CN117802138A

CN117802138A - Rinacipran soluble intermediate and preparation method of linaclotran

Info

Publication number: CN117802138A
Application number: CN202410229264.8A
Authority: CN
Inventors: 洪浩; 詹姆斯·盖吉; 张娜; 赵军旗; 焦学成; 岳卓; 李奕铭; 关莹; 李艳玲
Original assignee: Tianjin Kailaiying Biotechnology Co ltd
Current assignee: Tianjin Kailaiying Biotechnology Co ltd
Priority date: 2024-02-29
Filing date: 2024-02-29
Publication date: 2024-04-02
Anticipated expiration: 2044-02-29
Also published as: CN117802138B

Abstract

The invention provides a linaclotide soluble intermediate and a preparation method of linaclotide. The preparation method comprises the following steps: expressing linaclotide soluble intermediates using host cells containing recombinant plasmids containing a gene capable of expressing a Sumo tag protein and a gene capable of expressing linaclotide; host cells include Shuffle T7-B cells or Origami B (DE 3) cells. Can solve the problem of low activity of linaclotide prepared and obtained in the prior art, and is suitable for the field of medicine biosynthesis.

Description

Rinacipran soluble intermediate and preparation method of linaclotran

Technical Field

The invention relates to the field of medicine biosynthesis, in particular to a linaclotide soluble intermediate and a preparation method of linaclotide.

Background

Linaclotide (Linaclotide), a GC-C (guanylate cyclase-C) agonist drug useful for clinical local intestinal onset, is useful for treating constipation-predominant irritable bowel syndrome (IBS-C) and Chronic Idiopathic Constipation (CIC) adult patients.

At present, linaclotide is mainly obtained by chemical coupling synthesis and inclusion body expression, wherein the chemical coupling synthesis process is relatively complex, impurities are easy to generate in the synthesis process, and the purity and the recovery rate are relatively low. The inclusion body expression has higher expression quantity, can avoid protease degradation, but the inclusion body is subjected to subsequent denaturation and renaturation to purify the inclusion body to obtain a final product, and a large amount of a modifier such as urea or guanidine hydrochloride is used in the process, so that the purification process is complex, the problem of folding inside the linaclotide is easily caused, and the activity of the final linaclotide is low.

Disclosure of Invention

The invention mainly aims to provide a linaclotide soluble intermediate and a preparation method of linaclotide, which are used for solving the problem of low activity of the linaclotide prepared and obtained in the prior art.

In order to achieve the above object, according to one aspect of the present invention, there is provided a method for preparing a linaclotide soluble intermediate, the method comprising: expressing linaclotide soluble intermediates using host cells containing recombinant plasmids containing a gene capable of expressing a Sumo tag protein and a gene capable of expressing linaclotide; host cells include Shuffle T7-B cells or Origami B (DE 3) cells.

Further, the recombinant plasmid contains a gene capable of expressing Sumo tag protein and a gene capable of expressing linaclotide which are sequentially connected in the direction of 5'-3' end.

Further, genes capable of expressing the Sumo tag protein include those comprising SEQ ID NO:1, or a polynucleotide having a nucleotide sequence set forth in SEQ ID NO:1 and a disulfide bond-containing structure, wherein the nucleotide sequence has a identity of 70% or more; genes capable of expressing linaclotide include those comprising SEQ ID NO:2, or a polynucleotide having a nucleotide sequence set forth in SEQ ID NO:2, and a polynucleotide having a nucleotide sequence having a identity of more than 70%.

Further, the recombinant plasmid contains SEQ ID NO:3, and a nucleotide sequence shown in 3.

In order to achieve the above object, according to a second aspect of the present invention, there is provided a method for producing linaclotide, comprising: linaclotide is obtained by excision of the Sumo tag protein on the linaclotide soluble intermediate obtained by the preparation method of any one of the above-mentioned linaclotide soluble intermediates.

Further, removing Sumo tag protein on the linaclotide soluble intermediate to obtain crude linaclotide; purifying the crude linaclotide to obtain linaclotide.

Further, the resecting comprises: the enzyme is used for cutting the soluble intermediate of the linaclotide to obtain the separated Sumo tagged protein and the crude linaclotide.

Further, the purification includes: regulating the pH of a system containing crude linaclotide to the isoelectric point of Sumo tag protein to obtain a tag protein precipitation system; mixing the tagged protein precipitation system with an organic solution and centrifuging, and separating the supernatant to obtain linaclotide.

Further, the isoelectric point is pH 5.4 to 5.8, more preferably pH 5.6.

Further, the organic solution comprises one or more of acetonitrile solution, hexafluoroisopropanol solution or acetone solution; preferably, the acetonitrile solution is an aqueous acetonitrile solution having a volume fraction of acetonitrile of 50% to 70%, more preferably 60%.

By applying the technical scheme of the invention, a preparation method of the linaclotide soluble intermediate is provided, recombinant plasmids containing genes capable of expressing Sumo tag proteins and genes capable of expressing linaclotide are constructed, the linaclotide soluble intermediate prepared by the preparation method has good solubility, and the linaclotide is not required to be denatured, renatured or reduced by adding components such as protein denaturant, renaturation agent or reducing agent when being prepared later, so that the final product linaclotide has high activity.

Drawings

The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the invention. In the drawings:

FIG. 1 is a graph showing the results of electrophoresis detection of precipitation and supernatant after induced centrifugation of a strain expressing a soluble intermediate of linaclotide according to example 1 of the present invention.

FIG. 2 shows a graph of the results of electrophoresis detection after purification of a fusion protein comprising a soluble intermediate of linaclotide according to example 1 of the present invention.

FIG. 3 shows a graph of the results of electrophoresis detection of a soluble intermediate of linaclotide after purification according to example 1 of the present invention.

FIG. 4 shows a graph of LC-MS detection mass spectrum of linaclotide according to example 1 of the present invention.

Detailed Description

It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other. The present invention will be described in detail with reference to examples.

As mentioned in the background art, the preparation of linaclotide in the prior art adopts a chemical synthesis preparation method, which not only has complicated process and high cost, but also can generate raceme, thus leading to longer preparation period and difficult quality control of products. When the fusion protein is prepared to express the linaclotide, the expression quantity is low, inclusion bodies are easy to form, and the misfolding of the inclusion bodies needs to be corrected and purified by adding a protein denaturing agent and a renaturation agent, so that the preparation process is complex, and the activity of the final product linaclotide is low. In this application the inventors have tried to develop a soluble intermediate of linaclotide and a process for preparing linaclotide, and thus proposed a series of protection schemes of this application.

In a first exemplary embodiment of the present application, there is provided a process for preparing a soluble intermediate of linaclotide, the process comprising: expressing a linaclotide soluble intermediate using a host cell comprising a recombinant plasmid comprising a gene capable of expressing a Sumo tag protein and a gene capable of expressing a linaclotide; such host cells include Shuffle T7-B cells or Origami B (DE 3) cells.

Protein tag (Protein tag) refers to an application technology of fusing a polypeptide, a Protein domain or an intact Protein with a specific function with a target Protein by using a gene cloning means so as to realize expression purification, detection, tracing and the like of the target Protein. The Sumo (Small ubiquitin-like modifier) tag protein is a Small molecule ubiquitin-like modified protein, and can be used as a protein tag and a molecular chaperone expressed by recombinant proteins for increasing the solubility, stability and purity of target proteins. The method for carrying out fusion expression on Sumo tag protein and linaclotide realizes high-efficiency soluble expression on polypeptides containing three pairs of disulfide bonds, and improves the soluble expression of the linaclotide.

Inclusion bodies refer to high-density, insoluble protein particles formed by membrane encapsulation when exogenous genes are expressed in prokaryotic cells, the formation of which is related to the rate of formation of cytoplasmic proteins, the concentration of newly produced polypeptides is high, and there is insufficient time to fold, thereby facilitating the formation of amorphous, amorphous protein aggregates. Therefore, in the existing method, protein denaturants and renaturation agents are usually required to be added to correct and renature the misfolded inclusion bodies, and excessive reagents are required to be removed after purification, so that linaclotide can be prepared and obtained.

In the prior art, in order to increase the yield of linaclotide, a method of increasing the copy number of linaclotide (including but not limited to using one fusion tag to express multiple target proteins in series) is generally adopted to prepare a fusion protein, but there is no mention of a fusion protein obtained by combining multiple copies of linaclotide with a protein tag to express the fusion protein, so that a soluble expression effect can be achieved. Meanwhile, all fusion proteins after tandem expression in the prior art need to be treated by urea, which indicates that the fusion proteins expressed by the method are insoluble and the fusion proteins need to be treated by a protein denaturant. Since the linaclotide contains multiple pairs of disulfide bonds, cyclization is easy to occur, the purification efficiency of the obtained polypeptide Q Sepharose is low, so that in the prior art, in order to avoid the problem, linearization is also needed for fusion proteins, the influence caused by disulfide bond cyclization in the linaclotide is eliminated, then purification is carried out, and cyclization treatment is carried out after the purified linearized linaclotide is obtained, so that the final product can be obtained. Although disulfide bond cyclization inside the linaclotide is avoided in the prior art, the linaclotide is obtained by multiple denaturation and purification of reactants, so that the final product has low activity, and the method has the advantages of more purification times, complicated steps, high technical requirements on operators and relatively high preparation cost.

In the examples of the present application, various tags of Sumo (Small ubiquitin-like modifier), trx (Thioredoxin), fh8 (Fasciola hepatica putative calcium-Binding protein), ffu209 (Fluorescent Fusion Ubiquitin 209) and CBM (Carbohydrate-Binding Module) were fused with a target polypeptide sequence, and constructed in pET-28a (+) expression vector. pET-28a-Sumo, pET-28a-Trx, pET-28a-Fh8, pET-28a-Ffu209 and pET-28a-CBM vector frameworks are respectively connected with genes capable of expressing linaclotide and respectively transformed into a host cell of Shuffle T7-B, origamiB (DE 3) and BL21 (DE 3) for expression. Experiments show that compared with other labels, sumo can express and obtain fusion protein with better solubility in a shuffleT7-B cell when being connected with a linaclotide gene, and can not form inclusion bodies, so that the problem of inclusion body renaturation can be avoided, the expression quantity is higher, and the soluble expression of the protein can be efficiently promoted, so that a linaclotide soluble intermediate can be directly prepared without adding a protein denaturing agent and a renaturation agent, and further, a target product linaclotide with high activity and high purity can be obtained through simple purification steps.

In a preferred embodiment, the recombinant plasmid contains a gene capable of expressing a Sumo tag protein and a gene capable of expressing linaclotide, linked in sequence in the 5'-3' direction.

The gene capable of expressing Sumo label protein and the gene capable of expressing linaclotide are connected, and a linaclotide soluble intermediate formed by connecting Sumo protein and linaclotide can be obtained in subsequent expression, wherein the Sumo label is positioned at the N end of the linaclotide.

Preferably, on a recombinant plasmid, a gene capable of expressing the Sumo tag protein is ligated to only a gene capable of expressing linaclotide.

In a preferred embodiment, the gene capable of expressing the Sumo tag protein comprises a gene comprising SEQ ID NO:1, or a polynucleotide having a nucleotide sequence set forth in SEQ ID NO:1 and a disulfide bond-containing structure, wherein the nucleotide sequence has a identity of 70% or more; genes capable of expressing linaclotide include those comprising SEQ ID NO:2, or a polynucleotide having a nucleotide sequence set forth in SEQ ID NO:2, and a polynucleotide having a nucleotide sequence having a identity of more than 70%.

SEQ ID NO：1：

ATGCATCATCATCATCATCATGGCAGTCTGCAAGATAGCGAAGTGAATCAAGAAGCGAAGCCAGAAGTGAAACCGGAAGTTAAACCGGAGACCCACATCAATCTGAAGGTGAGCGACGGCAGCAGCGAGATCTTCTTCAAGATCAAGAAGACGACCCCGCTGCGTCGTCTGATGGAAGCCTTCGCCAAACGCCAAGGCAAAGAAATGGACAGTCTGCGCTTTCTGTACGATGGTATCCGCATCCAAGCCGATCAAGCCCCGGAAGATCTGGACATGGAGGACAACGACATCATCGAGGCGCATCGCGAACAGATCGGCGGC。

SEQ ID NO：2：

TGCTGCGAATATTGCTGCAACCCGGCGTGCACCGGCTGCTAT。

In a preferred embodiment, the recombinant plasmid comprises the sequence of SEQ ID NO:3, and a nucleotide sequence shown in 3.

SEQ ID NO：3：

ATGCATCATCATCATCATCATGGCAGTCTGCAAGATAGCGAAGTGAATCAAGAAGCGAAGCCAGAAGTGAAACCGGAAGTTAAACCGGAGACCCACATCAATCTGAAGGTGAGCGACGGCAGCAGCGAGATCTTCTTCAAGATCAAGAAGACGACCCCGCTGCGTCGTCTGATGGAAGCCTTCGCCAAACGCCAAGGCAAAGAAATGGACAGTCTGCGCTTTCTGTACGATGGTATCCGCATCCAAGCCGATCAAGCCCCGGAAGATCTGGACATGGAGGACAACGACATCATCGAGGCGCATCGCGAACAGATCGGCGGCTGCTGCGAATATTGCTGCAACCCGGCGTGCACCGGCTGCTATTAA。

Identity (Identity) in the present application refers to "Identity" between amino acid sequences or nucleic acid sequences, i.e. the sum of the ratios of amino acid residues or nucleotides of the same kind in an amino acid sequence or a nucleic acid sequence. The identity of amino acid sequences or nucleic acid sequences can be determined using alignment procedures such as BLAST (Basic Local Alignment Search Tool), FASTA, etc.

The above-mentioned sequence is identical to SEQ ID NO:2, and the active site, the active pocket, the active mechanism, the protein structure and the like of the protein which have the same identity and have the same functions are the same as those provided by the above sequences, and have 70%, 75%, 80%, 85%, 90%, 95%, 99% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 98.5%, 99%, 99.5%, 99.6%, 99.7%, 99.8% or more, and even 99.9% or more).

As used herein, amino acid residues are abbreviated as follows: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gln; Q), glycine (Gly; G), histidine (His; H), isoleucine (Ile; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y) and valine (Val; V).

The rules of substitution, replacement, etc., generally, which amino acids are similar in nature, and the effect after replacement is similar. For example, in the above homologous proteins, conservative amino acid substitutions may occur. "conservative amino acid substitutions" include, but are not limited to:

the hydrophobic amino acid (Ala, cys, gly, pro, met, val, ile, leu) is substituted by other hydrophobic amino acids;

the hydrophobic amino acid (Phe, tyr, trp) with large side chain is replaced by other hydrophobic amino acids with large side chain;

the amino acid (Arg, his, lys) with positive side chain is replaced by other amino acids with positive side chains;

the amino acid (Ser, thr, asn, gln) with the side chain having the polarity uncharged is substituted with other amino acids with the side chain having the polarity uncharged.

The amino acids may also be conservatively substituted by those skilled in the art according to amino acid substitution rules well known to those skilled in the art as the "blosum62 scoring matrix" in the art.

By the above preparation method, a linaclotide soluble intermediate including Sumo tagged protein and linaclotide can be obtained.

Wherein the Sumo tag protein comprises SEQ ID NO:4, or an amino acid sequence as set forth in SEQ ID NO:4, and has a disulfide bond-containing structure, wherein the amino acid sequence of the polypeptide has a identity of 70% or more; linaclotide comprises SEQ ID NO:5, or an amino acid sequence as set forth in SEQ ID NO:5, and the amino acid sequence has more than 70% of identity. The linaclotide soluble intermediate is SEQ ID NO:6, and a polypeptide having the amino acid sequence shown in FIG. 6.

SEQ ID NO：4：

HHHHHHGSLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGG。

SEQ ID NO：5：

CCEYCCNPACTGCY。

SEQ ID NO：6：

HHHHHHGSLQDSEVNQEAKPEVKPEVKPETHINLKVSDGSSEIFFKIKKTTPLRRLMEAFAKRQGKEMDSLRFLYDGIRIQADQAPEDLDMEDNDIIEAHREQIGGCCEYCCNPACTGCY。

In a second exemplary embodiment of the present application, there is provided a method for preparing linaclotide, the method comprising: linaclotide is obtained by excision of the Sumo tag protein on the soluble intermediate of linaclotide prepared by any of the above preparation methods.

In a preferred embodiment, the Sumo tag protein on the soluble intermediate of linaclotide is cleaved to obtain crude linaclotide; purifying the crude linaclotide to obtain linaclotide.

In a preferred embodiment, the resecting comprises: the enzyme is used for cutting the soluble intermediate of the linaclotide to obtain the separated Sumo tagged protein and the crude linaclotide.

Since there are many cysteines in linaclotide, disulfide bonds are easily formed in linaclotide to affect the digestion effect, it is difficult to effectively separate tag proteins from linaclotide when preparing soluble intermediates of linaclotide. In the prior art, crude linaclotide products are separated and purified by adding a reducing agent for decyclization and then enzyme digestion, and the purified linear polypeptides need to be cyclized again to form functional target polypeptides. The method has a plurality of steps, and complicated process flows such as decyclization, cyclization and the like are required to easily cause the problems of reduction or even loss of the activity of the polypeptide and possible reduction of the activity and purity. In the application, when the label protein is subjected to enzyme digestion, a reducing agent is not required to be added, and the target polypeptide linaclotide can be obtained only through one-step enzyme digestion and one-step purification. The purification method utilizes an organic solvent for precipitation, can realize the separation and purification of the target polypeptide without using methods such as column chromatography and the like, has simple purification steps and low cost, and is beneficial to industrialized mass production. Compared with the prior art, the preparation method has the advantages of fewer steps, simple operation, high enzyme digestion and purification efficiency, and capability of preparing the linaclotide with high activity and high purity.

Enzymes herein refer to enzymes capable of acting on the Sumo protein tag to separate it from the polypeptide of interest, such enzymes including but not limited to Ulp1 enzymes.

In a preferred embodiment, the purification comprises: regulating the pH of a system containing crude linaclotide to the isoelectric point of Sumo tag protein to obtain a tag protein precipitation system; mixing the tagged protein precipitation system with an organic solution and centrifuging, and separating the supernatant to obtain linaclotide.

In a preferred embodiment, the isoelectric point is between pH 5.4 and 5.8, including but not limited to 5.4, 5.5 or 5.6; more preferably pH 5.6.

In a preferred embodiment, the organic solution includes, but is not limited to, one or more of acetonitrile solution, hexafluoroisopropanol solution, or acetone solution; preferably, the acetonitrile solution is 50% -70% by volume, including but not limited to 50%, 55%, 60%, 65% or 70%; more preferably 60% acetonitrile in water.

In the method, the pH is adjusted to the isoelectric point of Sumo tag protein, so that the Sumo tag protein is precipitated, an organic solvent acetonitrile precipitation method is used, the Sumo tag protein and the linaclotide are combined, the final product linaclotide is obtained, the purity and the activity of the product obtained through the purification method are high, the yield is relatively high, and the linaclotide is precipitated and separated by using the organic solvent, so that the preparation process is simple and efficient, and the cost is reduced.

In the preparation method, the crude linaclotide is treated by using acetonitrile and other organic solvents, and the target product linaclotide is separated by precipitating the tag protein. The purification by using chromatographic purification and other methods is avoided, the purification steps are simple and convenient, the loss rate of the target product is low, and the activity of the final product linaclotide is high.

The advantageous effects of the present application will be explained in further detail below in connection with specific examples.

Example 1

A. Construction of Sumo-tag fusion expression of linaclotide Gene engineering Strain

The DNA capable of expressing Sumo protein tag and the DNA sequence capable of expressing target polypeptide are fused, and expression vector is constructed in pET-28a (+) plasmid.

1. And (3) connecting the Sumo protein tag with a pET-28a (+) plasmid to construct a pET-28a-Sumo and a vector skeleton.

2. The target polypeptide sequence is shown in SEQ NO: 1.

DNA capable of expressing the target polypeptide fragment was ligated to pET-28a-Sumo vector backbone by homologous recombination, and the ligation product was transformed into Sheffle T7-B (available from Beijing bang nationality biological gene technologies Co., ltd., cat# zc 1229-2) and subjected to monoclonal sequencing analysis to select a strain containing the correct clone expression vector pET-28a-Sumo-lina (lina is an abbreviation of Linaclotide).

3. 3 strains are selected and inoculated in 250mL shake flasks for screening, different clone bacterial sludge is crushed, centrifuged, sediment is subjected to SDS-PAGE detection, and the group with better expression is screened by electrophoresis band depth, so that the final expression strain in each strain is screened. 4mL of the strain containing the recombinant plasmid is inoculated into a 2L triangular flask containing 400mL of LB culture medium, when the culture is carried out at 37 ℃ and 200rpm in a shaking way until the OD600 is 1.0, IPTG with the final concentration of 0.2mM is added, induction is carried out overnight at 25 ℃, after the induction is finished, the thalli are collected by centrifugation. Supernatants were collected by sonication and detected by SDS-PAGE with 17% separation gel. The electrophoresis detection result is shown in FIG. 1, wherein lane M represents Marker band, lane 1 represents band of supernatant for electrophoresis detection, lane 2 represents band of sediment for electrophoresis detection, wherein the band indicated by arrow represents position of target protein Sumo-lina fusion protein in lane, sumo-lina is Sumo tag protein-linaclotide soluble intermediate in the figure.

B. Purification of fusion proteins

1. The expressed Sumo-lina strain was resuspended in 20% bacterial concentration, sonicated (5 s sonicated, 5s interval, 30% power) and centrifuged, and the supernatant was filtered through a 0.45 μm filter to obtain a crude enzyme solution.

2. The crude enzyme solution obtained was purified by affinity chromatography (AKTA system assembled with 5mL HisTrap HP):

the filter samples were loaded at a flow rate of 5mL/min and then rinsed with binding buffer (50 mM Tris-HCl,200mM NaCl,pH 8.0) until unbound protein was completely eluted. The heteroprotein was eluted for 4 column volumes using buffer 50mM Tris-HCl,200mM NaCl,50mM imidazole at pH 8.0. Finally, the target protein is eluted under the conditions of 50mM Tris-HCl,200mM NaCl,300mM imidazole and pH=8.0 of an elution buffer, the detection result is shown in a graph in fig. 2, wherein a lane M represents a Marker band, a lane 1 represents a band after electrophoresis detection of the eluted target protein, a band indicated by an arrow represents that the target protein contains a Sumo-lina fusion protein, and Sumo-lina is a Sumo tag protein-linaclotide soluble intermediate in the graph.

It was calculated that 1g of bacterial sludge could yield 40.5mg of purified Sumo-lina fusion protein (which could be further improved by condition optimization).

C. Purification of linaclotide

The purified Sumo-Lina was digested with Ulp1, the mass ratio of Ulp1 to Sumo-Lina being 1:40 (mg/mg), and cleavage at 30℃for 16h. Then adjusting pH to 5.6 with hydrochloric acid, adding 60% acetonitrile into the reaction system, mixing uniformly, oscillating at 30 ℃ for 2 hours, centrifuging at 12000rpm to separate supernatant and precipitate, detecting polypeptide separation by using 17% SDS-PAGE, wherein the detection result is shown in figure 3, lane M represents a Marker band, lane 1 represents a band of electrophoresis detection of supernatant obtained after purification and separation of a Sumo-lina fusion protein, and the band indicated by an arrow represents that linaclotide is contained in the separated supernatant; lane 2 represents the band of the sediment detected by electrophoresis after purification and separation of the Sumo-lina fusion protein, the band indicated by the arrow represents the fact that the sediment contains Sumo-tagged protein, in which lina refers to linaclotide and Sumo refers to Sumo-tagged protein.

pH5.6 is the isoelectric point of His-Sumo, and in the above preparation method, since pET28a (+) contains a His tag, the protein obtained by actual expression is His-Sumo-linaclotide.

D. Mass spectrometric detection of polypeptide molecular weight

The molecular weight of the prepared polypeptide was analyzed by LC-MS.

1. Samples were first separated by HPLC column: agilent ZORBAX Edipse Plus C18, 4.6X100 mm,3.5 μm, mobile phase A:0.1% trifluoroacetic acid, mobile phase B:0.1% acetonitrile solution of trifluoroacetic acid, adopting a gradient elution mode: 0min 10%B,9min 95%B,12min 100%B,12.1min 10%B,15min 10%B, column temperature 50 ℃, UV detector 210nm, flow rate 0.3mL/min.

2. The components separated by HPLC are subjected to Q exact HF combined quadrupole Orbitrap mass spectrometer, an electrospray ion source (Dual AJS ESI) is used, positive ion mode detection is set, the sheath air flow rate is 35arb, the auxiliary air flow rate is 8arb, the spray voltage is 3800V, the temperature of an ion transmission tube is 320 ℃, the scanning range is 200-3000 m/z, and mass spectrum data are processed by BioPharma Finder software. Finally, linaclotide has a theoretical molecular weight of 1526.8Da, and mass spectrum analysis shows that Linaclotide has a molecular weight of 1526.4, and a mass spectrum is shown in fig. 4, wherein Linaclotide is Linaclotide. The protein tag-linaclotide expression amounts in this example are shown in table 1.

Example 2

The difference from example 1 is that the host cell is Origami B (DE 3) (available from Shanghai Biotechnology Co., ltd.; cat# EC 1020) and the remaining steps are identical to those of example 1. The protein tag-linaclotide expression amounts in this comparative example are shown in table 1.

Comparative example 1

The difference from example 1 is that the host cell is Origami B (DE 3), the tag protein is Trx, and the remaining steps are identical to those of example 1. The protein tag-linaclotide expression amounts in this comparative example are shown in table 1.

Comparative example 2

The difference from example 1 is that the host cell is Origami B (DE 3), the tag protein is Ffu209, and the remaining steps are identical to those of example 1. The protein tag-linaclotide expression amounts in this comparative example are shown in table 1.

Comparative example 3

The difference from example 1 is that the host cell is Origami B (DE 3), the tag protein is Fh8, and the remaining steps are identical to those of example 1. The protein tag-linaclotide expression amounts in this comparative example are shown in table 1.

Comparative example 4

The difference from example 1 is that the host cell is Origami B (DE 3), the tag protein is CBM, and the remaining steps are identical to those of example 1. The protein tag-linaclotide expression amounts in this comparative example are shown in table 1.

Comparative example 5

The difference from example 1 is that the tag protein is Trx and the remaining steps are identical to example 1. The protein tag-linaclotide expression amount and the theoretical yield of the pure product in this comparative example are shown in Table 1.

Comparative example 6

The difference from example 1 is that the tag protein is Ffu209 and the remaining steps are identical to example 1. The protein tag-linaclotide expression amount and the theoretical yield of the pure product in this comparative example are shown in Table 1.

Comparative example 7

The difference from example 1 is that the tag protein is Fh8, the remaining steps are identical to example 1. The protein tag-linaclotide expression amount and the theoretical yield of the pure product in this comparative example are shown in Table 1.

Comparative example 8

The difference from example 1 is that the tag protein is a CBM and the remaining steps are identical to those of example 1. The protein tag-linaclotide expression amount and the theoretical yield of the pure product in this comparative example are shown in Table 1.

Comparative example 9

The difference from example 1 is that the host cell is BL21 (DE 3) (product number zc121 from the company Utility biological gene technology Co., ltd., beijing) and the rest of the procedure is identical to that of example 1. The protein tag-linaclotide expression amounts in this comparative example are shown in table 1.

Comparative example 10

The difference from example 1 is that the host cell is BL21 (DE 3), the tag protein is Trx, and the remaining steps are identical to those of example 1. The protein tag-linaclotide expression amounts in this comparative example are shown in table 1.

Comparative example 11

The difference from example 1 is that the host cell is BL21 (DE 3) and the tag protein is Ffu209; the remaining steps are identical to those of example 1. The protein tag-linaclotide expression amounts in this comparative example are shown in table 1.

Comparative example 12

The difference from example 1 is that the host cell is BL21 (DE 3) and the tag protein is Fh8; the remaining steps are identical to those of example 1. The protein tag-linaclotide expression amounts in this comparative example are shown in table 1.

Comparative example 13

The difference from example 1 is that the host cell is BL21 (DE 3) and the tag protein is CBM; the remaining steps are identical to those of example 1. The protein tag-linaclotide expression amounts in this comparative example are shown in table 1.

TABLE 1

。

By comparing the Shuffle T7-B, origami B (DE 3) and BL21 (DE 3) hosts with different pET-28a-Tag-lina, wherein the expression level of the lina fusion protein is highest in the Shuffle T7-B host cells and can reach 40.5mg/g wet cells, the soluble expression of linaclotide containing three pairs of disulfide polypeptides is realized.

From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: compared with other protein tags for enhancing the solubility of target proteins in the prior art, the method for preparing the linaclotide soluble intermediate in the Sheffle T7-B host cell by utilizing the Sumo tag protein has the advantages that the linaclotide soluble intermediate containing the Sumo tag protein expressed in the Sheffle T7-B host cell is not limited by the problem of inclusion body renaturation, the inclusion body is not required to be treated by using an additional reagent, the expression quantity is high, and the linaclotide can be more efficiently and effectively expressed. When the linaclotide is purified, only a simple organic solvent precipitation method is needed to purify the target polypeptide, so that the step of purifying the product is simplified, and compared with the method for enzyme digestion of the polypeptide after enzyme digestion of a protein tag in the prior art, the method for enzyme digestion of the polypeptide after cyclization is relatively low in cost and high in efficiency, and the activity of the final product linaclotide can be further improved.

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

Claims

1. A process for the preparation of a linaclotide soluble intermediate, comprising:

expressing the linaclotide soluble intermediate using a host cell containing a recombinant plasmid containing a gene capable of expressing a Sumo tag protein and a gene capable of expressing linaclotide;

the host cells include Shuffle T7-B cells or Origami B (DE 3) cells.

2. The method according to claim 1, wherein the recombinant plasmid contains the gene capable of expressing Sumo tag protein and the gene capable of expressing linaclotide, which are sequentially linked in the 5'-3' direction.

3. The method of claim 1, wherein the gene capable of expressing a Sumo tag protein comprises a polypeptide comprising SEQ ID NO:1, or a polynucleotide having a nucleotide sequence set forth in SEQ ID NO:1 and a disulfide bond-containing structure, wherein the nucleotide sequence has a identity of 70% or more;

the gene capable of expressing linaclotide comprises a gene comprising SEQ ID NO:2, or a polynucleotide having a nucleotide sequence set forth in SEQ ID NO:2, and a polynucleotide having a nucleotide sequence having a identity of more than 70%.

4. A method according to any one of claims 1 to 3, wherein the recombinant plasmid comprises the sequence set forth in SEQ ID NO:3, and a nucleotide sequence shown in 3.

5. A process for the preparation of linaclotide, comprising:

the linaclotide is obtained by excision of a Sumo tag protein on the linaclotide soluble intermediate obtained by the preparation method of any one of claims 1-4.

6. The method of claim 5, wherein the Sumo tag protein on the soluble intermediate of linaclotide is cleaved to obtain crude linaclotide;

purifying the crude linaclotide to obtain the linaclotide.

7. The method of preparing according to claim 5 or 6, wherein the excision comprises: cleaving the linaclotide soluble intermediate with an enzyme to obtain the separated Sumo tagged protein and crude linaclotide.

8. The method of preparing according to claim 6, wherein the purifying comprises:

regulating the pH of a system containing the crude linaclotide to the isoelectric point of the Sumo tag protein to obtain a tag protein precipitation system;

mixing and centrifuging the tagged protein precipitation system with an organic solution, and separating the supernatant to obtain the linaclotide.

9. The method according to claim 8, wherein the isoelectric point is pH 5.4 to 5.8.

10. The method of claim 9, wherein the isoelectric point is pH 5.6.

11. The method of claim 8, wherein the organic solution comprises one or more of an acetonitrile solution, a hexafluoroisopropanol solution, or an acetone solution.

12. The method of claim 11, wherein the acetonitrile solution is an aqueous acetonitrile solution having a volume fraction of acetonitrile of 50% to 70%.

13. The method of claim 12, wherein the aqueous acetonitrile solution is a 60% aqueous acetonitrile solution.