CN106589051B

CN106589051B - Protein with chemical modification group and preparation method thereof

Info

Publication number: CN106589051B
Application number: CN201510682996.3A
Authority: CN
Inventors: 戴旭东; 刘相钦
Original assignee: Individual
Current assignee: Individual
Priority date: 2015-10-20
Filing date: 2015-10-20
Publication date: 2020-01-03
Anticipated expiration: 2035-10-20
Also published as: CN106589051A

Abstract

The invention relates to a technology for site-specific modification of a chemical group to the C-terminal of a protein by utilizing a protein trans-splicing technology. Specifically, the invention provides a technology for modifying polyethylene glycol (PEG) to the C end of granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), human interferon alpha 2b (IFN alpha 2b), interleukin-15 (ILK-15) and Urate Oxidase (UOX) by utilizing a protein trans-splicing technology, and a method for preparing the medicine for site-specific C-end modification. Meanwhile, a protein intron sequence for site-directed modification of protein polypeptide drugs is also provided.

Description

Protein with chemical modification group and preparation method thereof

Technical Field

The present invention relates to the field of protein drug modification. The invention relates to a technology for site-specific modification of a chemical group to the C-terminal of a protein by utilizing a protein trans-splicing technology. Also provides a plurality of pegylation drugs and a preparation method thereof. Meanwhile, a protein intron sequence for site-directed modification of protein polypeptide drugs is also provided.

Background

Chemical modification of polyethylene glycol (PEG) is an effective way to prolong the half-life of protein polypeptide drugs in vivo. The physicochemical properties such as conformation, electrostatic adsorption, hydrophobicity, and the like of the protein modified with polyethylene glycol are changed. These physical and chemical changes make pegylated drug proteins significantly superior to conventional protein polypeptide drugs. The advantages of pegylated protein polypeptide drugs are mainly reflected in: the solubility of the drug is increased, the stability of the drug is improved, the immunogenicity is reduced, the filtration of the kidney is reduced, and the protein polypeptide drug is effectively protected from being decomposed by proteolytic enzyme.

One of the biggest technical challenges in pegylation is specificity. In many cases, it is desirable to modify polyethylene glycol to a specific site on a protein, and the molecular ratio of the modified polyethylene glycol molecule to the protein should be 1: 1. however, a plurality of modification sites (-SH, -NH2, etc.) are usually present in a protein molecule, and since random modification is performed, the modification sites are not uniform, and the number of modified polyethylene glycols is also not uniform, resulting in a mixture of PEG-modified products. Moreover, the biological activities of the isomers are different, so that the batch stability is difficult to realize in large-scale preparation, and sometimes the requirements of drug production and new drug declaration are difficult to achieve.

To solve this problem, some studies have been conducted to modify the site of interest. For example, some methods are polyethylene glycol modification of the N-terminus of the protein (e.g., U.S. Pat. No. 6077939; U.S. Pat. No. 7090835; U.S. Pat. No. 5621039). In addition, there is a method of introducing free cysteine into a protein drug for site-directed modification (U.S. Pat. No. 7214779). Yet another approach has been to introduce unnatural amino acids into proteins as sites for site-directed modification (U.S. Pat. No. 7230068).

However, these methods have limitations, for example, the steps of these methods are complicated and time and labor consuming. Moreover, the modification efficiency is low, reducing the activity of the drug protein (the protein is not folded correctly). At the same time, these methods also produce by-products, which can affect the activity of the drug, and the removal of these by-products is also enormous. Furthermore, to optimize these processes, it is possible to mutate some amino acids as targets for the polymer. However, mutated amino acids may result in loss of activity (e.g., changes in secondary structure and protein conformation) of the drug polypeptide.

Therefore, a new method of site-directed modification is needed to overcome the above difficulties. Based on the protein trans-splicing technology, the invention provides a technology for site-directed modification of the C-terminal of a protein. Protein trans-splicing (protein trans-splicing) is mediated by an intron (intein) of the protein. Protein introns are autocatalytically active proteins, and after translation, protein intron fragments are spliced out of the protein precursor and joined by peptide bonds to flanking protein exons (extins) to form the mature protein. The split protein intron (split intein) is split at a specific site IN the middle region of the protein intron to form an N-terminal fragment (IN, N part of intein) and a C-terminal fragment (IC, C part of intein), and the intron can be combined and reconstructed to form a functional protein intron and can also catalyze trans-splicing reaction between protein molecules. In addition, protein trans-splicing can be performed not only under physiological conditions but also at relatively low protein concentrations. Trans-splicing provides a novel approach to protein specific site modification.

Disclosure of Invention

In order to solve the problem of nonspecific modification in the field of protein polypeptide drug modification and make up the defects of the prior art, the invention provides a technology for site-specific modification of a chemical group to the multi-C-terminal of a protein by using a protein trans-splicing technology. The invention firstly utilizes the protein trans-splicing technology to modify polyethylene glycol (PEG) to a specific site of the protein polypeptide drug, so as to obtain the long-acting recombinant protein drug with uniform components. These modified drugs include granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), human interferon alpha 2b (IFN alpha 2b), interleukin-15 (ILK-15), and Urate Oxidase (UOX). The invention also provides a method for preparing the medicine modified by the fixed-point C-terminal. Meanwhile, a protein intron sequence for site-directed modification of protein polypeptide drugs is also provided.

In one aspect of the present application, there is provided a method for chemically modifying a protein having only one chemical modification site, which can provide a uniform site-directed modified protein.

The method uses the site-specific modification of a split protein intron, and comprises the following specific steps:

i. preparing a C-protein (comprising the sequence of the C-stretch of the split intein and the first amino acid sequence) and chemically modifying said C-protein;

preparing an N-protein (comprising the N-stretch sequence of the split intein and a second amino acid sequence);

connecting the C-protein with the N-protein by a trans-shearing technology, wherein the first amino acid sequence and the second amino acid sequence are connected by peptide bonds, and the C-segment sequence is shown as SEQ ID NO. 1, and the N-segment sequence is shown as SEQ ID NO. 2. It should be noted that the split intein in the present application is not limited to the above sequence, and other possible sequences include:

a. an Ssp DnaB artificial cleavage protein intron and a mutant thereof with protein splicing activity; b. a Sce VMA artificially disrupted protein intron and a mutant thereof having protein splicing activity; c. an Npu DnaE split protein intron, and mutants thereof having protein splicing activity; d. a Ter ThyX artificial broken protein intron and a mutant thereof with protein splicing activity; e. an Ssp DnaX artificial cleavage protein intron and a mutant thereof with protein splicing activity; f. an artificially broken protein intron of Ter DnaE-3 and a mutant thereof with protein splicing activity; g. an artificial fragment of protein intron Cne PRP8, and its mutant with protein splicing activity; h. rma DnaB artificial broken protein intron and mutant thereof with protein splicing activity

Wherein, the sequence of the first amino acid sequence is shown as SEQ ID NO. 3, the fourth site is the only chemical modification site, and the second amino acid sequence represents the amino acid sequence of a protein polypeptide drug. These protein polypeptide drugs include granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), human interferon alpha 2b (IFN alpha 2b), interleukin-15 (ILK-15), and Urate Oxidase (UOX). However, it will be appreciated by those skilled in the art that the present application is directed to a method and corresponding sequence (i.e., first amino acid sequence) having one and only one site of chemical modification, and that proteins linked to the first amino acid sequence by cleavage of the intein and trans cleavage techniques are not limited to these protein polypeptide drugs. Those skilled in the art can freely design other protein drugs and link these polypeptides to the first amino acid sequence according to routine technical means. These polypeptides are, for example, fibrinogen, albumin, Erythropoietin (EPO), human coagulation factor VIII.

Wherein the C-protein further comprises a histidine tag and a tag protein selected from thioredoxin (T protein), maltose binding protein (MBP protein), SUMO protein, and glutathione mercaptotransferase (GST). Meanwhile, as mentioned above, since protein C has a chemical modification site, the modification site can be modified with chemical groups commonly used in the art for improving protein performance or stability, including but not limited to: polyethylene glycol or polyethylene glycol derivatives, MMAE and DM 1. Thus the C (precursor) protein can be designated as H (histidine-tagged) S (tagged protein) I_C(Ssp GyrB cleavage protein C-segment sequence) -SAGC (first amino acid sequence) -mPEG (modified chemical group)

Wherein the N-protein further comprises a histidine tag, i.e.the N (precursor) protein may be designated G-CSF (protein polypeptide, second amino acid sequence) -I_N(Ssp GyrB cleavage protein N-stretch) -H (histidine tag).

In a second aspect of the present application, there is provided a protein modified with a chemical group, which is obtained by the above method and has excellent homogeneity.

The protein consists of a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence and the second amino acid sequence are connected through a peptide bond, the sequence of the first amino acid sequence is shown as SEQ ID NO. 3, the fourth site is a chemical modification site (and is unique), and the second amino acid sequence represents the amino acid sequence of a protein polypeptide drug.

As described above, the protein polypeptide drugs include granulocyte colony stimulating factor (G-CSF), human growth hormone (hGH), human interferon alpha 2b (IFN alpha 2b), interleukin-15 (ILK-15), and Urate Oxidase (UOX). However, it will be understood by those skilled in the art that the second amino acid sequence in the present application is not limited thereto.

Wherein the fourth site is modified with a water-soluble compound. The water soluble compound is polyethylene glycol or polyethylene glycol derivative, MMAE and DM 1.

In another aspect of the present application, the nucleotide sequence of the aforementioned C-segment sequence is designed, and specifically shown in SEQ ID NO. 4.

In another aspect of the present application, a nucleotide sequence of the aforementioned N-segment sequence is designed, specifically as shown in SEQ ID NO. 5.

Drawings

FIG. 1 is SDS-PAGE of the PEGylation product of protein C in example 3 (FIG. 1A is a gel including the S protein,

FIG. 1C including the T protein) and MALDI-TOF MS (FIG. 1B including the S protein and FIG. 1D including the T protein).

In FIG. 1A, the lanes are:

lane 1C-protein (HSI)_C-SAGC)。

Lane 2 Maleimide PEG (20 kD).

Lane 3, sample after completion of PEGylation reaction.

Lane 4 purified PEGylated protein C (HSI)_C-SAGC-mPEG)。

In fig. 1C, the lanes are:

lane 1C-protein (HTI)_C-SAGC)。

Lane 2 Maleimide PEG (20 kD).

Lane 3, sample after completion of PEGylation reaction.

Lane 4 purified PEGylated protein C (HTI)_C-SAGC-mPEG)。

FIG. 2 is the SDS-PAGE (FIG. 2A) and MALDI-TOF MS (FIG. 2B) identification of protein trans-splicing (protein trans-splicing) -mediated Granulocyte Colony-Stimulating Factor (G-CSF) C-terminal pegylation products of example 4.

Lane 1N-precursor protein (G-CSF-I)_N-H)。

Lane 2C-precursor protein (HSI)_C-SAGC-mPEG)。

Lane 3, samples after cleavage of N-and C-precursor proteins.

Lane 4, purified splicing product (G-CSF-SAGC-mPEG).

FIG. 3 is the SDS-PAGE (FIG. 3A) and MALDI-TOF MS (FIG. 3B) identification of the protein trans-splicing (protein trans-splicing) -mediated C-terminal pegylation product of human Growth Hormone (hGH) in example 5.

Lane 1N-precursor protein (hGH-I)_N-H)。

Lane 2C-precursor protein (HSI)_C-SAGC-mPEG)。

Lane 3, samples after cleavage of N-and C-precursor proteins.

Lane 4, purified splicing product (hGH-SAGC-mPEG).

FIG. 4 is the SDS-PAGE identification of the protein trans-splicing (protein trans-splicing) -mediated C-terminal PEGylation product of Interleukin-15 (Interleukin-15, ILK-15) in example 6.

Lane 1N-precursor protein (ILK-15-I)_N-H)。

Lane 2C-precursor protein (HTI)_C-SAGC-mPEG)。

Lane 3 sample after the cleavage of N-precursor protein and C-precursor protein, the cleavage product was ILK-15-SAGC-mPEG

FIG. 5 is the SDS-PAGE (FIG. 5A) and MALDI-TOF MS (FIG. 5B) identification of the transsplicing (protein trans-splicing) mediated Uricase (UOX) C-terminal PEGylation product of protein in example 7.

Lane 1N-precursor protein (UOX-I)_N-H)。

Lane 2C-precursor protein (HSI)_C-SAGC-mPEG)。

Lane 3, samples after cleavage of N-and C-precursor proteins.

Lane 4 purified splicing product (UOX-SAGC-mPEG).

FIG. 6 is the SDS-PAGE (FIG. 6A) and MALDI-TOF MS (FIG. 6B) identification of the protein trans-splicing (protein trans-splicing) -mediated C-terminal pegylation product of interferon alpha 2B (interferon alpha 2B, IFN alpha 2B) in example 8.

Lane 1N-precursor protein (IFN. alpha.2b-I)_N-H)。

Lane 2C-precursor protein(HSI_C-SAGC-mPEG)。

Lane 3, samples after cleavage of N-and C-precursor proteins.

Lane 4, purified splicing product (IFN. alpha.2b-SAGC-mPEG).

Detailed Description

Example 1: preparation of protein C

We selected the genetically engineered Ssp GyrB split protein intron for construction of the C-protein. The C-protein belongs to the fusion protein, which in this example comprises the 6 × histidine tag, the C-segment of the protein intron (SGsplit I) of the SUMO (SmallUbiquitin-like Modifier protein), SG (Ssp GyrB) in sequence_CSEQ ID NO:1), and an amino acid sequence SAGC containing only one cysteine (i.e., the first amino acid sequence, SEQ ID NO: 3). Furthermore, another option for the C-protein is the C-segment (SGsplit I) comprising in order the 6 × His-tag, the thioredoxin (T-protein), SG (Ssp GyrB) protein intron_CSEQ ID NO:1), and an amino acid sequence SAGC containing only one cysteine (i.e., the first amino acid sequence, SEQ ID NO: 3). Of course, it will be understood by those skilled in the art that other tag proteins may be selected from maltose binding protein (MBP protein), glutathione mercaptotransferase (GST), and the like.

Specifically, the 6 × histidine tag was used for affinity chromatography purification of C-protein. The SUMO protein or T protein is used for increasing the expression level of C-protein and improving the solubility of C-protein. C-segment of the intron of SG proteins (SGsplit I)_C) In subsequent examples 4 to 8, the N-segment was bound to each other and trans-splicing of the protein occurred. SAGC is considered to be a "protein exon" and is joined to the C-terminus of another "exon" after the splicing reaction is completed. Among them, S is involved in trans-splicing of proteins and is an essential element in splicing. C is a chemical group for modifying the activity of sulfhydryl. AG separates S and C as a segment of amino acid sequence to prevent chemical groups from affecting protein trans-splicing reaction.

The nucleotide sequence of the C-protein was cloned into the PET-28 vector, i.e., pE-HS (or T) I_C-SAGC. Plasmid pE-HS was transformed using standard transformation methods(or T) I_CSAGC was transformed into BL21(DE3) competent cells. The colonies containing the plasmid of interest were transferred to LB (10g peptone, 5g yeast extract, 10g NaCl/L, 50. mu.g/mL) medium. Incubate at 37 ℃ for 6h on a shaker at a rate of 1: 100(v/v) was inoculated in LB medium. Cultured to form A₆₀₀About 0.6, Isopropyl-beta-D-Thiogalactopyranoside (IPTG) is added to the mixture at a final concentration of 0.1-0.8 mM, and the mixture is induced at 16-25 ℃ for 12 hours. C-protein (HSI)_CSAGC) with Ni²⁺NTA agarose column (Qiagen) purification. First, the cells were collected by centrifugation (6000r/min, 10 min). Using a lysis buffer (50mM NaH)₂PO₄300mM NaCl, 10mM imidazole, pH 8.0), and disrupted by French Press (14,000 PSI). Centrifuging at low temperature (15000r/min, 30min), and collecting supernatant. The supernatant was poured into Ni²⁺In NTA agarose column, Wash buffer (50mM NaH)₂PO₄300mM NaCl, 20mM imidazole, pH 8.0). Using an Elution buffer (250mM NaH)₂PO₄300mM NaCl, 250mM imidazole, pH 8.0) elute C-protein (HS (or T) I_C-SAGC)。

Example 2: preparation of N-protein

In order to verify the universality of the method, a plurality of protein polypeptide drugs are respectively taken as target proteins. These protein polypeptide drugs include Granulocyte Colony-Stimulating Factor (G-CSF), human Growth Hormone (hGH), human interferon alpha 2b (interferon alpha 2b, IFN alpha 2b), Interleukin-15 (Interleukin 15, ILK-15), and Urate Oxidase (UOX). Various protein polypeptide drugs (namely a second amino acid sequence, the specific sequence can be seen in SEQ ID NO:6-10 in the sequence table) are sequentially combined with an N segment (SGsplit I) of a protein intron_N) (SEQ ID NO:2) and 6 XHis tag fusion expression, i.e.N protein (N)_drugI_NH)。

The nucleotide sequence of each N-protein was cloned into the PET-28 vector, i.e., pE-N_drugI_NH. Each N was separately converted according to standard transformation methods_drugI_NH was transformed into BL21(DE3) competent cells. Will be provided withThe colonies containing the plasmid of interest were transferred to LB (10g peptone, 5g yeast extract, 10g NaCl/L, 50. mu.g/mL) medium. Incubate at 37 ℃ for 6h on a shaker at a rate of 1: 100(v/v) was inoculated in LB medium. Cultured to form A₆₀₀About 0.6, Isopropyl-beta-D-Thiogalactopyranoside (IPTG) is added to the mixture at a final concentration of 0.2-0.8 mM, and the mixture is induced at 16-25 ℃ for 12 hours. All N-proteins (N)_drugI_NH) All adopt Ni²⁺NTA agarose column (Qiagen) purification. First, the cells were collected by centrifugation (6000r/min, 10 min). Using a lysis buffer (50mM NaH)₂PO₄300mM NaCl, 10mM imidazole, pH 8.0), and disrupted by French Press (14,000 PSI). Centrifuging at low temperature (15000r/min, 30min), and collecting supernatant. The supernatant was poured into Ni²⁺In NTA agarose column, Wash buffer (50mM NaH)₂PO₄300mM NaCl, 20mM imidazole, pH 8.0). Using an Elution buffer (250mM NaH)₂PO₄300mM NaCl, 250mM imidazole, pH 8.0) elute the N-protein (N)_drugI_NH)。

Example 3: PEGylation and purification of C-proteins

A schematic of the PEGylation and purification of the C-protein is shown in FIG. 1. The C-protein (HS (or T) I) obtained in example 1 was first isolated_CSAGC) dialysis against a labeling buffer (140mM NaCl, 2.7mM KCl, 10mM Na₂HPO₄，1.8mM KH₂PO₄pH 7.3) and tris (2-carbonylethyl) phosphonium hydrochloride (TCEP) was added to a final concentration of 1 to 10 mM. Adding thiol-active polyethylene glycol (PG1-ML-20k, NANOSC), wherein the molar ratio of the polyethylene glycol to the protein is 3-10: 1, standing at room temperature for 2.5-12 h. After the labeling reaction is finished, Dithiothreitol (DTT) is added to the solution to a final concentration of 1-10 mM. Due to the presence of C-protein (HS (or T) I)_CSAGC) only one cysteine is present, so that PEG with a thiol-reactive functional group is modified to the C-protein (HS (or T) I_CSAGC) at a specific site, forming the C-precursor protein (HS (or T) I)_C-SAGC-mPEG). The polyethylene glycol modifications were detected on SDS-PAGE gels (FIG. 1A, C).

For purification from the mixture of the above modification reactions to obtain the C-precursor protein (HS (or T) I)_C-SAGC-mPEG), the mixture was loaded onto an anion exchange column (1 × 10cm) and subsequently eluted. The elution buffer is Tris buffer (pH 7.0) containing 30 to 400mm nacl, and the elution peak of the modified product is collected. The modified product was identified by SDS-PAGE and MALDI-TOF-MS, respectively (as shown in FIG. 1).

The results of PEGylation of C-protein and purification of the C precursor protein are shown in FIGS. 1B and D, where it can be seen that a band with a larger molecular weight is formed after the modification reaction is completed, indicating that the C-protein is successfully modified by PEG (PG1-ML-20k, NANOCS). And again this is demonstrated by the MALDI-TOF-MS results.

Example 4: protein trans-splicing (protein trans-splicing) -mediated C-terminal pegylation of Granulocyte Colony-Stimulating Factor (G-CSF).

To achieve C-terminal PEGylation of Granulocyte Colony-Stimulating Factor (G-CSF), the N-protein (G-CSFI) obtained in example 2 was purified_NH) and C-precursor protein (HSI) from example 3_C-SAGC-mPEG) in a molar ratio of 1: 1 and mixing. The mixture was dialyzed into a splicing buffer (20mM Tris-HCl,150mM NaCl,1mM DTT,1mM EDTA, pH 8.0) and incubated overnight at 4-25 ℃. Samples were taken before and after the splicing reaction, and the protein trans-splicing reaction was detected using SDS-PAGE gels.

To purify the spliced product (G-CSF-SAGC-mPEG) from the mixture of the above-mentioned trans-splicing reaction of proteins, the mixture was applied to an anion exchange column (1X 10cm) and then eluted. The elution buffer is Tris buffer (pH 8.0) containing 30 to 400mM NaCl, and the elution peak of the modified product is collected. The modified product was identified by SDS-PAGE and MALDI-TOF-MS, respectively (as shown in FIG. 2).

The results of PEGylation of the C-terminal of granulocyte colony stimulating factor mediated by trans-splicing of protein and purification of the spliced product are shown in FIG. 2A, in which it can be seen that the protein is formed after the trans-splicing reaction of protein is completed3 new bands which can be judged from the molecular weight of the bands to be splicing products (G-CSF-SAGC-mPEG) and N, C fragments including protein introns (I)_NH and HSI_C) This indicates that successful trans-splicing of the protein has occurred. And again this is demonstrated by the MALDI-TOF-MS results. By analyzing the change in the density of the C-protein band in the SDS-PAGE patterns before and after the protein trans-splicing reaction, 80% of the granulocyte colony stimulating factor was site-specifically modified by PEG.

Example 5: protein trans-splicing (protein trans-splicing) mediated C-terminal pegylation of human Growth Hormone (hGH).

To achieve C-terminal pegylation of human Growth Hormone (hGH), the N-protein (hGH-I) obtained was purified in example 2_NH) and C-precursor protein (HSI) from example 3_C-SAGC-mPEG) in a molar ratio of 1: 1 and mixing. The mixture was dialyzed into a splicing buffer (20mM Tris-HCl, pH 8.0,150mM NaCl,1mM DTT,1mM EDTA) and incubated overnight at 4-25 ℃. Samples were taken before and after the splicing reaction, and the protein trans-splicing reaction was detected using SDS-PAGE gels.

To purify the spliced product (hGH-mPEG) from the mixture of the above-mentioned trans-splicing reaction of proteins, the mixture was applied to an anion exchange column (1X 10cm) and then eluted. The elution buffer is Tris buffer (pH 8.0) containing 30 to 400mM NaCl, and the elution peak of the modified product is collected. The modified product was identified by SDS-PAGE and MALDI-TOF-MS, respectively (as shown in FIG. 3).

The results of protein trans-splicing mediated C-terminal PEGylation of human growth hormone and purification of the spliced product are shown in FIG. 3A, in which it can be seen that 3 new bands are formed after the end of the protein trans-splicing reaction, and the molecular weights of these bands can be determined as splicing product (hGH-SAGC-mPEG) and N, C fragment including protein intron (I)_NH and HSI_C) This indicates that successful trans-splicing of the protein has occurred. And again this is demonstrated by the MALDI-TOF-MS results. By passingBy analyzing the change of the density of the C-protein band in the SDS-PAGE pattern before and after the protein trans-splicing reaction, 85% of human growth hormone was site-specifically modified by PEG.

Example 6: protein trans-splicing (protein trans-splicing) mediated C-terminal pegylation of Interleukin-15 (Interleukin-15, ILK-15).

To achieve C-terminal PEGylation of Interleukin-15 (Interleukin-15, ILK-15), the N-protein (ILK-15-I) obtained in example 2 was purified_NH) and C-precursor protein (HTI) from example 3_C-SAGC-mPEG) in a molar ratio of 1: 1 and mixing. The mixture was dialyzed into a splice buffer (20mM Tris-HCl,150mM NaCl,1mM DTT,1mM EDTA, pH 8.0) and incubated overnight at 4-25 ℃. Samples were taken before and after the splicing reaction, and the protein trans-splicing reaction was detected using SDS-PAGE gels.

The results of protein trans-splicing mediated interleukin-15C-terminal PEGylation and purification of the spliced product are shown in FIG. 4, in which it can be seen that 3 new bands are formed after the protein trans-splicing reaction is completed, and the molecular weights of these bands can be determined as the spliced product (ILK-15-SAGC-mPEG) and the N, C fragment including the protein intron (I)_NH and HTI_C) This indicates that successful trans-splicing of the protein has occurred. By analyzing the change of the density of the C-protein band in the SDS-PAGE pattern before and after the protein trans-splicing reaction, 65% of the interleukin-15 was site-specifically modified by PEG.

Example 7: protein trans-splicing (protein trans-splicing) mediated C-terminal pegylation of Urate Oxidase (UOX).

To achieve C-terminal PEGylation of Urate Oxidase (UOX), the resulting N-protein (UOX-I) was purified in example 2_NH) and C-precursor protein (HSI) from example 3_C-SAGC-mPEG) in a molar ratio of 1: 1 and mixing. The mixture was dialyzed into a splice buffer (20mM Tris-HCl, pH 8.0,150mM NaCl,1mM DTT,1mM EDTA) and incubated overnight at 4-25 ℃. Samples were taken before and after the splicing reaction and SDS-PAGE gels were usedDetecting the protein trans-splicing reaction.

To purify the spliced product (UOX-SAGC-mPEG) from the mixture of the above protein trans-splicing reactions, the mixture was applied to an anion exchange column (1X 10cm) and then eluted. The elution buffer is Tris buffer (pH 8.0) containing 30 to 400mM NaCl, and the elution peak of the modified product is collected. The modified product was identified by SDS-PAGE and MALDI-TOF-MS, respectively (as shown in FIG. 5).

The results of the PEGylation of the C-terminal of urate oxidase mediated by protein trans-splicing and the purification of the spliced product are shown in FIG. 5A, in which it can be seen that 3 new bands are formed after the end of the protein trans-splicing reaction, and the molecular weights of these bands can be determined as splicing product (UOX-SAGC-mPEG) and N, C fragment including protein intron (I)_NH and HSI_C) This indicates that successful trans-splicing of the protein has occurred. And again this is demonstrated by the MALDI-TOF-MS results. By analyzing the change in the density of the C-protein band in the SDS-PAGE pattern before and after the protein trans-splicing reaction, 95% of the urate oxidase was fixed-point modified with PEG.

Example 8: protein trans-splicing (protein trans-splicing) mediated C-terminal pegylation of interferon alpha 2b (interferon alpha 2b, IFN alpha 2 b).

To achieve C-terminal pegylation of interferon alpha 2b (interferon alpha 2b, IFN alpha 2b), the N-protein (IFN alpha 2b I) obtained was purified in example 2_NH) and C-precursor protein (HSI) from example 3_C-SAGC-mPEG) in a molar ratio of 1: 1 and mixing. The mixture was dialyzed into a splice buffer (20mM Tris-HCl, pH 8.0,150mM NaCl,1mM DTT,1mM EDTA) and incubated overnight at 4-25 ℃. Samples were taken before and after the splicing reaction, and the protein trans-splicing reaction was detected using SDS-PAGE gels.

To purify the spliced product (IFN. alpha.2b-SAGC-mPEG) from the mixture of the above-mentioned trans-splicing reactions of proteins, the mixture was applied to an anion exchange column (1X 10cm) and then eluted. The elution buffer is Tris buffer (PH 8.0) containing 30-400 mM NaCl, and the elution peak of the modified product is collected. The modified products were identified by SDS-PAGE and MALDI-TOF-MS, respectively (as shown in FIG. 6).

The purification results of interferon alpha 2bC terminal PEGylation and splicing products mediated by protein trans-splicing are shown in FIG. 6A, in which it can be seen that 3 new bands are formed after the protein trans-splicing reaction is finished, and the molecular weight of these bands can be used to judge that these new bands are respectively splicing products (IFN alpha 2b-SAGC-mPEG) and N, C fragments (I) including protein intron_NH and HSI_C) This indicates that successful trans-splicing of the protein has occurred. And again this is demonstrated by the MALDI-TOF-MS results. By analyzing the change in the density of the C-protein band in the SDS-PAGE pattern before and after the protein trans-splicing reaction, it was calculated that 95% of interferon alpha 2b was site-specifically modified by PEG.

Claims

1. A method for site-directed modification of a protein using a split intein comprising:

i. preparing a C-protein comprising a sequence of the C-stretch of the split intein and a first amino acid sequence and chemically modifying said C-protein;

preparing an N-protein comprising the sequence of the N-stretch of the split intein and a second amino acid sequence;

connecting the C-protein with the N-protein by a trans-shearing technology, wherein the first and the second amino acid sequences are connected by peptide bonds, and the C-segment sequence is shown as SEQ ID NO. 1, the N-segment sequence is shown as SEQ ID NO. 2,

wherein, the sequence of the first amino acid sequence is shown as SEQ ID NO. 3, the fourth site of the first amino acid sequence is a chemical modification site, and the second amino acid sequence represents the amino acid sequence of a protein polypeptide drug.

2. The method of claim 1, wherein said C-protein further comprises a histidine tag and a tag protein selected from thioredoxin (T protein), maltose binding protein (MBP protein), SUMO protein (S protein), and glutathione mercaptotransferase (GST).

3. The method of claim 1, wherein the N-protein further comprises a histidine tag.

4. The protein with the chemical modification group is composed of a first amino acid sequence and a second amino acid sequence, wherein the first amino acid sequence and the second amino acid sequence are connected through a peptide bond, the sequence of the first amino acid sequence is shown as SEQ ID NO. 3, the fourth site of the first amino acid sequence is a chemical modification site, and the second amino acid sequence represents the amino acid sequence of a protein polypeptide drug.

5. The protein of claim 4, wherein said protein polypeptide drug comprises granulocyte colony-stimulating factor (G-CSF), human growth hormone (hGH), human interferon alpha 2b (IFN alpha 2b), interleukin-15 (ILK-15), and Urate Oxidase (UOX).

6. The protein of claim 4, wherein said fourth site is modified with a water-soluble compound.

7. The protein of claim 6, wherein the water soluble compound is polyethylene glycol or a polyethylene glycol derivative, MMAE and DM 1.

8. A split intein comprising a C-segment sequence and an N-segment sequence, wherein the C-segment sequence is shown as SEQ ID NO. 1, and the nucleotide sequence is shown as SEQ ID NO. 4; the N-segment sequence is shown as SEQ ID NO. 2, and the nucleotide sequence is shown as SEQ ID NO. 5.