CN115747241A - Preparation method and application of nicotinamide phosphoribosyl transferase - Google Patents
Preparation method and application of nicotinamide phosphoribosyl transferase Download PDFInfo
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Abstract
The invention provides a preparation method and application of nicotinamide phosphoribosyltransferase, which comprises cloning nicotinamide phosphoribosyltransferase gene (NAMPT) in Xanthomonas campestris (Xanthomonas campestris pv. Campestris 8004) of Cruciferae, and constructing expression pRSFDuet-1: xccNAMPT, the successfully constructed expression vector is transformed into escherichia coli BL21 (DE 3), soluble phosphoribosyltransferase is obtained by induced expression of the escherichia coli, the yield of the recombinant nicotinamide phosphoribosyltransferase protease is high, the enzyme activity of the recombinant protease is 7 times of that of humanized NAMPT, and the application value is provided for the industrial high-efficiency production of Nicotinamide Mononucleotide (NMN).
Description
Technical Field
The invention relates to the technical field of bioengineering, and particularly relates to a purification and extraction method of nicotinamide phosphoribosyl transferase with high enzyme activity.
Background
Nicotinamide adenine dinucleotide (NAD +) is an important class of coenzymes that plays a crucial role in a variety of biological processes. Nicotinamide phosphoribosyl transferase is the rate-limiting enzyme in the NAD + biosynthetic pathway, and can catalyze Nicotinamide (NAM) and phosphoribosyl pyrophosphate (PRPP) to generate NAD + precursor NMN, thereby playing an important role in the NAD + biosynthetic pathway. It is also a widely accepted fact that NAD + is also closely related to aging or longevity, that NAD + levels in cells and tissues of biological organisms decrease with age, and that the organisms age, and that NAD + supplementation can prolong life cycle and delay aging.
In present-stage research data, it was shown that NMN, a NAD + precursor, increases NAD + biosynthesis in tissues and organs such as pancreas, adipose tissue, heart, skeletal muscle, liver, kidney and eye. At present, at least in a considerable part of the research, it has been demonstrated that NMN, a precursor of NAD + supplementation, can offer a wide range of applications and that it has the potential for disease treatment. Long-term oral NMN supplementation is safe, has no obvious toxic or other side effects after the NMN supplementation, and can improve Alzheimer's disease, heart failure, aging and the like related to the reduction of NAD + levels. In conclusion, NMN can improve physiological functions of a plurality of tissues and organs and can alleviate the development of a plurality of diseases, and most importantly, NMN has good expression on delaying senescence.
At present, NMN can be obtained by chemical synthesis and biological enzyme synthesis, but the chemical synthesis process has too many impurities, high cost and low recovery rate. Therefore, the method for synthesizing NMN by catalyzing NAM and PRPP through the biological enzyme NAMPT becomes a mainstream method. However, in view of the low enzyme activity of the human NAMPT widely used at present, even if a large amount of cost is invested to develop the NAMPT activator at present, the effect of improving the enzyme activity of the NAMPT activator in practical application is still not obvious. And although recombinant NAMPT has been reported at present, the purity and yield of the NAMPT still need to be improved.
Disclosure of Invention
Aiming at the defects of the prior art, the invention aims to provide a method for improving the NMN biosynthesis efficiency, in particular to provide a recombinant expression vector and a recombinant bacterium for coding high-efficiency NAMPT protein, and the recombinant bacterium is used for expressing the high-enzyme-activity XccNAMPT protease. The enzyme activity of the XccNAMPT protease is greatly improved compared with the reported human NAMPT, and the industrialized large-scale production can be realized.
The purpose of the invention is realized by the following technical scheme.
A nicotinamide phosphoribosyltransferase gene vector, which inserts a nicotinamide phosphoribosyltransferase gene with the length of 1407bp into a pRSFDuet-1 vector to form a vector pRSFDuet-1: xccNAMPT.
The NAMPT gene is derived from the family Cruciferae Ustilago virens (Xanthomonas campestris. Campesris 8004).
The vector pRSFDuet-1: the XccNAMPT is transferred into host bacteria to obtain the nicotinamide phosphoribosyltransferase engineering bacteria.
The host bacterium is Escherichia coli BL21 (DE 3).
The invention relates to a construction method of nicotinamide phosphoribosyltransferase engineering bacteria, which comprises the following steps:
and 3, carrying out double enzyme digestion on the gene amplification product in the step 2 and the pRSFDuet-1 vector by using restriction enzymes BamHI and XhoI, connecting the two enzyme digestion products at 16 ℃ for 4 hours by using T4 ligase, transferring the connection product into escherichia coli DH5 alpha competent cells, extracting a plasmid, determining pRSFDuet-1 after sequencing is successful: the construction of the expression vector XccNAMPT is successful.
The invention also provides an extraction and purification method of the XccNAMP recombinase, which comprises the following steps:
and 4, centrifuging the crushed bacterium liquid at 12000rpm for 60min at 4 ℃, and collecting supernatant.
Step 5, the supernatant was affinity purified using a nickel column and repeated three times.
Step 6, washing the nickel column with Tris-HCl buffer containing 30mM imidazole, and eluting nicotinamide phosphoribosyltransferase XccNAMPT from the nickel column with Tris-HCl buffer containing 300mM imidazole and collecting.
And 7, concentrating the collected eluent, and replacing the concentrated eluent with Tris-HCl buffer solution without imidazole to obtain the high-purity XccNAMPT.
The invention has the beneficial effects that:
the invention clones the nicotinamide phosphoribosyltransferase gene from microorganisms by using a genetic engineering technology, successfully expresses the nicotinamide phosphoribosyltransferase gene, effectively purifies the XccNAMPT by using the N segment of the recombinase with 6 histidine tags through the principle of His tag and nickel column affinity, and finally obtains the XccNAMPT recombinase with high purity and high yield.
The XccNAMPT recombinase solves the problem of low enzyme activity of NAMPT in production, and the enzyme activity of the XccNAMPT recombinase is 7 times of that of human NAMPT, thereby providing application value for industrial large-scale production of NMN.
Drawings
FIG. 1 shows the result of PCR amplification of the gene fragment XccNAMPT, where M is DNAmarker and 1 is the target gene fragment XccNAMPT;
FIG. 2 is an SDS-PAGE analysis of the XccNAMPT recombinase, wherein M is a protein Marker,1 is a supernatant after cell disruption centrifugation, 2 is a precipitate after cell disruption centrifugation, 3 is a nickel medium eluted by a Tris-HCl buffer containing 300mM imidazole, and 4 is an XccNAMPT recombinase solution eluted by a Tris-HCl buffer containing 300mM imidazole;
FIG. 3 shows the results of enzyme activity detection of human NAMPT and XccNAMPT recombinases. The NAMPT colorimetric detection kit (cyclexnamptcolometricacassaykit) was used for the experiments.
Detailed Description
The technical solution of the present invention is further described below with reference to specific examples. Unless otherwise specified, all technical means used in the present invention are well known to those skilled in the art.
The test materials used in the examples of the present invention are all conventional test materials in the art, and are commercially available without specific description. Wherein the specific operation is as follows
1. Construction of Nicotinamide phosphoribosyltransferase engineering bacteria
The PCR reaction system is as follows: 5 xPCLBuffer (Mg) 2+ plus) 10. Mu.L, dNTPmix (2.5 mM) 4. Mu.L, 1. Mu.L of each of the upstream and downstream primers, 1. Mu.L of the template, 0.5. Mu.L of DNA polymerase, and sterile water to 50. Mu.L.
The PCR reaction conditions are as follows: pre-denaturation at 98 deg.C for 10min, denaturation at 98 deg.C for 30s, annealing at 53 deg.C for 30s, extension at 72 deg.C for 2min, reaction for 30 cycles, and extension at 72 deg.C for 10min. The complete target gene fragment obtained after PCR is shown in figure 1, and the sequence is shown in sequence 1.
TABLE 1 primer sequences used
And 2, recovering the amplified XccNAMPT gene fragment with the enzyme cutting site by using a DNA purification kit, carrying out double enzyme cutting on the gene fragment and pRSFDuet-1 vector by using BamHI and XhoI, recovering enzyme cutting products, connecting the two enzyme cutting products at 16 ℃ for 4 hours by using T4 ligase after the enzyme cutting products are recovered, transferring the connecting products into Escherichia coli DH5 alpha competent cells, uniformly coating the connecting products on an LB solid culture medium with kanamycin resistance (100 mu g/mL), culturing for 14 hours at 37 ℃, picking single clones, inoculating the single clones into a 5mLLB liquid culture medium containing kanamycin resistance (100 mu g/mL), and culturing overnight at 37 ℃ at 200 rpm. And on the next day, collecting thalli, extracting plasmids, carrying out enzyme digestion identification, sequencing, and determining pRSFDuet-1: the construction of the expression vector XccNAMPT is successful.
And 3, successfully constructing pRSFDuet-1: the XccNAMPT vector is transformed into BL21 (DE 3) competent cells, and is uniformly coated on an LB solid culture medium with kana resistance (100 mu g/mL), cultured for 14 hours at 37 ℃, and a single clone is picked up and inoculated into the LB culture medium with the kana resistance (100 mu g/mL) for culture, so that the nicotinamide phosphoribosyltransferase engineering bacteria can be obtained.
2. Expression purification of XccNAMPT recombinase
Tris-HCl buffer formulation: 20mM Tris-HCl (pH 8.0), 150mM NaCl. The buffer was used after suction filtration.
And 4, balancing the nickel column by using Tris-HCl (pH8.0) buffer solution in advance, and pouring the supernatant obtained in the step 3 into the nickel column for affinity purification.
Step 5, washing the nickel column with Tris-HCl (pH 8.0) buffer containing 30mM imidazole until the eluate hardly allows G250 to turn blue, and eluting the nickel column with Tris-HCl (pH 8.0) buffer containing 300mM imidazole.
And 6, concentrating the collected eluent, and replacing the solution with a Tris-HCl buffer solution without imidazole to finally obtain the high-purity XccNAMPT. Protein purification was followed by SDS-PAGE as shown in FIG. 2.
3. Enzyme activity comparison and determination of XccNAMPT recombinase
The enzymatic activity of NAMPT is determined by means of a CyclexNAMPT colorimetric kit by detecting NAD converted from NMN + In the assay, NAD + The more the amount produced, i.e. the darker the yellow color.
And 2, adding 19 mu L of detection reagent mixed liquor into a 384-well plate, sucking 1 mu L of 50 mu g/mL enzyme solution, and adding the enzyme solution into the corresponding well. Monitoring the absorbance of the mixed solution at 450nm wavelength with a microplate reader at a constant temperature of 30 DEG C
The enzymatic activity of NAMPT was calculated from the A450 and reaction time curves. The enzyme activity was obtained by dividing the difference between the highest a450 value and the lowest a450 value in the monitoring curve by the time span between these two values, as shown in figure 3. The formula is as follows:
Claims (4)
1. a recombinant nicotinamide phosphoribosyltransferase NAMPT gene expression vector is characterized in that: inserting said nicotinamide phosphoribosyltransferase gene into a vector pRSFDuet-1 to form a vector pRSFDuet-1: xcc NAMPT.
2. A composition comprising a nucleotide sequence encoding the recombinant protease of claim 1, wherein: the recombinant protease is formed by combining nicotinamide phosphoribosyltransferase NAMPT and a histidine tag, wherein the NAMPT sequence is derived from cruciferae black rot pathogen, and the histidine tag is derived from pRSFDuet-1 vector. The sequence of the recombinant protease Xcc NAMPT gene is shown as a sequence 1.
3. The Nicotinamide phosphoribosyltransferase engineered bacteria prepared from the Nicotinamide phosphoribosyltransferase gene vector of claim 1 or 2, wherein: the recombinant vector pRSFDuet-1: and (3) transferring the Xcc NAMPT into host bacteria, wherein the host bacteria are escherichia coli, and finally obtaining the nicotinamide phosphoribosyltransferase engineering bacteria.
4. The method for preparing high-enzyme activity and high-purity nicotinamide phosphoribosyltransferase (Xcc NAMPT) according to the nicotinamide phosphoribosyltransferase engineering bacteria of claim 3, which is characterized in that: the process comprises the following steps:
step 1, inoculating nicotinamide phosphoribosyltransferase engineering bacteria into an LB culture medium, carrying out amplification culture on the engineering bacteria for 4-5 hours at 37 ℃, adding 0.5mM IPTG into a bacterial liquid at 16 ℃, and carrying out induction overnight;
step 2, centrifuging the bacterial liquid at 5000rpm for 15min at 4 ℃, and collecting thalli;
step 3, re-suspending the thalli by using a precooled Tris-HCl buffer solution, and crushing the re-suspended thalli by using a high-pressure ultrasonicator;
step 4, centrifuging the crushed bacterial liquid at 12000rpm for 60min at the temperature of 4 ℃, and collecting supernatant;
step 5, pouring the supernatant into a gravity column filled with a nickel medium, performing affinity purification, and repeating for three times;
step 6, the nickel column was washed with Tris-HCl buffer containing 30mM imidazole, and nicotinamide phosphoribosyltransferase Xcc NAMPT was eluted from the nickel column with Tris-HCl buffer containing 300mM imidazole.
And 7, concentrating the collected enzyme solution, and replacing the enzyme solution with Tris-HCl buffer solution without imidazole to obtain the Xcc NAMPT recombinase with high purity and high enzyme activity.
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| Title |
|---|
| "A0A0H2X5R2 • A0A0H2X5R2_XANC8，Nicotinamide phosphoribosyltransferase", pages 7 - 9, Retrieved from the Internet <URL:https://www.uniprot.org/uniprotkb/A0A0H2X5R2/entry> * |
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