CN111848774B - Preparation method of metreleptin - Google Patents

Abstract

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.

Description

Preparation method of metreleptin

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, a 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 general purification techniques including ammonium sulfate precipitation, ultrafiltration, ion exchange chromatography, hydrophobic chromatography, gel filtration chromatography, preparative reverse phase chromatography and the like can be adopted to ensure that the purity of the target protein metreleptin is more than 95%, and more preferably, the purity of the metreleptin is more than 98%.

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 solution, the bacteria-breaking buffer solution and the cleaning solution are all aqueous solutions containing 20mM PB, 0.15M NaCl, 1mM EDTA and 0.1% TritonX-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. The sample No.1 is a whole bacterial liquid cultured for 4 hours in an induction way, the sample No.2 is a precipitate after the bacteria breaking in the induction way for 4 hours, the sample No.3 is a whole bacterial liquid cultured for 8 hours in the induction way, the sample No.4 is a precipitate after the bacteria breaking in the induction way for 8 hours, the sample No.5 is a whole bacterial liquid cultured for 16 hours in the induction way, the sample No.6 is a precipitate after the bacteria breaking in the induction way 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.

Example 1: construction of Metridia recombinant expression vector

1.1 optimization and Synthesis of Mettricin expression Gene sequences

By referring to a prokaryotic escherichia coli codon usage frequency table, combining factors such as mRNA secondary structure stability and GC content balance, and the like, on the premise of not changing the metreleptin amino acid sequence shown in SEQ ID No.1, a base sequence is optimized so as to improve the expression quantity of the target protein to the maximum extent, and the optimized metreleptin expression gene sequence is shown in SEQ ID No. 2. The sequence was synthesized by chemical synthesis.

1.2 construction of Mettricuspid recombinant expression vector

And constructing a metreleptin recombinant expression vector by adopting a prokaryotic expression vector. In this example, the recombinant expression vectors were constructed by a seamless cloning method. The genes for the protein of interest (metreleptin) are all immediately preceded by the start codon ATG and the stop codon after the last amino acid codon TGC uses the optimal stop codon TAA; in order to enhance the termination efficiency, an additional T is added after the termination codon TAA, thereby improving the termination efficiency to the maximum extent. The following are enumerated for illustration:

1.2.1 construction of recombinant expression vector of pET32a (+) -leptin

The pET32a (+) vector is a fusion protein expression vector, contains a T7 promoter, and sequentially contains a lac operon, a Ribosome Binding Site (RBS), a thioredoxin tag (Trx-tag), a 6 XHis tag (N-6 XHis), a thrombin cleavage site (N-thrombin), an S-tag (S-tag), an EK enzyme cleavage site (N-EK), a Multiple Cloning Site (MCS), a 6 XHis tag (C-6 XHis) and a T7 terminator at the downstream of the T7 promoter; the vector contains an ampicillin resistance gene to facilitate selection of recombinants.

(1) Obtaining target gene fragment and linearized vector

The forward primer pET32a (+) -lept F (SEQ ID NO.3) and the reverse primer pET32a (+) -lept R (SEQ ID NO.4) of the target gene were designed, and the target gene fragment was amplified by PCR using the gene sequence chemically synthesized in example 1.1 as a template. On one hand, the target gene primer introduces an optimal termination codon sequence TAAT (underline TAAT complementary sequence ATTA) of escherichia coli, can efficiently terminate translation, and on the other hand, introduces a partial sequence (lower case letters) of a vector to facilitate seamless cloning:

pET32a(+)-lept F：5'-aggagatatacatatgGTGCCGATCCAGAAGG-3'；

pET32a(+)-lept R：5'-agcagccggatcATTAGCAGCCCGGGCTCAG-3'。

the vector forward primer pET32a (+) F (SEQ ID NO.5) and reverse primer pET32a (+) R (SEQ ID NO.6) were designed and the pET32a (+) vector was linearized by PCR amplification. The vector primer can simultaneously remove the sequence of Trx-tag, N-6 XHis, N-thiombin, S-tag, N-EK, MCS and C-6 XHis after the initiation codon of pET32a (+) vector, and can shorten the size of the vector sequence:

pET32a(+)F：5'-TAATgatccggctgctaac-3'；

pET32a(+)R：5'-catatgtatatctccttc-3'。

as will be understood by those skilled in the art, shortening the size of the vector sequence is beneficial to reducing the energy required for vector replication, is beneficial to shortening the replication time of the vector, and is helpful to improving the expression efficiency of the target protein.

(2) Seamless clonal joining

The operation can be performed according to the instruction of a commercial seamless cloning kit, and in this embodiment, the seamless cloning reaction system can be: 0.03pmol of linearized vector, 0.06pmol of target gene fragment, 5 XBuffer 4. mu.L, and 1. mu.L of Exnase; after the reaction is carried out for 0.5h at 37 ℃, ice bath is carried out for 5min, then the Top10 strain is transformed, the culture is carried out for 13-16 h at 37 ℃ on an LB solid culture medium containing ampicillin with the final concentration of 100 mu g/ml, recombinant clones are obtained by screening, and after the correctness is identified by PCR, the accuracy of the sequence is confirmed by sequencing. And extracting the recombinant plasmid which is identified by PCR and sequenced to be correct to obtain a recombinant expression vector which is marked as pET32a (+) -leptin.

1.2.2 construction of pCold IV-leptin recombinant expression vector

The pCold IV vector contains the cold shock gene CSPA promoter, downstream of which contains the 5 'untranslated region (5' UTR), the Translation Enhancing Element (TEE), the 6 xhis tag sequence, the factor Xa cleavage site, the Multiple Cloning Site (MCS) and the 3 'untranslated region (3' UTR); the promoter also contains lac operon downstream, and the expression of the lac operon can be induced by IPTG or lactose or analogues thereof. The vector contains an ampicillin resistance gene to facilitate selection of recombinants.

(1) Obtaining target gene fragment and linearized vector

A forward primer pCold-lept F (SEQ ID NO.7) and a reverse primer pCold-lept R (SEQ ID NO.8) of the target gene were designed, and the target gene fragment was obtained by PCR amplification using the gene sequence chemically synthesized in example 1.1 as a template. On one hand, the target gene primer introduces an optimal termination codon sequence TAAT (TAAT complementary sequence ATTA shown by underline) of escherichia coli, can efficiently terminate translation, and on the other hand, introduces a partial sequence (lower case letters) of a vector, so as to facilitate the seamless cloning:

pCold-lept F：5'-gaggtaataccatatgGTGCCGATCCAGAAGG-3'；

pCold-lept R：5'-gggtaccgagctcATTAGCAGCCCGGGCTCAG-3'。

designing a forward primer pCold F (SEQ ID NO.9) and a reverse primer pCold R (SEQ ID NO.10) of the vector, and amplifying a linearized pCold IV vector by adopting PCR. The vector forward primer introduces a termination codon sequence TAAT expressed by a target gene:

pCold F：5'-TAATgagctcggtaccctcgagggatc-3'；

pCold R：5'-catatggtattacctcttaataat-3'。

(2) seamless clonal joining

The target gene fragment obtained by PCR amplification of 1.2.2(1) and a linearized pCold IV vector are subjected to seamless cloning connection according to the instruction of a commercial seamless cloning kit, then a Top10 strain is transformed, the target gene fragment is cultured for 13-16 hours at 37 ℃ on an LB solid culture medium containing ampicillin with the final concentration of 100 mu g/ml, a recombinant clone is obtained by screening, and the accuracy of the sequence is confirmed by sequencing after the PCR identification is correct. And extracting the recombinant plasmid which is verified to be correct through PCR identification and sequencing to obtain a recombinant expression vector which is marked as pCold IV-leptin.

1.2.3 construction of pBV220-leptin recombinant expression vector

The pBV220 vector is an escherichia coli temperature control expression vector constructed by the institute of viral research of national academy of preventive medicine science, and contains an SD sequence; a Multiple Cloning Site (MCS) is followed to facilitate the insertion of a foreign gene with an initiation codon ATG; contains strong transcription terminator rrnB T1 and T2 terminator; contains lambda phage pL/pR promoter and cIts857 regulatory gene cI encoding cI protein gene which has inhibitory effect on the promoter and is temperature sensitive, so that the transcription of foreign gene inserted therein can be controlled by temperature; and contains ampicillin resistance gene to facilitate recombinant screening.

(1) Obtaining target gene fragment and linearized vector

A forward primer pBV220-leptin F (SEQ ID NO.11) and a reverse primer pBV220-leptin R (SEQ ID NO.12) of the target gene were designed, and the target gene fragment was obtained by PCR amplification using the gene sequence chemically synthesized in example 1.1 as a template. On one hand, the target gene primer introduces an optimal termination codon sequence TAAT (underline TAAT complementary sequence ATTA) of escherichia coli, can efficiently terminate translation, and on the other hand, introduces a partial sequence (lower case letters) of a vector to facilitate seamless cloning:

pBV220-lept F：5'-ttcccggggatccATGGTGCCGATCCAGAAGG-3'；

pBV220-lept R：5'-ctgcaggtcgacATTAGCAGCCCGGGCTCAG-3'。

the vector forward primer pBV 220F (SEQ ID NO.13) and the reverse primer pBV 220R (SEQ ID NO.14) were designed, and the pBV220 vector was linearized by PCR amplification. The vector forward primer introduces a termination codon sequence TAAT expressed by a target gene, and the reverse primer introduces a translation initiation codon ATG (a reverse complementary sequence CAT of the ATG shown in bold uppercase) expressed by the target gene:

pBV220 F：5'-TAATgtcgacctgcagccaagcttc-3'；

pBV220 R：5'-CATggatccccgggaattcctcct-3'。

(2) seamless clonal joining

In the same way as 1.2.2(2), the target gene fragment obtained by PCR amplification of 1.2.3(1) and the linearized pBV220 vector are subjected to seamless cloning connection, a Top10 strain is transformed, and a recombinant expression vector is obtained by screening, identifying and extracting and is recorded as pBV 220-leptin.

Example 2: construction and induced expression of metreleptin gene engineering bacteria

The recombinant expression vector constructed in example 1.2 was passed through CaCl₂Transferring the recombinant strain into an adapted Escherichia coli expression strain cell by a heat shock transformation method or an Electroporation method (Electroporation), and screening to obtain a recombinant, namely the genetic engineering strain. The following are enumerated for illustration:

2.1 construction and inducible expression of pET-DE3 engineering strains

The pET32a (+) -leptin recombinant expression vector constructed in the embodiment 1.2.1 is transformed into BL21(DE3) escherichia coli expression strain, and is cultured on LB solid culture medium containing ampicillin with the final concentration of 100 mug/ml at 37 ℃ for 13-16 hours to obtain recombinants through screening, namely the metreleptin genetic engineering strain, which is marked as pET-DE3 engineering strain.

(1) Inducer IPTG concentration screening

The activated pET-DE3 engineered strain was inoculated in LB medium containing 100. mu.g/ml Amp at a ratio of 1:100(V/V), and cultured in a shake flask at 37 ℃ until OD600 became 0.6, and IPTG was added to the culture medium at final concentrations of 0.25mM, 0.5mM, 0.75mM and 1mM, respectively. After the induction culture for 22h, 20 mul of bacterial liquid is taken for SDS-PAGE detection, and the expression level of the target protein is detected.

As shown in FIG. 1, the target protein has the highest expression level and less impurity protein expression level when the final concentration of IPTG is 1 mM.

(2) Induction culture time selection

The activated pET-DE3 engineered strain was inoculated in LB medium containing 100. mu.g/ml Amp at a ratio of 1:100(V/V), and when the strain was cultured in a shake flask at 37 ℃ until OD600 became 0.6, IPTG was added to the strain at a final concentration of 1 mM. The induction was carried out for 4 hours, 8 hours and 16 hours, respectively. Collecting thallus and ultrasonic breaking. Taking the precipitate and the whole bacteria liquid to carry out SDS-PAGE detection.

As shown in FIG. 2, the target protein in the whole bacterial liquid and the precipitate gradually increased in expression amount with time after 4 hours, 8 hours and 16 hours of induction expression.

2.2 construction and inducible expression of pCold-BL21 engineering strains

The pCold IV-leptin recombinant expression vector constructed in the example 1.2.2 is transformed into BL21 escherichia coli expression strain, and cultured on LB solid culture medium containing ampicillin with the final concentration of 100 mu g/ml at 37 ℃ for 13-16 hours to obtain recombinants through screening, namely the metreleptin genetic engineering strain which is marked as pCold-BL21 engineering strain.

The activated pCold-BL21 engineered strain was inoculated in LB medium containing 100. mu.g/ml Amp at a ratio of 1:100(V/V), and when shaking cultured at 37 ℃ until OD600 became 0.8, the temperature was lowered to 16 ℃ and IPTG was added to a final concentration of 1mM, and the induction culture was carried out for 22 hours. Collecting thalli, carrying out ultrasonic disruption, taking precipitate, and carrying out SDS-PAGE detection on supernatant and whole bacterial liquid. The result shows that the major part of the target protein metreleptin exists in the bacteria-breaking sediment and is expressed as an inclusion body.

2.3 construction and inducible expression of pBV-BL21 engineering strains

The pBV220-leptin recombinant expression vector constructed in the example 1.2.3 is transformed into BL21 escherichia coli expression strain, and cultured on LB solid culture medium containing ampicillin with the final concentration of 100 mug/ml at 37 ℃ for 13-16 hours to obtain recon through screening, namely the metreleptin genetic engineering strain which is marked as pBV-BL21 engineering strain.

The activated pBV-BL21 engineered strain was inoculated in LB medium containing 100. mu.g/ml Amp at a ratio of 1:100(V/V), and when shaking cultured at 37 ℃ until OD600 became 0.4, the temperature was raised to 42 ℃ and IPTG was added to a final concentration of 0.5mM, and the induction culture was carried out for 4 hours. Collecting thalli, carrying out ultrasonic disruption, taking precipitate, and carrying out SDS-PAGE detection on supernatant and whole bacterial liquid. The result shows that the target protein metreleptin is mostly present in the bacteria-breaking precipitate and is expressed by inclusion bodies.

Example 3: small-scale fermentation of pET-DE3 engineering bacteria, preparation and washing of inclusion body

The frozen pET-DE3 engineering bacteria constructed in example 2.1 were activated, expanded and inoculated in 5L of mediumThe fermentation culture solution (the components of the fermentation culture solution are 5.0g/L of glucose, 6.0g/L of peptone, 12.0g/L of yeast powder, 1.0g/L of NaCl and NH₄Cl 2g/L，Na₂HPO₄·12H₂O 30.0g/L，KH₂PO₄ 3.0g/L，MgSO₄·7H₂O1.0 g/L) was cultured in a fermentor (temperature 37 ℃, rotation speed 200rpm, aeration rate 1.8Nm³H, pot pressure 0.2 bar); controlling dissolved oxygen at above 30% in early stage of fermentation, gradually adjusting rotation speed to 600rpm when oxygen is insufficient, and adjusting ventilation amount to 2.5Nm³Finally, supplementing carbon source (glucose 600g/L) and nitrogen source (peptone 150g/L, yeast powder 150g/L) and controlling dissolved oxygen at about 30%; in the early stage of fermentation, ammonia water is used for regulating and controlling the pH value to be 7.0, and in the subsequent fermentation process, the pH value is controlled by supplementing materials as much as possible. When the OD600 of the fermentation broth was 40, the temperature was lowered to 30 ℃ and IPTG was added to the final concentration of 0.35mM, and after 8 hours of induction culture, the cells were collected by centrifugation.

The collected thalli is washed, broken and centrifuged to obtain the inclusion body, and then the inclusion body is cleaned by a proper method to obtain the purer inclusion body. Illustratively, inclusion bodies can be prepared and washed by the following method:

(1) washing of bacterial cells

Resuspending the thalli in a thalli washing buffer solution (containing 20mM PB, 0.15M NaCl, 1mM EDTA, 0.1% TritonX-100 and pH 7.4) according to the ratio of 1: 15-1: 30(V/V), uniformly stirring by using a magnetic stirrer, centrifuging, removing supernatant, and collecting the thalli; repeating the step for 1-2 times;

(2) preparation of inclusion body by breaking bacteria

Resuspending the washed thallus in the step (1) in a bacteria breaking buffer solution (containing 20mM PB, 0.15M NaCl, 1mM EDTA, 0.1% TritonX-100 and pH 7.4) according to the ratio of 1:15(V/V), placing the thallus in an ice bath, and breaking the thallus by using a high-pressure homogenizer (the breaking condition is that the pressure is less than 900bar, and the thallus is homogenized for 4 times); after the bacteria are broken, the sediment is centrifugally collected to obtain the inclusion body.

(3) Inclusion body washing

And (3) suspending the inclusion body obtained in the step (2) in an inclusion body washing buffer solution (containing 20mM PB, 0.15M NaCl, 1mM EDTA, 0.1% TritonX-100 and pH 7.4), washing the inclusion body, centrifuging to remove an inclusion body washing solution, and repeatedly washing for 2-4 times to obtain the inclusion body with high purity.

Example 4: inclusion body solubilization and hydrophobic chromatography renaturation

This example used relatively mild means to solubilize inclusion bodies and used hydrophobic chromatography (HIC) for renaturation and primary purification. A metreleptin related protein impurity appears in the purification process, which is an oxidation product of the first methionine residue at the N terminal (Liu JL, Lu KV, Eris T, etc. in vitro methylation of recombinant human leptin Pharm Res 1998,15(4):632-640.), and the proportion of the oxidation impurity can be detected by using a C18 column RP-HPLC method; antioxidants and/or metal chelators may also be added during the preparation process to inhibit oxidation. Illustratively, the following are listed:

4.1 Inclusion body solubilization and hydrophobic chromatography renaturation (with antioxidant)

The inclusion bodies prepared and washed in example 3 were taken, and a dissolution solution (containing 0.5M Arg and 2mg/ml Met, pH12) was added to each 1g of inclusion bodies at a ratio of 30ml of the dissolution solution (containing 0.5M Arg and 2mg/ml Met, pH12), stirred at 4 ℃ for 12 hours, centrifuged at 12000g for 10min, and the supernatant was collected, i.e., an inclusion body dissolution sample. To the inclusion body-solubilized sample, an equal volume of a 4M NaCl-containing solubilization solution (containing 0.5M Arg-2mg/ml Met, pH12) was added, and then a Phenyl Fast Flow 6(high) column of 3 bed volumes was equilibrated in advance with equilibration solution 1; after the sample loading is finished, the chromatographic column is re-balanced by 3 column bed volumes by using a balance liquid 1; then, 5 bed volumes are eluted in a gradient manner from the equilibrium solution 1 to the equilibrium solution 2, 5 bed volumes are eluted in a gradient manner from the equilibrium solution 2 to the renaturation solution 1, and 5 bed volumes are eluted in a gradient manner from the renaturation solution 1 to the renaturation solution 2; finally eluting with eluent, and collecting target peak to obtain crude product of metreleptin;

the equilibrium solution 1 is an aqueous solution containing 0.5M Arg and 2mg/ml Met-2M NaCl, and has a pH value of 12;

the equilibrium solution 2 is an aqueous solution containing 0.5M Arg and 2mg/ml Met-2M NaCl, and has a pH value of 10;

the renaturation solution 1 is an aqueous solution containing 20mM PB, 2mg/ml Met, 0.5M NaCl, 5% glycerol (w/v) and 0.01% Tween80(w/v), and the pH value is 8.0;

the renaturation solution 2 is an aqueous solution containing 20mM PB, 0.15M NaCl and 0.01% Tween80(w/v) and has the pH value of 8.0;

the eluent was 0.01% Tween80 (w/v).

4.2 Inclusion body solubilization and hydrophobic chromatography renaturation (with addition of Metal chelator)

The inclusion bodies prepared and washed in example 3 were taken, 50ml of a dissolving solution (containing 1M Arg (arginine), 1mM EDTA, pH12) was added to 1g of the inclusion bodies, the dissolving solution (containing 1M Arg, 1mM EDTA, pH12) was added, the mixture was stirred at 8 ℃ for 3 hours, and after centrifugation at 12000g for 10min, the supernatant was collected, which was an inclusion body-solubilized sample. To the inclusion body-solubilized sample, an equal volume of a 4M NaCl-containing solubilization solution (containing 1M Arg and 1mM EDTA, pH12) was added, and then a Phenyl Fast Flow 6(high) column of 5 bed volumes was equilibrated in advance with equilibration solution 1; after the sample loading is finished, the chromatographic column is re-balanced by 5 column bed volumes by using a balance liquid 1; sequentially carrying out gradient elution on 3 bed volumes from the equilibrium solution 1 to the equilibrium solution 2, carrying out gradient elution on 3 bed volumes from the equilibrium solution 2 to the renaturation solution 1, and carrying out gradient elution on 3 bed volumes from the renaturation solution 1 to the renaturation solution 2; finally eluting with eluent, and collecting target peak to obtain crude product of metreleptin;

the equilibrium solution 1 is an aqueous solution containing 0.5M Arg, 1mM EDTA and 2M NaCl, and has a pH value of 12;

the equilibrium solution 2 is an aqueous solution containing 0.5M Arg, 1mM EDTA and 2M NaCl, and has a pH value of 10;

the renaturation solution 1 is an aqueous solution containing 20mM PB, 1mM EDTA, 0.5M NaCl, 5% glycerol (w/v) and 0.01% Tween80(w/v), and the pH value is 8.0;

the renaturation solution 2 is an aqueous solution containing 20mM PB, 0.15M NaCl and 0.01% Tween80(w/v) and has the pH value of 8.0;

the eluent is an aqueous solution containing 0.01% Tween80 (w/v).

4.3 Inclusion body solubilization and hydrophobic chromatography renaturation (with addition of antioxidants and metal chelators)

The inclusion bodies prepared and washed in example 3 were taken, and a dissolution solution (containing 0.5M Arg, 2mg/ml Met, 1mM EDTA, pH12) was added to each 1g of inclusion bodies at a ratio of 30ml of the dissolution solution (containing 0.5M Arg, 2mg/ml Met, 1mM EDTA, pH12), stirred at 4 ℃ for 12 hours, centrifuged at 12000g for 10min, and the supernatant was collected, which was an inclusion body dissolution sample. To the inclusion body-solubilized sample was added an equal volume of a 4M NaCl-containing solubilization solution (containing 0.5M Arg, 2mg/ml Met, 1mM EDTA, pH12), and then a Phenyl Fast Flow 6(high) column of 3 bed volumes was equilibrated in advance with equilibration solution 1; after the sample loading is finished, the chromatographic column is re-balanced by 3 column bed volumes by using a balance liquid 1; then, 5 bed volumes are eluted in a gradient manner from the equilibrium solution 1 to the equilibrium solution 2, 5 bed volumes are eluted in a gradient manner from the equilibrium solution 2 to the renaturation solution 1, and 5 bed volumes are eluted in a gradient manner from the renaturation solution 1 to the renaturation solution 2; finally eluting with eluent, and collecting target peak to obtain crude product of metreleptin;

the equilibrium solution 1 is an aqueous solution containing 0.5M Arg, 2mg/ml Met, 1mM EDTA and 2M NaCl, and has a pH value of 12;

the equilibrium solution 2 is a water solution containing 0.5M Arg, 2mg/ml Met, 1mM EDTA and 2M NaCl, and has a pH value of 10;

the renaturation solution 1 is an aqueous solution containing 20mM PB, 2mg/ml Met, 1mM EDTA, 0.5M NaCl, 5% glycerol (w/v) and 0.01% Tween80(w/v), and the pH value is 8.0;

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

the eluent is an aqueous solution containing 0.01% Tween80 (w/v).

4.4 Inclusion body solubilization and hydrophobic chromatography renaturation (without addition)

The inclusion bodies prepared and washed in example 3 were taken, 40ml of a dissolving solution (containing 0.75M Arg (arginine), pH12) was added to each 1g of inclusion bodies, the dissolving solution (containing 0.75M Arg, pH12) was stirred at 2 ℃ for 18h, and after centrifugation at 12000g for 10min, the supernatant was collected, which was the inclusion body dissolved sample. To the inclusion body-solubilized sample, an equal volume of a 4M NaCl-containing solubilization solution (containing 0.75M Arg, pH12) was added, and then a Phenyl Fast Flow 6(high) column was loaded in which 4 bed volumes had been equilibrated in advance with equilibration solution 1; after the sample loading is finished, the chromatographic column is re-balanced by 4 column bed volumes by using balance liquid 1; sequentially carrying out gradient elution on 10 bed volumes from the equilibrium solution 1 to the equilibrium solution 2, carrying out gradient elution on 10 bed volumes from the equilibrium solution 2 to the renaturation solution 1, and carrying out gradient elution on 10 bed volumes from the renaturation solution 1 to the renaturation solution 2; finally eluting with eluent, and collecting target peak to obtain crude product of metreleptin;

the equilibrium solution 1 is an aqueous solution containing 0.5M Arg and 2M NaCl, and has a pH value of 12;

the equilibrium solution 2 is an aqueous solution containing 0.5M Arg and 2M NaCl, and has a pH value of 10;

the renaturation solution 1 is an aqueous solution containing 20mM PB, 0.5M NaCl, 5% glycerol (w/v) and 0.01% Tween80(w/v), and the pH value is 8.0;

the renaturation solution 2 is an aqueous solution containing 20mM PB, 0.15M NaCl and 0.01% Tween80(w/v), and is pH8.0;

the eluent is an aqueous solution containing 0.01% Tween80 (w/v).

4.5 comparison of crude Metriptolide Oxidation impurities content

The proportions of the crude oxidized impurities of metreleptin prepared in examples 4.1, 4.2, 4.3 and 4.4 were measured by using a C18 column RP-HPLC method (pre-equilibrated with 95% of phase A (1% TFA) and 5% of phase B (100% acetonitrile-1% TFA) C18 analytical column (product of Waters, Specification: 4.6X 250mm, 5 μm, cat # WAT106151), UV detection was performed at 214nm, column temperature was normal temperature, loading was 50 μ g, and phase B was eluted with a linear gradient from 5% to 95% for 30 minutes at a flow rate of 1 ml/min.) and the results are shown in Table 1, where antioxidants (2mg/ml Arg) and/or metal chelators (1mMEDTA) were effective in suppressing the formation of oxidized impurities of metreleptin, reducing the oxidative degradation of the target protein and contributing to the improvement of the final yield of the target protein.

Table 1 comparison of crude metreleptin oxidation impurity content

Preparation method of crude metreleptin	Additive material	Content of oxidized impurities (%)
			Example 4.1	2mg/ml Met	12.5
Example 4.2	1mM EDTA	13.7
			Example 4.3	2mg/ml Met and 1mM EDTA	12.8
Example 4.4	Is free of	30.8

Example 5: metrectin protein purity and quality identification thereof

In the embodiment, the crude product of metreleptin is finely purified by adopting a method combining anion exchange chromatography (DEAE) and reverse phase preparative chromatography (C8), and the purity of the obtained pure product of metreleptin is more than 98% by RP-HPLC method and SEC-HPLC method. Illustratively, the specific method is as follows:

taking the crude product of the metreleptin obtained by the hydrophobic chromatography (HIC) renaturation and the primary purification of the example 4, adding 1/9 volumes of 0.5M Tris (pH8.5) to ensure that the metreleptin protein is in a buffer system of 50mM Tris-0.01% Tween80(w/v) and pH8.5; loading a DEAE-Sepharose Fast Flow column equilibrated in advance with 50mM Tris-0.01% Tween80(w/v), pH 8.5; after loading, re-equilibrating with the same buffer [50mM Tris-0.01% Tween80(w/v), pH8.5 ]; the protein of interest was then eluted with eluent [50mM Tris-0.01% Tween80(w/v) -0.1M Arg, pH7.5 ].

The above DEAE-Sepharose Fast Flow chromatography eluate collection was loaded on a C8 column previously pre-equilibrated with 95% of phase A (1% TFA) and 5% of phase B (100% acetonitrile-1% TFA); then setting the linear gradient elution of the phase B from 5% to 95%, wherein the elution volume is 2 column volumes; and collecting the target protein elution peak when the gradient is 65% of phase B, thus obtaining the pure metreleptin product. The organic solvent in the protein sample can be further removed by lyophilization, ion exchange chromatography, or hydrophobic chromatography, and the sample can be replaced with an appropriate solvent.

Taking the Inclusion Bodies (IBs) prepared and washed in the example 3, dissolving the inclusion bodies according to the method described in the example 4.1, renaturing by hydrophobic chromatography (HIC) and primarily purifying to obtain crude metreleptin; then the pure metreleptin product is prepared by anion exchange chromatography (DEAE) and reverse phase preparative chromatography (C8) according to the method. The experiments are repeated for three times, the collection and purification yield is shown in table 2, and the final obtained pure metreleptin product per liter of fermentation liquid is over 500 mg.

TABLE 2 summary of the purification yields of metreleptin

The resulting pure metreleptin was subjected to purity detection by RP-HPLC (pre-equilibration of C18 analytical column (product of Waters, specifications: 4.6X 250mm, 5 μ M, cat # WAT106151) with 95% of phase A (1% TFA) and 5% of phase B (100% acetonitrile-1% TFA), UV detection set at 214nm, column temperature at room temperature, loading 50 μ G, and elution of phase B from 5% to 95% linear gradient for 30 minutes at a flow rate of 1 ml/min.) and SEC-HPLC (equilibration solution: 85% 0.25M PB buffer-15% acetonitrile, pH 7.0. equilibration solution balance Kgel G2000SWxl gel column (product of TOSO, specifications: 7.8X 300mm, 5 μ M, cat # 08540) to pressure stabilization, loading 80 μ G to 100 μ G, and elution with UV equilibration solution for 40 minutes at 280nm at a flow rate of 0.3 ml/min), respectively.

The purity test results show that the purity is 100 percent by RP-HPLC (figure 3) and 99.25 percent by SEC-HPLC (figure 4).

And (3) entrusting the pure metreleptin product to Shanghai Zhongke new life biotechnology and science and technology Limited company to detect 15 amino acid sequences at the N terminal by adopting an Edman degradation method, detecting non-reduction and reduction relative molecular weights by adopting a mass spectrometry, and carrying out disulfide bond pairing analysis.

The detection of the N-terminal sequence after 15 cycles of N-terminal sequencing is as follows: NH (NH)₂-Met-Val-Pro-Ile-Gln-Lys-Val-Gln-Asp-Asp-Thr-Lys-Thr-Leu-Ile, corresponding to the theoretical sequence; the detection results of the non-reducing and reducing relative molecular weights are shown in FIG. 5, the detected non-reducing metreleptin has the relative molecular weight of 16156.00Da, while the theoretical relative molecular weight is 16157.64Da, which is within the error range and has a difference of less than 2 Da. The relative molecular weight after reduction is 16158.00Da, which is different from 16156.00Da before reduction by 2Da, which is exactly the molecular weight of 2 hydrogen atoms, and the prepared metreleptin is proved to have a disulfide bond; the disulfide bond analysis results are shown in fig. 6, and the comprehensive results prove that 1 disulfide bond (C97-C147) which is consistent with the theoretical pairing mode exists in the samples to be detected through the liquid chromatography-mass spectrometry analysis after the proteolysis and the combination of software and manual analysis.

The biological activity of the pure metreleptin product is detected by referring to the method recorded in the specification of the original medicine (Myalept, product of Amylin company), and the result shows that the biological activity (EC50 value: 2.875ng) of the pure metreleptin product is equivalent to the biological activity (EC50 value: 3.381ng) of the commercial recombinant human leptin protein (product of R & D company, product number: 398-LP-01M), which indicates that the prepared metreleptin has good biological activity.

The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.

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Claims (6)

1. A preparation method of metreleptin is characterized in that the 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 the 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 eluting 3-10 bed volumes from the equilibrium solution 1 to the equilibrium solution 2 in a gradient manner, eluting 3-10 bed volumes from the equilibrium solution 2 to the renaturation solution 1 in a gradient manner, and eluting 3-10 bed volumes from the renaturation solution 1 to the renaturation solution 2 in a gradient manner;

(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 eluent contained 0.01% Tween80 (w/v);

the expression vector used by the prokaryotic expression system is a recombinant expression vector, and the construction method of the recombinant expression vector comprises the following steps:

respectively designing a forward primer pET32a (+) -lept F of a target gene as shown in SEQ ID NO.3 and a reverse primer pET32a (+) -lept R as shown in SEQ ID NO.4, and carrying out PCR amplification by taking a target gene sequence shown in SEQ ID NO.2 as a template to obtain a target gene fragment; designing a forward primer pET32a (+) F of the vector as shown in SEQ ID NO.5 and a reverse primer pET32a (+) R as shown in SEQ ID NO.6, and amplifying a linearized pET32a (+) vector by PCR; carrying out seamless cloning connection on the linearized pET32a (+) vector and the target gene fragment to obtain a recombinant expression vector;

the PB is PB buffer solution consisting of NaH₂PO₄And Na₂HPO₄Adding purified water to prepare the product.

2. The method for preparing metreleptin according to claim 1, wherein an antioxidant is further added to at least one of the solution, equilibrium solution 1, equilibrium solution 2 and renaturation solution 1, wherein the antioxidant is Met, and the final concentration of Met after addition is 2 mg/ml.

3. The method for preparing metreleptin according to claim 1, wherein EDTA, a metal ion chelating agent, is further added to at least one of the solution, the equilibrium solution 1, the equilibrium solution 2 and the renaturation solution 1, and the final concentration of the EDTA after the addition of EDTA is 1 mM.

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

5. 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.

6. 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.

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