antibody-toxin conjugate through a 50kDa ultrafiltration centrifugal tube (Millipore, the product number is UFC905024#), and removing unreacted toxin raw materials. The replacement buffer was 20mM histidine buffer (pH 6.0). The protein solution was divided into three portions, adjusted to pH4.0, pH6.0, and pH8.0 with 1M citric acid solution or 1M Tris solution, respectively, placed in a water bath at 25 deg.C, sampled for 24h, 48h, 72h, 96h, and 120h, and subjected to HIC-HPLC (detection conditions as above), and the results are shown in Table 2.

TABLE 2 stability of NBOK containing trastuzumab modified with DM1 at 25 ℃ at various pHs

The results in table 2 show that the oxime bond formed in the DM 1-modified trastuzumab containing unnatural amino acids provided by the invention is relatively stable at low pH.

EXAMPLE 11 reactive group reduction of Lys-azido

(1) High resolution mass spectrometry of product molecular weight

The secretory growth hormone expression method of reference literature (Song Shenhua et al, J.biol., 16 (1): 6-8,1999) comprises adding OmpA signal peptide coding sequence on the N-terminal of rhGH on the expression vector according to the signal peptide sequence provided in the literature, combining with auxiliary plasmid pSupAR-MbPylRS in example 8, constructing origamB (DE3) expression strain of rhGH with K140 codon mutated to amber codon (TAG), and expressing natural rhGH with 140 th position mutated to Lys-azido by adding Lys-azido during fermentation, and numbering rhGH-Lys-azido-140; through adding NBOK in the fermentation process, the natural rhGH with the No. 140 mutation position being NBOK is expressed, and the rhGH-NBOK-140 is numbered, and the purification is carried out by referring to the corresponding purification means of the reference. The complete molecular weight was analyzed by LC-MS (high resolution Mass Spectrometry: XevoG2-XS Q-Tof, Waters; ultra high Performance liquid chromatography: UPLC (acquisition UPLC I-Class), Waters) using rhGH-Lys-azido-140 and rhGH-NBOK-140.

As shown in FIG. 10A, a 26Da less than theoretical molecular weight (22265Da) component appeared in the rhGH-Lys-azido-140 sample, which was deduced to be due to the azido structure (-N) at the Lys-azido terminus₃) Is reduced to (-NH)₂) The product of (a) shows that the currently commonly used unnatural amino acid Lys-azido is unstable in azide group when inserted into protein and is easily reduced to form a reduction product, so that the coupling activity is lost. As shown in FIG. 10B, rhGH-NBOK-140The sample has no impurity with higher proportion, which shows that NBOK is more stable than Lys-azido in the prepared recombinant protein product.

(2) Comparison of coupling efficiencies

As shown in the above reaction scheme, 30KD BCN-PEG (made by Chinese patent CN 112279906A) is site-specifically coupled with rhGH-Lys-azido-140 (wherein R is R)₁To R₂In the N-terminal to C-terminal direction of the amino acid sequence). The target protein obtained above was adjusted to 0.5mg/ml with PBS buffer pH7.0, according to the ratio of 1:15 and 1:25 (molar ratio, recombinant protein: 30KD BCN-PEG), putting the BCN-PEG solid into rhGH-Lys-azido solution, fully shaking for dissolution to obtain clear and transparent solution, sealing the reaction solution, and shaking for reaction in a constant temperature shaking table (25 ℃, 70 rpm). Samples were taken at intervals and the reaction results were checked by SDS-PAGE, and after 72h the reaction was stopped with a conversion of about 50% -70%. The reaction results are shown in FIG. 11A.

Referring to example 8, 30kD aminoxy PEG was site-directed coupled to rhGH-NBOK-140 by an oximation reaction. Samples were taken at intervals and the reaction was checked by SDS-PAGE, and after 48h the reaction was stopped with a conversion of about 90%. The reaction results are shown in FIG. 11B.

The reduction of Lys-azido can be judged to cause that the product can not be coupled with BCN-PEG by combining the molecular weight detection result of high resolution mass spectrum, thereby reducing the coupling rate. When the recombinant protein containing the unnatural amino acid is coupled with PEG, the conversion rate is obviously higher than that of the recombinant protein containing Lys-azido, and the reaction time is also obviously shortened, so that the reaction efficiency is obviously improved.

Unless otherwise defined, all terms used herein have the meanings commonly understood by those skilled in the art.

The described embodiments of the present invention are for illustrative purposes only and are not intended to limit the scope of the present invention, and those skilled in the art may make various other substitutions, alterations, and modifications within the scope of the present invention, and thus, the present invention is not limited to the above-described embodiments but only by the claims.

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Unnatural amino acid, application thereof, recombinant protein containing unnatural amino acid and recombinant protein conjugate

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