Process for the preparation and use of recombinant cystathionine-beta-lyase

Abstract

The application discloses a preparation method and application of a recombinant cystathionine-beta-lyase. The application provides a preparation method of the recombinant cystathionine-beta-lyase based on a prokaryotic expression system. The gene sequence coding the cystathionine-beta-lyase is optimized in different ways of synonymous codon bias preference, the obtained optimized codon constructs a vector, a large amount of soluble protein can be stably expressed in E. coli, the yield is improved, and the purification step is simplified, the obtained recombinant cystathionine-beta-lyase has high activity, and is suitable for industrial production.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of gene detection, in particular to a preparation method and application of recombinant cystathionine-β-lyase. BACKGROUND

[0002] Homocysteine (Hcy), also known as homocysteine, is a kind of thiol-containing amino acid, which is an important intermediate in the metabolism of methionine and cysteine. Recent studies have shown that the level of Hcy metabolism is closely related to cardiovascular diseases, diabetes and other diseases. High levels of homocysteine in plasma will lead to hyperhomocysteinemia, and Hcy and hyperlipidemia are considered to be two major causes of cardiovascular disease. The determination of homocysteine in plasma has become a common method for diagnosing vitamin B12, folate deficiency and early cardiovascular diseases. Determining Hcy is an important prerequisite for diagnosing various diseases.

[0003] At present, the detection of homocysteine mainly includes isotope method, chromatography method, immunization method and fluorescence polarization technology, which has the disadvantages of complicated operation, expensive instrument and high experimental cost, and cannot be fully popularized. The cyclic enzyme method has the characteristics of rapidity, simplicity, high sensitivity and easy automation. The principle of enzyme cycle method is to use cystathionine-β-lyase (CBS) to catalyze L-serine and homocysteine to form L-cystathionine, and L-cystathionine can form homocysteine, pyruvic acid and ammonia under the catalysis of cystathionine β-lyase (CBL). Therefore, obtaining high-activity cystathionine-β-lyase is the key to using enzyme cycle method to determine homocysteine.

[0004] In the prior art, a method for preparing cystathionine-β-lyase based on genetic engineering has been developed. In 1982, Chandra M. Dwivedi et al. firstly cloned, purified and characterized cystathionine-β-lyase from Escherichia coli. The general method for producing cystathionine-β-lyase at present is to amplify the cystathionine-β-lyase gene from the microorganism producing cystathionine-β-lyase in nature, connect it to an expression vector, and then introduce it into a relevant expression host to express cystathionine-β-lyase. However, the cystathionine-β-lyase obtained in this way generally has a small protein expression amount and poor stability. In 1995, Arno C. Alting et al. obtained CBL from Lactococcus lactis subsp. cremoris B78 by three-step purification, with an enzyme specific activity of 2.1 U / mg, a recovery rate of 21%, and a purification fold of 150. In 2000, Nada Dobric et al. cloned the CBL gene of Lactococcus lactis ssp. cremoris MG1363 into E. coli BL21 (DE3) to obtain CBL with a specific activity of 11.5 U / mg. However, the cystathionine-β-lyase from these sources has low activity, which is difficult to adapt to the reagent application of homocysteine, and the stability of cystathionine-β-lyase also greatly affects the accuracy of the reagent. Therefore, there is still a need in the art to develop a method for preparing cystathionine-β-lyase with high expression amount, high activity and high stability of the expression product. SUMMARY

[0005] The purpose of the present application is to provide a method for preparing cystathionine-β-lyase.

[0006] Another purpose of the present application is to provide a polynucleotide sequence encoding cystathionine-β-lyase.

[0007] Another purpose of the present application is to provide a vector adapted to the polynucleotide sequence encoding cystathionine-β-lyase.

[0008] Another purpose of the present application is to provide a kit containing the polynucleotide sequence encoding cystathionine-β-lyase.

[0009] To solve the above technical problems, the first aspect of the present application provides a polynucleotide encoding cystathionine-β-lyase, which is codon-optimized, and the polynucleotide is selected from any one of the following:

[0010] (i) a polynucleotide of the sequence shown in SEQ ID NO. 1-4;

[0011] (ii) a polynucleotide having more than 95% homology to the sequence as shown in SEQ ID NO. 1-4; and

[0012] (iii) a polynucleotide complementary to the polynucleotide sequence as described in (i) or (ii).

[0013] In a second aspect of the present application, an expression vector is provided, wherein the expression vector comprises the polynucleotide provided in the first aspect of the present application.

[0014] In some preferred embodiments, the expression vector is an Escherichia coli expression vector, more preferably pET-28a(+).

[0015] In a third aspect of the present application, a host cell is provided, wherein the host cell comprises the expression vector provided in the second aspect of the present application; or

[0016] the genome of the host cell is integrated with the polynucleotide provided in the first aspect of the present application.

[0017] In some preferred embodiments, the host cell is Escherichia coli.

[0018] In some preferred embodiments, the host cell is Escherichia coli BL21(DE3) strain.

[0019] In a fourth aspect of the present application, a method for preparing cystathionine-β-lyase is provided, wherein the method comprises the steps of: culturing the host cell provided in the third aspect of the present application to express the target protein; and

[0020] isolating the target protein, thereby obtaining the cystathionine-β-lyase.

[0021] In some preferred embodiments, the host cell is obtained by transforming Escherichia coli with the plasmid containing the polynucleotide provided in the first aspect of the present application.

[0022] In some preferred embodiments, the host cell is cultured in TB or LB medium.

[0023] In some preferred embodiments, the host cell is cultured in a shaking environment.

[0024] In some preferred embodiments, the host cell is cultured at a temperature of 16-19℃ or 35-39℃, more preferably at a temperature of 16-19℃.

[0025] In some preferred embodiments, kanamycin resistance gene is contained in the culture medium when the host cell is cultured.

[0026] In some preferred embodiments, the host cell is cultured and induced with IPTG to express the protein of interest.

[0027] In some preferred embodiments, the host cell is cultured to an OD600 of 0.6 to 0.8, and then induced with IPTG to express the protein of interest.

[0028] In some preferred embodiments, the step of isolating the protein of interest comprises:

[0029] The supernatant of the broken protein of interest is eluted from the chromatography column with the flow phase, and the eluate is collected.

[0030] In some preferred embodiments, the chromatography column is a Ni-column affinity chromatography column (Ni-NTA).

[0031] The fifth aspect of the present application provides a kit comprising: a polynucleotide as provided in the first aspect of the present application; or

[0032] an expression vector as provided in the second aspect of the present application; or

[0033] a host cell as provided in the third aspect of the present application; or

[0034] or a cystathionine-β-lyase prepared according to the method of the fourth aspect of the present application.

[0035] The present application has at least the following advantages over the prior art:

[0036] The present application provides a method for preparing recombinant cystathionine-β-lyase based on a prokaryotic expression system. By optimizing the codon usage bias of the gene sequence encoding cystathionine-β-lyase in different ways, the optimized codons are used to construct a vector that can stably express a large amount of soluble protein in E. coli, thereby improving the yield and simplifying the purification process. The recombinant cystathionine-β-lyase produced has high activity and is suitable for industrial production.

[0037] It should be understood that, within the scope of the present application, each of the technical features of the present application described above and in the following (e.g., in the examples) can be combined with each other to form new or preferred technical solutions. Due to the limited space, they will not be listed one by one here. BRIEF DESCRIPTION OF DRAWINGS

[0038] One or more embodiments are illustrated by way of example in the accompanying drawings that are not intended to be limiting of the embodiments.

[0039] Figure 1is a schematic diagram of SDS-PAGE identification result of expression product of optimized codon I and optimized codon II in an embodiment of the present application;

[0040] Figure 2 is a schematic diagram of SDS-PAGE identification result of expression product of optimized codon III and optimized codon IV in an embodiment of the present application;

[0041] Figure 3 is a SDS-PAGE identification result diagram of cystathionine-β-lyase after purification in an embodiment of the present application;

[0042] Figure 4 is a standard curve diagram of cystathionine-β-lyase in an embodiment of the present application. DETAILED DESCRIPTION

[0043] The present inventors have made extensive and in-depth research, screened a large number of heterologous cystathionine-β-lyase codons suitable for expression in the E. coli expression system, and optimized the synonymous codon preference for several times, and obtained an optimized codon sequence with suitable expression amount and high soluble protein content. In the present application, a prokaryotic system-based expression system of cystathionine-β-lyase is also developed, and the expressed cystathionine-β-lyase has high activity and good stability.

[0044] The optimized codon sequence in the present application is obtained by optimizing the synonymous codon of the polynucleotide sequence encoding the target protein. Specifically, the steps of obtaining the optimized codon include: 1) obtaining the nucleic acid sequence related to the target gene / obtaining the target protein, 2) optimizing the synonymous codon preference of the sequence obtained in step 1).

[0045] Obtaining the nucleic acid sequence related to the target gene / obtaining the target protein

[0046] The nucleotide full-length sequence or fragment of the target protein or its element in the present application can generally be obtained by PCR amplification method, recombination method or artificial synthesis method. For the PCR amplification method, primers can be designed according to the published nucleotide sequence related to the open reading frame sequence, and a commercially available cDNA library or a cDNA library prepared according to the conventional method known to those skilled in the art is used as a template for amplification to obtain the related sequence. When the sequence is long, it is often necessary to perform two or more times of PCR amplification, and then the fragments amplified in each time are spliced together in the correct order.

[0047] Once the related sequence is obtained, the recombination method can be used to obtain the related sequence in large quantities. This is usually to clone it into a vector, then transfer it into cells, and then separate the related sequence from the proliferated host cells by conventional methods.

[0048] In addition, the relevant sequences can also be synthesized by artificial synthesis, especially when the length of the fragment is short. Generally, a long sequence can be obtained by synthesizing a plurality of small fragments first and then connecting them.

[0049] The method of amplifying DNA / RNA by PCR is preferably used to obtain the gene of the present application. The primers for PCR can be appropriately selected based on the sequence information of the present application disclosed herein, and can be synthesized by conventional methods. The amplified DNA / RNA fragments can be separated and purified by conventional methods such as gel electrophoresis.

[0050] Different proteins of the same kind from different biological sources have different amino acid sequences. The gene sequence obtained based on the target protein of different sources usually has unpredictable functional activity of the recombinant expression product. In an embodiment of the present application, the amino acid sequence of the target protein from different sources is analyzed by NCBI database to obtain the sequence information of the target gene from different sources. In some examples, the cystathionine-β-lyase of Enterobacter is analyzed by NCBI database to obtain the sequence information of the cystathionine-β-lyase gene of Enterobacter.

[0051] Synonymous codon preference optimization

[0052] In order to overcome the potential problem of reduced yield in the expression of heterologous proteins in E. coli, the present application relates to a polynucleotide sequence optimized by synonymous codon preference. The obtained target gene sequence is optimized by synonymous codon preference, and the target gene sequence optimized by synonymous codon preference can express the same amino acid sequence as the target protein. In some embodiments of the present application, the optimized codons shown as SEQ ID NO: 1-4 are obtained by E. coli synonymous codon preference optimization of the cystathionine-β-lyase gene of Enterobacter. The homologous target protein is optimized by different synonymous codon preferences to obtain several codons. Generally, these codons can express active target proteins, but the expression amount of different codons introduced into E. coli, especially the soluble expression amount, is significantly different. In some embodiments of the present application, the optimized codon I, the optimized codon II, the optimized codon III and the optimized codon IV all have soluble expression, but the soluble protein expression amount is significantly different, and the vector containing the optimized codon I has significantly higher soluble expression amount when introduced into E. coli.

[0053] The present application also relates to a polynucleotide having a homology of greater than 80%, preferably greater than 85%, more preferably greater than 90%, more preferably greater than 91%, more preferably greater than 95% with the sequence shown as SEQ ID NO: 1-4; and a polynucleotide complementary to the sequence shown as SEQ ID NO: 1-3.

[0054] Vector of target gene

[0055] The present application also relates to vectors comprising the polynucleotides of the present application. "Vectors" in the present application mean linear or circular DNA molecules comprising a segment encoding a protein of interest operably linked to additional segments that provide for its transcription. Such additional segments can include promoter and terminator sequences, and can optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, vectors, etc. The vector segment can be derived from a host organism, another organism, a plasmid, or viral DNA, or can be synthetic. The vector can be any expression vector, synthetic or conveniently manipulated by recombinant DNA techniques, the choice of which generally depends on the host cell into which the vector is to be introduced. Thus, the vector can be an autonomously replicating vector, i.e., a vector, which exists as an extrachromosomal entity, the replication of which is independent of chromosomal replication, e.g., a plasmid. Alternatively, the vector can be one which, when introduced into a host cell, is integrated into the DNA of the cell, and replicated together with the cellular DNA. In one embodiment, the vector of the present application is an expression vector. In one embodiment of the present application, pET-28a(+) is selected as the vector to achieve higher expression efficiency.

[0056] Methods well known to those skilled in the art can be used to construct expression vectors containing the DNA sequences of the present application and appropriate transcriptional / translational control signals. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, etc. The DNA sequences described can be operably linked to a suitable promoter in an expression vector to direct mRNA synthesis. The expression vector also includes a ribosome binding site for translation initiation and a transcription terminator. Illustratively, the vector DNA molecule is cleaved by DNA endonuclease to a linear molecule that can be ligated with the foreign gene, and then the codon-optimized gene fragment of interest is ligated to the vector, which can be achieved by using a single enzyme cleavage site for sticky end ligation, directional cloning of double enzyme cleavage fragments, sticky end ligation of different restriction enzyme cleavage sites, blunt end ligation, artificial linker ligation, or ligation with oligonucleotide ends to achieve the insertion of the foreign DNA fragment.

[0057] Transformation of host cells with vectors containing the gene of interest

[0058] The present application also relates to host cells genetically engineered with the vector or the fusion protein coding sequence of the present application. The vector containing the codon-optimized gene of interest can be inserted, transfected or otherwise transformed into host cells by known methods, so as to obtain transformants containing the codon-optimized gene of interest of the present application and capable of expressing the protein of interest. In the present application, "host cell" refers to a cell into which a foreign polynucleotide and / or vector is introduced. The host cell can be a eukaryotic host cell or a prokaryotic host cell, and the host cell is preferably bacteria, and more preferably Escherichia coli, and even more preferably Escherichia coli Rosetta (DE3) strain.

[0059] Method for preparing protein of interest

[0060] The present application also relates to a method for preparing a protein of interest, which can express or produce a recombinant protein by using the polynucleotide sequence of the present application. Generally, the method comprises the following steps:

[0061] (1) transforming or transducing a suitable host cell with the polynucleotide of the present application encoding the protein of the present application (or variant), or with a recombinant expression vector containing the polynucleotide;

[0062] (2) culturing the host cell in a suitable medium;

[0063] (3) isolating and purifying the protein from the medium or the cell.

[0064] In step (1), the transformation or transduction of a suitable host cell with a recombinant expression vector containing the polynucleotide can be performed by conventional techniques well known to those skilled in the art, and when the host is Escherichia coli, heat shock and electroporation can be used.

[0065] The obtained transformants can be cultured by conventional methods to express the polypeptide encoded by the gene of the present application. Depending on the host cell used, the medium used in the culture can be selected from various conventional media, such as SB, TB, LB or SOC medium. The culture is carried out under conditions suitable for the growth of the host cell. After the host cell grows to an appropriate cell density, the selected promoter is induced by a suitable method (such as temperature shift or chemical induction), and the cell is cultured for a further period of time. In order to promote the expression of the protein of interest and increase the expression amount of soluble protein, in a preferred embodiment of the present application, the host cell is cultured in TB or LB medium, and the medium used contains kanamycin resistance gene.

[0066] In order to further promote the soluble expression of the protein of interest, in a preferred embodiment of the present application, the host cell is cultured to an OD 600After 0.6-0.8, IPTG is used for induction, and the culture is continued at 17 to 19°C or 35 to 39°C for about 8 to 12 hours. In the preferred embodiment of the present application, the host cells are cultured at 17 to 19°C using TB medium, and the culture medium contains a kanamycin resistance gene, and the resulting expression product soluble protein accounts for a higher proportion than the proportion of soluble protein induced by other culture media (such as LB, etc.) at other temperatures (such as 25°C or 37°C).

[0067] The protein in the above method can be expressed in the cell, on the cell membrane, or secreted outside the cell. If necessary, the protein can be isolated and purified by various separation methods using its physical, chemical and other properties. Therefore, in the present application, after successfully culturing the target protein, a step of isolating and purifying it is also involved, for example, in step (3), the protein is isolated and purified from the culture medium to obtain a high-purity target protein. Although the method of purifying the target protein can be a conventional means familiar to those skilled in the art, including but not limited to: conventional renaturation treatment, treatment with a protein precipitant (salting-out method), centrifugation, osmotic lysis, ultra-treatment, ultra-centrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and other various liquid chromatography techniques, and combinations of these methods. In the preferred embodiment of the present application, a method for isolating the target protein is provided, the steps of which include: passing the supernatant of the broken target protein and the mobile phase through the chromatography column at the same time to elute, and collecting the eluate; the mobile phase includes Buffer A, Buffer B and / or Buffer C; wherein Buffer A includes a solution of Tris and NaCl (concentration 1M); Buffer B includes a solution of Tris, NaCl (concentration 1M) and imidazole; and Buffer C includes a solution of Tris and NaCl (concentration 1M).

[0068] As a preferred scheme for the ratio between the components in the mobile phase, for example, the volume ratio of Tris and NaCl solution (concentration 1M) in Buffer A is 1:1; in Buffer B, the volume ratio of Tris, NaCl solution (concentration 1M) and imidazole is 1:1:10; and in Buffer A, the volume ratio of Tris and NaCl solution (concentration 1M) is 1:1; and in Buffer B.

[0069] Preferably, in the elution step, the procedure of the elution comprises a first stage, a second stage; in the first stage, the mobile phase used is Buffer A; in the second stage, the mobile phase used is a mixture of Buffer A and Buffer B, wherein the volume percentage content of Buffer A gradually decreases from 100% to 40%, and the volume percentage content of Buffer B gradually increases from 0% to 60%.

[0070] More preferably, the procedure of the elution further comprises a third stage, in which the mobile phase used is Buffer B.

[0071] The target protein product after elution purification is dialyzed, and the dialysis sample is collected. The concentration of the dialysis sample can be measured by the BCA method, and the yield is calculated.

[0072] In the present application, the use of any example or exemplary language provided herein for certain embodiments (e.g., "for example") is merely intended to better illuminate the application, and not to limit the scope of the application otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the application.

[0073] If the definition or use of a term in a cited document is inconsistent or not consistent with the definition of the term described herein, the definition of the term described herein is used, and the definition of the term in the cited document is not used.

[0074] Various terms used herein are shown as follows. If the term used in the claims is not defined in the following, the most broad definition of the term given by the person skilled in the art should be given to reflect the publication printed at the time of application or the issued patent.

[0075] As used herein, the term "isolated" refers to a nucleic acid or polypeptide that is separated from at least one other component with which the nucleic acid or polypeptide is found in nature (e.g., nucleic acid or polypeptide). In one embodiment, the nucleic acid or polypeptide is found only (if at all) in the solvent, buffer, ion or other component normally present in its solution. The terms "isolated" and "purified" do not include nucleic acid or polypeptide present in its natural source.

[0076] As used herein, the terms "polynucleotide" and "polynucleotide sequence" can be in the form of DNA or RNA. The DNA form includes cDNA, genomic DNA or artificially synthesized DNA. The DNA can be single-stranded or double-stranded. The DNA can be a coding strand or a non-coding strand.

[0077] The present application also relates to variants of the above polynucleotides which encode protein fragments, analogs and derivatives having the same amino acid sequence as the present application. Such variants of the polynucleotide can be naturally occurring allelic variants or non-naturally occurring variants. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is an alternative form of a polynucleotide which can arise from one or more nucleotide substitutions, deletions or insertions, but will not be so substantial as to substantially alter the functional properties of the polypeptide it encodes.

[0078] As used herein, the term "codon optimization" refers to a means of improving the efficiency of gene synthesis by avoiding the use of lowly utilized or rare codons, according to the codon usage bias exhibited by the organism in which the protein is actually expressed or produced, including E. coli, yeast, mammalian blood cells, plant cells, insect cells, and the like.

[0079] As used herein, the terms "homology" and "identity" are used interchangeably to refer to the percentage of identical (i.e., identical) nucleotides or amino acids between two or more polynucleotides or polypeptides. Sequence identity between two or more polynucleotides or polypeptides can be measured by the following method. The nucleotide or amino acid sequences of the polynucleotides or polypeptides are aligned, the number of positions at which the aligned polynucleotides or polypeptides contain identical nucleotides or amino acid residues is counted, and this is compared to the number of positions at which the aligned polynucleotides or polypeptides contain different nucleotides or amino acid residues. A polynucleotide can differ at one position, for example, by containing a different nucleotide (i.e., a substitution or variation) or a deletion of a nucleotide (i.e., an insertion of one or two nucleotides into or a deletion of one or two nucleotides from the polynucleotide). A polypeptide can differ at one position, for example, by containing an amino acid (i.e., a substitution or variation) or a deletion of an amino acid (i.e., an insertion of one or two amino acids into or a deletion of one or two amino acids from the polypeptide). Sequence identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide. For example, percent identity can be calculated by dividing the number of positions containing identical nucleotides or amino acid residues by the total number of nucleotides or amino acid residues in the polynucleotide or polypeptide, and then multiplying by 100.

[0080] As used herein, the terms "sequence complement" and "reverse sequence complement" are used interchangeably to refer to a sequence that is complementary to the original polynucleotide sequence, but in the opposite direction to the original polynucleotide sequence. For example, if the original polynucleotide sequence is ACTGAAC, its reverse complement is GTTCAT.

[0081] As used herein, the term "expression" includes any step involved in the production of a polypeptide in a host cell, including but not limited to, transcription, translation, post- translational modification, and secretion. Expression can be followed by harvesting, i.e., recovering, the host cell or the expression product.

[0082] In order to make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the present application will be further described below with specific examples. It should be understood that these examples are only used to illustrate the present application and not intended to limit the scope of the present application. The experimental methods in the following examples, if not otherwise specified, are usually carried out according to the conventional conditions or the conditions suggested by the manufacturers. Unless otherwise specified, the percentages and parts are weight percentages and weight parts. The experimental materials and reagents used in the following examples, if not otherwise specified, can be obtained from the commercial channels.

[0083] Unless otherwise indicated, the technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. It should be noted that the terms used herein are only for the purpose of describing the specific embodiments and are not intended to limit the exemplary embodiments of the present application.

[0084] Example 1

[0085] In this embodiment, high-purity cystathionine-β-lyase is obtained by genetic engineering.

[0086] (1) Construction of cystathionine-β-lyase plasmid

[0087] The gene sequence of Enterobacter cystathionine-β-lyase is obtained, and after optimization of the Escherichia coli synonymous codon bias, the optimized codon I is obtained. The recombinant plasmid is synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd.

[0088] (2) Introduction of recombinant plasmid into host Escherichia coli

[0089] Take 1 μL of the expression plasmid, add it to 30 μL of Escherichia coli competent BL21(DE3) under ice bath conditions, place it in ice bath for 30 min, 42℃ water bath for 45 seconds, immediately place it on ice for 2 minutes, add 400 μL of SOC medium without antibiotics, and incubate it at 37℃, 230 rpm for 45 minutes. Take 100 μL of the bacterial solution and evenly spread it on an LB plate containing 100 μg / mL of kanamycin, and incubate it in a 37℃ incubator overnight.

[0090] (3) Expression process of the target gene

[0091] Pick the prepared monoclonal in step (2), and inoculate it in TB medium containing 100 μg / mL of kanamycin under sterile conditions, and incubate it at 37℃, 220 rpm until the OD600 is between 0.6-0.8. IPTG is used for induction, and the culture is incubated at 37℃ and 18℃ overnight. Take samples for ultrasonic crushing and SDS-PAGE identification. The identification results are shown in Figure 14-7. It can be seen that the expression product was present in the supernatant at 18°C, but not at 37°C.

[0092] (4) Purification of the expression product

[0093] About 4 g of the recombinant bacteria obtained in step (3) was weighed and added to 20 ml of Lysis Buffer and dispersed on ice using a disperser. The cells were broken up by ultrasonic treatment: Ф10 probe, power 10%, work 5.5 s, stop 9.9 s, ultrasonic treatment for 30 min. Centrifugation at 20,000 rpm and 4°C for 30 min, and the supernatant was taken and filtered through a 0.22 μm membrane. Purification was performed using 1 ml of Ni-NTA, and the components of the flow phase are shown in Table 1 below, and the flow rate was 0.5 ml / min. After the sample was applied, 20 ml of Lysis Buffer was used to rinse the UV and conductance to the baseline. The elution procedure included: Step 1: 0% B, 10 CV, 1.5 ml / min; Step 2: 0-60% B, 20 CV, 1.5 ml / min; Step 3: 100% B, 15 CV, 1.5 ml / min. The eluted product was dialyzed, and the dialysis buffer was 1 x PBS, pH 7.0.

[0094] Table 1

[0095] Reagents Buffer A Buffer B Buffer C Lysis Buffer Tris 50 mM 50 mM 50 mM 50 mM NaCl 50 mM 50 mM 1M 300 mM Glycerol - - - - Imidazole - 500 mM - - pH 7.0 7.0 7.0 7.0

[0096] The electrophoresis results after sample collection are shown in Figure 3 From Figure 3 it can be seen that the target protein can be hung on the column and eluted at a concentration of 70 mM imidazole. According to the SDS results, eluent 2A10-2B7 was selected for dialysis, and 14 ml of sample was obtained after dialysis. The concentration was measured by BCA, and the results were: R2=0.997, the concentration was 6.329 mg / ml, the yield was 88.599 mg, and the yield was 22.15 mg / g of bacteria.

[0097] Example 2

[0098] In this example, a cystathionine-β-lyase plasmid was constructed in substantially the same manner as in Example 1, except that different optimized codons were used in the step of constructing the cystathionine-β-lyase plasmid. In this example, optimized codon II was used. The recombinant plasmid was introduced into E. coli in the same way, and the crude product of cystathionine-β-lyase was obtained by culturing and expressing, and the purified cystathionine-β-lyase was obtained by purification.

[0099] The un-purified expression product was identified by SDS-PAGE, and the identification results are shown in Figure 1 Columns 10-13. From the figure, it can be seen that the expression product was present in the supernatant at 18°C. The purified expression product was collected in 13 mL. The concentration was measured by BCA, and the results were: R2 = 0.988, its concentration was 5.836 mg / ml, the yield was 75.868 mg, and the yield rate was 18.97 mg / g bacteria.

[0100] Example 3

[0101] In this example, the cystathionine-β-lyase plasmid was constructed in a manner substantially the same as in Example 1, except that different optimized codons were used in the step of constructing the cystathionine-β-lyase plasmid. In this example, optimized codon III was used. The recombinant plasmid was introduced into E. coli in the same manner, and the crude product of the cystathionine-β-lyase was obtained by culturing and expressing, and the purified cystathionine-β-lyase was obtained by purifying.

[0102] The unpurified expression product was identified by SDS-PAGE, and the identification results are shown in columns 4 to 7 of FIG. 4. As can be seen from the figure, expression was observed in the supernatant at 18°C. The purified expression product was collected in an amount of 14 mL. The concentration was measured by BCA, and the result was: R Figure 2 = 0.996, its concentration was 5.043 mg / ml, the yield was 70.602 mg, and the yield rate was 17.65 mg / g bacteria. 2 = 0.996, its concentration was 5.043 mg / ml, the yield was 70.602 mg, and the yield rate was 17.65 mg / g bacteria.

[0103] Example 4

[0104] In this example, the cystathionine-β-lyase plasmid was constructed in a manner substantially the same as in Example 1, except that different optimized codons were used in the step of constructing the cystathionine-β-lyase plasmid. In this example, optimized codon IV was used. The recombinant plasmid was introduced into E. coli in the same manner, and the crude product of the cystathionine-β-lyase was obtained by culturing and expressing, and the purified cystathionine-β-lyase was obtained by purifying.

[0105] The unpurified expression product was identified by SDS-PAGE, and the identification results are shown in columns 10 to 13 of FIG. 10. As can be seen from the figure, expression was observed in the supernatant at 18°C. The purified expression product was collected in an amount of 12 mL. The concentration was measured by BCA, and the result was: R Figure 2 = 0.996, its concentration was 5.043 mg / ml, the yield was 70.602 mg, and the yield rate was 17.65 mg / g bacteria. 2 = 0.996, its concentration was 5.043 mg / ml, the yield was 70.602 mg, and the yield rate was 17.65 mg / g bacteria.

[0106] Table 2 shows the sequence information of different optimized codons, and Table 3 shows the expression amounts of the recombinant plasmids constructed by using different optimized codons in host cells.

[0107] Table 2

[0108]

[0109]

[0110]

[0111] Table 3

[0112]

[0113]

[0114] The purified expression products in each example were subjected to activity and stability assays, and the test methods were as follows:

[0115]

Cystathionine-β-lyase activity assay

[0116] (1) Solution preparation

[0117] 12.5 mM pyridoxal phosphate solution (PLP): 0.03 g of pyridoxal phosphate was accurately weighed and dissolved in 10 mL of H2O.

[0118] 17.5 mM DL-cystathionine: 0.0388 g of DL-cystathionine and 0.0605 g of Tris were weighed and dissolved in water, pH adjusted to 8.0, and made up to 10 mL.

[0119] 150 mM NADH: 0.106 g of NADH powder was weighed and dissolved in 1 mL of deionized water, and stored at -20°C in the dark.

[0120] 50 mM Tris-HCl buffer at pH 8.0: 0.605 g of Tris was weighed and dissolved in water, pH adjusted to 8.0, and made up to 100 mL. The working solution was prepared according to Table 4, and 5 mL was taken as the working solution.

[0121] Table 4

[0122] Reagents Volume added Final concentration 12.5 mM Pyridoxal phosphate 20 μL 0.05 mM 150 mM NADH 5 μL 0.15 mM 17.5 mM DL-Cystathionine 1 mL 3.5 mM Lactate dehydrogenase (25 U / μL) 2 μL 10 U / mL 50 mM pH 8.0 Tris-HCl 3.75 mL

[0123] (2) Experimental procedure

[0124] Positive enzyme preparation: 840 U of positive enzyme was dissolved in 420 μL of PBS pH 7.4 buffer to prepare an enzyme solution of 2 U / μL, which was further diluted according to the gradient, and the dilution was PBS pH 7.4 buffer.

[0125] The microplate reader was preheated for 30 min, the temperature was set to 37°C, and the working solution was incubated at 37°C for 5 min. 100 μL of reaction solution was added, and the absorbance A1 was detected at 340 nm. Then 1 μL of enzyme solution of each concentration was added, shaken and mixed for 5 s, incubated at 37°C for 1 min, and the absorbance A2 was detected at 340 nm. The OD difference A2-A1 between the sample and the blank was calculated.

[0126] The purified expression products in each example were measured for absorbance according to the above method, and enzyme activity was calculated, and the results are summarized in Table 5.

[0127] Table 5

[0128]

[0129]

[0130] It is understood by those of ordinary skill in the art that the above embodiments are specific examples for implementing the present application, and in actual applications, various changes can be made in form and details without departing from the spirit and scope of the present application.

Claims

1. A polynucleotide encoding an isolated cystathionine-β-lyase, characterized in that, The polynucleotide is codon-optimized and is a polynucleotide as shown in SEQ ID NO.

1.

2. An expression carrier, characterized in that, The expression vector is an Escherichia coli expression vector, and the expression vector comprises the polynucleotide as described in claim 1.

3. The expression vector according to claim 2, characterized in that, The expression vector includes a polynucleotide sequence expressing a His×6 tag.

4. The expression vector according to claim 3, characterized in that, The expression vector is pET-28a(+).

5. A host cell, characterized in that, The host cell is Escherichia coli, and the host cell includes the expression vector as described in any one of claims 2 to 4; or the host cell genome integrates the polynucleotide as described in claim 1.

6. A method for preparing cystathionine-β-lyase, characterized in that, The method includes the following steps: Transform a host cell into the expression vector as described in any one of claims 2-4, wherein the host cell is Escherichia coli; The host cells were cultured to express the cystathionine-β-lyase.

7. The method according to claim 6, characterized in that, The host cells were cultured in TB or LB medium.

8. The method according to claim 6, characterized in that, When culturing the host cells, the target protein is expressed by IPTG induction.

9. The method according to claim 6, characterized in that, The host cells were cultured at a temperature of 16 to 19°C.

10. A reagent kit, characterized in that, The kit comprises: the polynucleotide as described in claim 1; or The expression vector as described in any one of claims 2 to 4; or The host cell as described in claim 5.

Images