阅读说明：本技术 一种美曲普汀的制备方法 (Preparation method of metreleptin ) 是由 汤华东 梅芸 董瑶 童齐金 陈亚 于 2020-08-05 设计创作，主要内容包括：本发明公开了一种美曲普汀的制备方法,具体是美曲普汀基因通过原核表达系统表达获得包涵体,包涵体进行溶解,再进行疏水层析复性,获得美曲普汀蛋白。包涵体溶解和疏水层析复性均采用温和的方式进行,过程中还可以添加抗氧化剂和/或金属螯合剂,以抑制目的蛋白的氧化、降解。该方法制备美曲普汀,易于实现产业化放大,复性效率高,收得率高,生物活性好。(The invention discloses a preparation method of metreleptin, and particularly relates to a preparation method of metreleptin protein, wherein metreleptin gene is expressed by a prokaryotic expression system to obtain an inclusion body, the inclusion body is dissolved, and then hydrophobic chromatography renaturation is carried out. The dissolution of the inclusion body and the renaturation of the hydrophobic chromatography are carried out in a mild mode, and an antioxidant and/or a metal chelating agent can be added in the process to inhibit the oxidation and degradation of the target protein. The method for preparing the metreleptin is easy to realize industrial amplification, and has high renaturation efficiency, high yield and good biological activity.)

1. A preparation method of metreleptin is characterized in that metreleptin gene is expressed by a prokaryotic expression system to obtain an inclusion body, the inclusion body is dissolved, and then hydrophobic chromatography renaturation is carried out to obtain metreleptin protein;

the dissolving solution used for dissolving is 0.5-1M of Arg, the pH value is 12, the dissolving temperature is 2-8 ℃, and the dissolving time is 3-18 hours;

the hydrophobic chromatography renaturation comprises the following steps:

(1) adding equal volume of 4M NaCl-containing solution into the dissolved inclusion body solution, and then loading the sample to a pre-balanced chromatographic column; the chromatographic column is a Phenylfast Flow 6(high) chromatographic column with 3-5 column bed volumes balanced by a balancing solution 1;

(2) after the sample loading is finished, the chromatographic column is rebalanced by balance liquid 1; sequentially carrying out gradient elution from the equilibrium solution 1 to the equilibrium solution 2 for 3-10 bed volumes, carrying out gradient elution from the equilibrium solution 2 to the renaturation solution 1 for 3-10 bed volumes, and carrying out gradient elution from the renaturation solution 1 to the renaturation solution 2 for 3-10 bed volumes;

(3) finally eluting with eluent, and collecting target peak to obtain crude product of metreleptin;

the equilibrium solution 1 contains 0.5 Magg, 2M NaCl, pH 12;

the equilibrium solution 2 contains 0.5 Magg, 2M NaCl, pH 10;

the renaturation solution 1 contains 20mM PB, 0.5M NaCl, 5% glycerol (w/v), 0.01% Tween80(w/v) and has the pH value of 8.0;

the renaturation solution 2 contains 20mM PB, 0.15M NaCl, 0.01% Tween80(w/v) and is pH 8.0;

the eluate contained 0.01% Tween80 (w/v).

2. The method for preparing metreleptin according to claim 1, wherein an antioxidant is further added to at least one of the dissolving solution, the equilibrium solution 1, the equilibrium solution 2 and the renaturation solution 1.

3. The method for preparing metreleptin according to claim 1, wherein the final concentration of the antioxidant after addition is 1-100 mg/ml.

4. The process for the preparation of metreleptin according to claim 3, wherein the antioxidant is Met, and the final concentration of Met after addition is 2 mg/ml.

5. The method for preparing metreleptin according to claim 1, wherein a metal ion chelating agent is further added to at least one of the dissolving solution, the equilibrium solution 1, the equilibrium solution 2 and the renaturation solution 1.

6. The method of claim 5, wherein the final concentration of the added metal ion chelating agent is 1-100 mM.

7. The method of claim 5, wherein the metal ion chelating agent is EDTA.

8. The method of claim 1, further comprising the step of purifying the crude metreleptin.

9. The method for preparing metreleptin according to claim 1, wherein the metreleptin gene sequence is shown as SEQ ID No.2, and the metreleptin amino acid sequence shown as SEQ ID No.1 is encoded; expressed by an E.coli expression system.

10. The method for preparing metreleptin according to claim 1, wherein the prokaryotic expression system is a genetically engineered bacterium containing a recombinant expression vector with metreleptin gene, and the expression condition is that the metreleptin is induced to be expressed in the genetically engineered bacterium when the genetically engineered bacterium grows to OD600 ═ 0.4-40.

11. The method for preparing metreleptin according to claim 1, wherein after the expression of the prokaryotic expression system is finished, the prokaryotic expression system is washed by a washing buffer solution, then the bacteria breaking buffer solution is used for breaking the bacteria to release the inclusion bodies, and the obtained inclusion bodies are washed by a cleaning solution for dissolving for later use;

the washing buffer, the bacteria-breaking buffer and the cleaning solution are all aqueous solutions containing 20mM PB, 0.15M NaCl, 1mM EDTA and 0.1% Triton X-100(w/v) and have the pH value of 7.4.

Technical Field

The invention relates to the technical field of protein engineering, in particular to a preparation method of metreleptin.

Background

Metreleptin (Metreleptin) is a recombinant human leptin derivative (rmetHuleptin) expressed by an Escherichia coli expression system, contains 147 amino acid residues, has one more methionine residue at the N-terminal compared with the natural human leptin protein, and is also called recombinant human methionyl leptin protein. The metreleptin is free of glycosylation modification, the 97 th cysteine residue and the 147 th cysteine residue form an intrachain disulfide bond, the C-terminal end of the metreleptin is blocked, and the relative molecular weight is about 16.15k Da. 24/2/2014, FDA approved metreleptin formulation (trade name: mylept, product of Amylin) to be marketed as a supplemental therapy for treating leptin deficiency complications in patients with congenital or acquired lipodystrophy, formulation specifications: 11.3 mg/count.

Coli (e.coli) expression system is widely used in academia and industry because of its clear genome information research and simple gene operation. However, the escherichia coli lacks a redox environment and a post-translational processing modification mechanism in cells, so that exogenous proteins recombinantly expressed by many escherichia coli expression systems cannot be correctly folded, and thus insoluble Inclusion Bodies (IBs) are formed. Inclusion bodies are predominantly aggregates of partially folded or misfolded proteins of interest. To obtain properly folded, biologically active proteins from inclusion bodies, a complex "denaturation-renaturation" process is generally used: the inclusion bodies are first solubilized with a high concentration of a denaturant such as 8M urea or 6M guanidine hydrochloride to completely open (denature) the secondary structure of the protein of interest, and then refolded by an appropriate solution to form the correct structure (renaturation).

When the recombinant human leptin protein or the derivative thereof is prepared by using an escherichia coli expression system, the target protein is mainly expressed in an inclusion body form, and the physiologically active leptin protein is obtained by a commonly used method which uses guanidine hydrochloride or urea for dissolving and then renaturation. However, the inclusion body is dissolved in a manner that the structure of the protein, including all secondary structures, is completely destroyed, so that during the renaturation (refolding) process of the protein, the protein is easy to be folded incorrectly, protein aggregation and precipitation are caused, and the renaturation fails, or the renaturation efficiency is low, so that the protein yield is low. The conventional renaturation adopts a dilution renaturation or dialysis renaturation mode to gradually remove a denaturant (urea or guanidine hydrochloride), and the target protein can be folded and form a correct conformation while the concentration of the denaturant is gradually reduced. Because the gradual reduction of the concentration of the denaturant is difficult to really realize in a dilution renaturation or dialysis renaturation mode, the target protein is not ready to be folded into a correct conformation when the concentration of the denaturant is suddenly reduced, an error conformation is often formed, and aggregation and precipitation are often caused, so that the renaturation efficiency is low; usually, the ratio of the inclusion body dissolving solution to the renaturation solution during dilution renaturation or dialysis renaturation is as high as 1: 50-1: 100, and the industrial amplification is not facilitated or the amplification cost is high. Therefore, there is a need for a process for the preparation of metreleptin which is easy to be scaled up industrially and has a high yield.

Disclosure of Invention

In order to solve the problems that the metreleptin in the prior art usually exists in the form of an inclusion body in the preparation process of bioengineering, is difficult to realize industrial amplification and has low yield and the like through complex denaturation and renaturation processes, the invention provides a novel preparation method of metreleptin. The method for preparing the metreleptin is easy to realize industrial amplification, and has high renaturation efficiency and high yield.

The technical scheme of the invention is detailed as follows:

a preparation method of metreleptin, metreleptin gene is expressed by a prokaryotic expression system to obtain an inclusion body, the inclusion body is dissolved, and then hydrophobic chromatography renaturation is carried out to obtain metreleptin protein;

the dissolving solution used for dissolving is 0.5-1M of Arg, the pH value is 12, the dissolving temperature is 2-8 ℃, and the dissolving time is 3-18 hours;

the hydrophobic chromatography renaturation comprises the following steps:

(1) adding equal volume of 4M NaCl-containing solution into the dissolved inclusion body solution, and then loading the sample to a pre-balanced chromatographic column; the chromatographic column is a Phenyl Fast Flow 6(high) chromatographic column with 3-5 column bed volumes balanced by an equilibrium liquid 1;

(2) after the sample loading is finished, the chromatographic column is rebalanced by balance liquid 1; sequentially carrying out gradient elution from the equilibrium solution 1 to the equilibrium solution 2 for 3-10 bed volumes, carrying out gradient elution from the equilibrium solution 2 to the renaturation solution 1 for 3-10 bed volumes, and carrying out gradient elution from the renaturation solution 1 to the renaturation solution 2 for 3-10 bed volumes;

(3) finally eluting with eluent, and collecting target peak to obtain crude product of metreleptin;

the equilibrium solution 1 contains 0.5M Arg and 2M NaCl, and has a pH value of 12;

the equilibrium solution 2 contains 0.5M Arg and 2M NaCl, and has a pH value of 10;

the renaturation solution 1 contains 20mM PB, 0.5M NaCl, 5% glycerol (w/v), 0.01% Tween80(w/v) and has the pH value of 8.0;

the renaturation solution 2 contains 20mM PB, 0.15M NaCl, 0.01% Tween80(w/v) and is pH 8.0;

the eluate contained 0.01% Tween80 (w/v).

Arg: arginine, 2-amino-5-guanidino-pentanoic acid; PB: PB buffer from NaH₂PO₄And Na₂HPO₄Adding purified water to prepare the product. The solvent of each solution is water, and the unit w/v of the percentage content of each component is weight volume ratio, for example, 5% glycerol, namely 5g glycerol in 100ml solution.

In order to avoid the damage effect of the conventional high-concentration urea or guanidine hydrochloride for dissolving the inclusion body on the secondary structure of the target protein, the invention adopts a milder mode for dissolving the inclusion body, namely the inclusion body is dissolved under the condition of high pH (0.5M-1M of Arg, pH12), and the inclusion body dissolving mode can keep the secondary structure of the protein, thereby increasing the renaturation efficiency; meanwhile, arginine can also help the dissolution of inclusion bodies and the folding and renaturation of target protein.

The hydrophobic chromatography renaturation adopts the mode of gradually reducing (gradient elution) the pH environment (pH12 → pH10 → pH8.0) of the target protein and gradually reducing (gradient elution) the salt ion concentration (2M NaCl → 0.5M NaCl → 0.15M NaCl) of the target protein, so that the target protein is fully folded into the correct conformation in time; the simultaneous addition of glycerol and Tween80 can improve the solubility of the temporary misfolded conformational protein, avoid aggregation and precipitation of the protein, and further improve the renaturation efficiency. One skilled in the art will appreciate that linear amplification is readily achieved for chromatographic processes once the process parameters are established.

The metreleptin gene is expressed by a prokaryotic expression system, namely, the metreleptin gene is inserted into a prokaryotic expression vector to construct a recombinant expression vector, the recombinant expression vector is introduced into a prokaryotic expression strain to construct a genetic engineering bacterium containing the recombinant expression vector, and then the genetic engineering bacterium is cultured under proper conditions to induce the metreleptin to express in the genetic engineering bacterium.

Prokaryotic expression vectors are vectors that can carry inserted gene sequences into prokaryotic cells for expression, and usually contain regulatory elements required for autonomous replication, expression of target genes, transcription, and the like of the vectors, including replication origin (Ori), promoters, transcription terminators, operators, operator repressor genes, and the like, and selection marker genes, such as ampicillin resistance gene, tetracycline resistance gene, kanamycin resistance gene, and the like; there are various types of promoters for regulating the expression of a target gene on a vector, such as a lambda phage T7 promoter (pET series vector), a pL/pR promoter (pBV220 vector), a cold shock gene CSPA promoter (pCold series vector), and the like. Alternatively or preferably, the prokaryotic expression vector is pET32a (+), pBV220 or pCold IV.

The prokaryotic expression strain is preferably E.coli, which should be selected taking into account the suitability of the prokaryotic expression vector chosen, as will be appreciated by those skilled in the art; for example, if the prokaryotic expression vector selected is a pET series vector, since the series vectors all use a lambda phage T7 promoter to start the transcription of the target gene, and the T7 promoter needs T7 RNA polymerase for the transcription, BL21(DE3) strain integrated with a lambda phage DE3 fragment (the fragment contains the gene coding for T7 RNA polymerase) can be selected; for example, a pBV220 vector in which pL/pR promoter initiates the transcription of a target gene or a pCold series vector in which CSPA promoter initiates the transcription of a target gene can be selected, and BL21 strain can be selected as an expression strain.

The technology of inserting gene sequences into prokaryotic expression vectors to construct recombinant expression vectors is common knowledge in the field, and can be inserted into a prokaryotic expression vector Multiple Cloning Site (MCS) in a mode of restriction enzyme digestion-connection by referring to molecular cloning experimental guidance compiled by J. SammBruke and M.R. Green; or inserted into the prokaryotic expression vector at an appropriate position by means of Seamless Cloning/In-Fusion Cloning. The seamless cloning is that 15-20 homologous base sequences are respectively carried on two ends of a gene sequence (target gene sequence) to be inserted and two ends of a linearized vector through PCR amplification, complementary pairing and cyclization are carried out by virtue of acting force between bases, the vector entering host bacteria can be directly used for transforming escherichia coli (host bacteria) without enzyme connection, and gaps are repaired by virtue of an escherichia coli repair system by virtue of the vector entering the host bacteria. Through seamless cloning, target gene sequences can be inserted without trace, and redundant sequences on the expression vector can be removed, so that the length of the sequences of the recombinant expression vector is shortest, the energy required by vector replication is reduced, and the effect of improving the expression efficiency is achieved to a certain extent.

The method for introducing the recombinant expression vector into the Escherichia coli expression strain is common knowledge in the field, and can be CaCl₂Heat shock transformation, Electroporation (Electroporation), and the like.

The induction expression means that the target gene expression is started by adjusting the culture temperature or adding an inducer, such as IPTG or lactose or the like, and the induction expression can be carried out by selecting the induction temperature or the inducer according to the induction conditions recorded in the specifications of different prokaryotic expression vectors, wherein the induction temperature can be 16-42 ℃, the concentration of the inducer can be 0.35-1 mM, and the culture time after the induction can be 4-22 hours.

Preferably, in the method for preparing metreleptin, an antioxidant is further added to at least one of the dissolving solution, the equilibrium solution 1, the equilibrium solution 2 and the renaturation solution 1. Is helpful for inhibiting the oxidative inactivation of the target protein metreleptin.

Preferably, in the preparation method of metreleptin, the final concentration of the antioxidant after addition is 1-100 mg/ml.

Preferably, in the method for preparing metreleptin, the antioxidant is Met (methionine), and the final concentration of Met after addition is 2 mg/ml.

Preferably, in the method for preparing metreleptin, at least one of the dissolving solution, the equilibrium solution 1, the equilibrium solution 2 and the renaturation solution 1 is further added with a metal ion chelating agent so as to inhibit the degradation of the target protein metreleptin by metalloprotease.

Preferably, in the method for preparing metreleptin, the final concentration of the added metal ion chelating agent is 1 to 100 mM.

Preferably, in the method for preparing metreleptin, the metal ion chelating agent is EDTA.

Preferably, the preparation method of metreleptin further comprises a step of purifying the crude metreleptin. The purification method is well known in the art, and the purity of the target protein metreleptin can be more than 95 percent by adopting general purification techniques such as ammonium sulfate precipitation, ultrafiltration, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, preparative reverse phase chromatography and the like, and more preferably, the purity of the metreleptin is more than 98 percent.

Preferably, in the preparation method of metreleptin, the metreleptin gene sequence is shown as SEQ ID NO.2, and the metreleptin amino acid sequence shown as SEQ ID NO.1 is encoded; expressed by an E.coli expression system. The nucleotide sequence is a metreleptin gene sequence optimized by codons, an escherichia coli preferred codon table is referred, and factors such as codon degeneracy, GC content, transcribed mRNA structure and stability are combined to optimize the DNA sequence so as to improve the expression quantity of the encoded protein. OptimumGene can be used^TMThe software performs DNA sequence optimization. It will be understood by those skilled in the art that, in order to facilitate molecular cloning operations such as insertion of the gene sequence into a cloning vector or an expression vector, a restriction enzyme recognition sequence may be further added to both ends of the gene sequence; in order to facilitate translation initiation and termination, a ribosome binding site sequence (RBS sequence)/Shine-Dalgarno (SD) sequence and/or a stop codon sequence may be added upstream of the 5' -end of the gene sequence.

Preferably, in the preparation method of metreleptin, the prokaryotic expression system is a genetically engineered bacterium containing a recombinant expression vector with metreleptin gene, and the expression condition is that the metreleptin is induced to be expressed in the genetically engineered bacterium when the genetically engineered bacterium grows to OD600 ═ 0.4-40.

Preferably, in the preparation method of metreleptin, after the expression of the prokaryotic expression system is finished, the prokaryotic expression system is washed by a washing buffer solution, then the bacteria are broken by a bacteria breaking buffer solution to release the inclusion body, and the obtained inclusion body is washed by a cleaning solution for dissolving for later use; the washing buffer, the bacteria-breaking buffer and the cleaning solution are all aqueous solutions containing 20mM PB, 0.15M NaCl, 1mM EDTA and 0.1% Triton X-100(w/v) and have the pH value of 7.4.

Compared with the prior art, the invention has the following beneficial effects:

the invention adopts a milder mode to dissolve the inclusion body, adopts hydrophobic chromatography to carry out renaturation, and further uses an antioxidant and/or a metal chelating agent in the preparation process to inhibit the oxidation and degradation of target protein. The method for preparing the metreleptin is easy to realize industrial amplification, has high renaturation efficiency and high yield, and the biological activity of the product is equivalent to that of a commercially available product.

Drawings

FIG. 1: SDS-PAGE patterns of the effect of different inducer IPTG concentrations on the expression of target protein of pET-DE3 engineering strain. Wherein the sample No.1 is inducer IPTG with final concentration of 1mM, the sample No.2 is inducer IPTG with final concentration of 0.75mM, the sample No.3 is inducer IPTG with final concentration of 0.5mM, the sample No.4 is inducer IPTG with final concentration of 0.25mM, and M is protein Marker.

FIG. 2: SDS-PAGE patterns of the influence of different induced expression time on the expression of target protein of pET-DE3 engineering strain. Wherein the sample No.1 is a whole bacterial liquid for inducing and culturing for 4 hours, the sample No.2 is a precipitate after the bacteria breaking for inducing and culturing for 4 hours, the sample No.3 is a whole bacterial liquid for inducing and culturing for 8 hours, the sample No.4 is a precipitate after the bacteria breaking for inducing and culturing for 8 hours, the sample No.5 is a whole bacterial liquid for inducing and culturing for 16 hours, the sample No.6 is a precipitate after the bacteria breaking for inducing and culturing for 16 hours, and M is a protein Marker.

FIG. 3: RP-HPLC analysis spectrum of the pure metreleptin.

FIG. 4: and (3) analyzing the spectrum of the pure metreleptin SEC-HPLC.

FIG. 5: and detecting the molecular weight of the pure metreleptin product by using a mass spectrometry. A is the non-reducing relative molecular weight result; b is the relative molecular weight result after reduction.

FIG. 6: and (5) analyzing the disulfide bond pairing of the pure metreleptin.

In the figure, leptin represents metreleptin.

Detailed Description

The present invention is further described with reference to specific examples to enable those skilled in the art to better understand the present invention and to practice the same, but the examples are not intended to limit the present invention.

In the present specification, unless otherwise specified, technical terms used are terms commonly used by those of ordinary skill in the art; the experimental method without specific conditions noted in the specification is a conventional experimental method; the test materials used in this specification are commercially available products unless otherwise specified, and the ingredients and preparation methods of various reagents and media can be referred to in the conventional laboratory manuals.