CN115896048A

CN115896048A - Recombinant human Cu, zn-SOD with high enzyme activity and good stability, and preparation method and application thereof

Info

Publication number: CN115896048A
Application number: CN202211658617.3A
Authority: CN
Inventors: 徐兰举; 齐磊; 于月欣; 刘鑫
Original assignee: Hebei Naco Biotechnology Co ltd
Current assignee: Hebei Naco Biotechnology Co ltd
Priority date: 2022-12-22
Filing date: 2022-12-22
Publication date: 2023-04-04
Anticipated expiration: 2042-12-22
Also published as: CN115896048B

Abstract

The invention relates to recombinant human Cu and Zn-SOD with high enzyme activity and good stability, a preparation method and application thereof, wherein the amino acid sequence of the coding gene is shown as SEQ ID NO. 3, and the nucleotide sequence is shown as SEQ ID NO. 2.

Description

Recombinant human Cu, zn-SOD with high enzyme activity and good stability, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to recombinant human Cu, zn-SOD with high enzyme activity and good stability, and a preparation method and application thereof.

Background

Superoxide dismutase (SOD, E.C. 1.15.1.1) is the first antioxidant enzyme in the process of organism active oxygen free radical scavenging reaction, and has good preventing and treating effects in delaying aging and other diseases caused by free radicals.

Currently known superoxide dismutase are of 4 types: copper-zinc superoxide dismutase (Cu/Zn-0 SOD), manganese superoxide dismutase (Mn-SOD), iron superoxide dismutase (Fe-SOD) and nickel superoxide dismutase (Ni-SOD), wherein Cu and Zn-SOD widely exist in cytoplasm, chloroplast and catalase of most eukaryotes such as mammals and fungi, are one of the superoxide dismutase which is most widely distributed in nature, and are also the superoxide dismutase which is most widely applied at present.

At present, most superoxide dismutase (SOD) products are mainly derived from animal blood, viscera and the like, and because of limited raw materials, difficult purification and the like, the SOD has low purity and low yield; especially, the risk of producing animal blood products is increased along with the occurrence of malignant infectious diseases such as mad cow disease, avian influenza, foot and mouth disease and SARS spread by animals all over the world; in addition, the increased purity requirements of the product also increase the production costs. With the development of genetic engineering technology, SOD genes from different sources such as microorganisms, plants and animals are cloned and expressed. However, recombinantly expressed SOD exists as inactive inclusion bodies, or has problems such as low activity, easy inactivation, or poor heat resistance.

SOD is obtained by means of genetic engineering, and mainly comprises several modes of fusion expression with other proteins, making point mutation on human SOD and screening high-performance SOD from other species.

CN103789278A discloses a novel PTD4-Cu, zn-SOD fusion protein and a preparation method thereof, the novel PTD4-Cu, zn-SOD fusion protein has very high efficiency of penetrating cell membranes, thereby leading Cu, zn-SOD which originally can not pass through the cell membranes to be transduced to the cell membranes to play a biological function.

CN102526712B discloses an application of SOD-TAT fusion protein in preparing medicine for preventing and treating radiation injury. The SOD-TAT can eliminate free radicals outside cells and free radicals inside cells, and can cross blood brain barriers, and the depth and the breadth of the radiation protection effect of the SOD-TAT are far stronger than those of the existing radiation protection agents used clinically.

CN101603048B discloses a molecular chaperone-free prokaryotic expression method of fungus Cu _ Zn-SOD. CN104450632 discloses a group of amino acid sequences capable of improving heat-resisting temperature and heat stability of SOD and application thereof. It is a group of N-terminal amino acid sequences of 13 special thermophilic Fe/Mn-SOD from Geobacillus.

CN101525600B discloses a method for increasing the yield of recombinant human Cu, zn-SOD active protein. The invention mutates the cysteine at the 6 th site and the 111 th site into serine, so that the proportion of recombinant protein in schizomycete supernatant to the total expression of the recombinant protein is increased from 40% to 80%, and the specific activity of the mutant rhCu, zn-SOD purified from the supernatant is equivalent to that of the non-mutant rhCu, zn-SOD purified from the supernatant. However, the patent does not investigate the stability of the product.

In view of the above, there is still a need in the art to continuously research recombinant human Cu and Zn-SOD with high enzyme activity and good stability.

Disclosure of Invention

The invention aims to provide recombinant human Cu, zn-SOD with high enzyme activity and good stability, and a preparation method and application thereof.

In order to realize the purpose, the technical scheme adopted by the invention is as follows:

on one hand, the invention provides a recombinant human Cu, zn-SOD, the amino acid sequence of the coding gene is shown as SEQ ID NO. 3.

The invention also provides a recombinant human Cu, zn-SOD, the nucleotide sequence of the coding gene is shown in SEQ ID NO. 2

The invention also provides a preparation method of the recombinant human Cu, zn-SOD, which comprises the following steps:

(1) Carrying out codon optimization by taking amino acid sequences of human Cu and Zn-SOD as templates to obtain a nucleotide sequence SEQ ID NO. 1, then carrying out whole-gene synthesis to obtain nucleic acid segments for coding the human Cu and Zn-SOD, constructing an expression vector of the gene and transforming host cells to obtain an expression strain.

(2) Selecting an upstream primer: 5' TAATACGACTCACTATAGGG-3', and 5' downstream primer 5' GCTGAGCAAATAACTAGC-3 ', wherein the expression vector of the gene in the step (1) is used as a template to perform error-prone PCR; constructing an expression vector of a mutant gene, transforming host cells, constructing a mutant library, and screening positive mutation to obtain a strain;

(3) After the strain is activated, inoculating the strain into an LB culture medium according to the proportion of 10 percent, inducing the expression of target protein by IPTG, and collecting thalli;

(4) And (4) carrying out thallus crushing on the bacteria liquid collected in the step (3), and purifying by using a Ni affinity chromatographic column and an ion exchange chromatographic column to obtain purified recombinant human Cu and Zn-SOD.

As a further improvement of the invention, the nucleic acid fragment coding the human Cu, zn-SOD in the step (1) is connected to pET30a (+) plasmid through Nde I and Kpn I enzyme cutting sites, pET30a (+) -SOD is transformed into BL21 (DE 3) competence, and an Escherichia coli expression strain expressing the human Cu, zn-SOD is obtained.

As a further improvement of the invention, in the step (2), the mutant gene is recovered by enzyme digestion, then is connected with a vector pET30a (+), is transferred into escherichia coli BL21 (DE 3), and is coated on LB solid medium containing kanamycin to obtain an expression strain.

In a final aspect of the invention there is provided the use of a recombinant human Cu, zn-SOD as described above in the medical or cosmetic field.

The technical scheme of the invention has the beneficial effects that:

(1) The enzyme activity of the recombinant human Cu, zn-SOD obtained by the invention is about one time higher than that of natural human Cu, zn-SOD.

(2) The recombinant human Cu and Zn-SOD obtained by the invention has high enzyme activity stability relative to the stability of natural human Cu and Zn-SOD.

Drawings

In order to more clearly illustrate the embodiments of the present invention, reference will now be made briefly to the accompanying drawings.

FIG. 1 shows electrophoretograms of fermented whole bacteria and purified samples;

m: protein Marker,1: natural Cu, zn-SOD whole bacteria, 2: natural Cu, zn-SOD bacteria-breaking precipitate, 3: breaking the bacteria supernatant of natural Cu and Zn-SOD, 4: affinity purification of natural Cu, zn-SOD, 5: ion exchange purification of natural Cu, zn-SOD, 6: BSA,7: mutant Cu, zn-SOD whole bacteria, 8: mutant Cu, zn-SOD precipitate, 9: mutant Cu, zn-SOD supernatant, 10: mutant Cu, zn-SOD affinity purification, 11: mutation search for ion exchange purification, 12: BSA.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the technical solutions of the present invention are described below in detail and completely with reference to the specific embodiments.

Example 1 expression construction of Natural human Cu, zn-SOD in E.coli

The amino acid sequence of human Cu and Zn-SOD (UniProtKB/Swiss-Prot: P00441.2) is taken as a template for codon optimization to obtain the following gene sequence:

(SEQ ID NO: 1), entrusted with Jinwei synthetic gene sequence, connected to pET30a (+) through Nde I and Kpn I restriction sites to obtain pET30a (+) -SOD, and transferred into BL21 (DE 3) competence to obtain Escherichia coli expression strain expressing human Cu, zn-SOD.

Example 2 Gene mutation

Error-prone PCR is carried out by taking pET30a (+) -SOD as a template, and an upstream primer: 5 'TAATACGACTCACTATAGGG-3', and 5 'downstream primer GCTGAGCAAATAACTAGC-3'.

The PCR system is as follows: 2 XTaq PCR Master Mix 25. Mu.l, plasmid template 1. Mu.l, upstream primer 1. Mu.l, downstream primer 1. Mu.l, manganese chloride solution 1. Mu.l, ddH2O to make up 50. Mu.l.

An error-prone PCR cycling program, namely dropping PCR, performing pre-denaturation at 95 ℃ for 5min, and cycling for 1 time; denaturation at 95 ℃ for 30s, denaturation at 58 ℃ for 30s and denaturation at 72 ℃ for 100s, reducing annealing temperature by 1 ℃ in each cycle, and circulating for 5 times; denaturation at 95 deg.C for 30s, denaturation at 55 deg.C for 30s, and denaturation at 72 deg.C for 100s, and circulating for 25 times; extension at 72 ℃ for 10min, and 1 cycle.

Example 3 preparation of super competence and construction of mutant library

The obtained target gene with a certain mutation rate is cut and recovered by Nde I and Kpn I enzyme and then is connected with a carrier pET30a (+), the connection product is added into E.coli Bl21 competent cells which are taken out from ice in advance, and the super competence with higher transformation efficiency is prepared according to the operation instruction of a super competence preparation kit. And (3) coating the reactivated bacterial liquid on an LB solid culture medium containing kanamycin, culturing at 37 ℃ for 30min, and then, inverting the culture dish for overnight culture to construct a mutant library with a high transformant number.

EXAMPLE 4 inducible expression of a Single clone and Positive mutation screening

The colonies on the solid medium were picked one by one into a 96-well bacterial culture plate (each well containing 200. Mu.l of LB medium to which antibiotics have been added) and cultured at 37 ℃ for 12-16 hours at 200 rpm; then, the bacterial suspension in each well was transferred to a new 96-well plate (each well containing 500. Mu.l of LB medium to which antibiotics had been added) at an inoculation amount of 2%, cultured for 6 hours, and then IPTG was added to induce expression. Centrifugally collecting the induced thallus, adding 40 cell lysate into each hole, and standing at 37 ℃ for 1-2h to crack the thallus; after centrifugation at 12000rpm for 10min at 4 ℃ the supernatant was collected, and the enzyme activity was measured by the method described in example 5 to screen for a forward mutation.

Mixing the rest bacteria liquid with glycerol at a ratio of 7: 3, freezing at-80 deg.C, and storing as seed.

Example 5 enzyme Activity determination and screening

1. Solutions of

Solution A: 0.01mol/L Tris-hydroxymethyl aminomethane hydrochloride buffer solution (Tris-HCl), pH8.20;

and B, liquid B: 4.5mmol/L pyrogallol hydrochloride solution.

2. Determination of pyrogallol autoxidation rate

2.35mL of solution A, 2.00mL of distilled water and 0.15mL of solution B were sequentially added to a 10mL colorimetric tube at about 25 ℃. Adding the solution B, mixing immediately, pouring into a cuvette, and measuring the absorbance values at the initial time and after 1min under the condition of 325nm wavelength respectively, wherein the difference is the self-oxidation rate delta A325 (min-1) of the pyrogallol.

3. Sample enzyme activity assay

Determination of SOD enzyme solution for inhibiting pyrogallol autoxidation rate adding enzyme solution according to 2 steps to make the rate of inhibiting pyrogallol autoxidation about 1/2 delta A325 (min-1),

TABLE 1SOD Activity measurement sample application Table

4. Calculation of results

U/mL-SOD enzyme activity unit;

delta A325-pyrogallol autoxidation rate;

delta A' 325-sample liquid or SOD enzyme liquid inhibits the auto-oxidation rate of pyrogallol;

v-volume of enzyme solution or sample solution added in milliliters (L);

d is the dilution multiple of the enzyme solution or the sample solution;

4.5-total volume of reaction in milliliters (mL).

The specific activity (U/mg) of the protein is obtained by dividing the activity of the SOD by the concentration of the protein.

EXAMPLE 6 rescreening of mutants

The system established by the high-throughput screening method is trace, has errors to a certain extent, and is commonly used for primary screening of directed evolution of target genes. In order to further determine the expression and activity of the mutant, carrying out shake flask amplification culture on the mutant obtained by primary screening, collecting thalli, and carrying out ultrasonic crushing on the thalli to obtain a target protein; the enzyme activity was measured by the method of example 5, and the protein expression was examined by SDS-PAGE. Further determining the mutant with high enzyme activity. Finally, the mutant of the mutants 2-A6, 8-F11, 14-D3, 14-E2 and 20-B3 is determined to have the highest enzyme activity. The stability of SOD obtained from each mutant was investigated later, and only the 8-F11 mutant was found to have good stability, so that only 8-F11 was shown in the following examples.

Example 7 high expression mutant plasmid extraction and sequencing

Coating the rescreened high-enzyme-activity mutant 8-F11 on an LB solid medium containing kanamycin, culturing for 30min at 37 ℃, and then inverting the culture dish for overnight culture. A single clone was picked and inoculated into a flask containing LB and cultured overnight at 37 ℃ and 180 rpm. Plasmids were extracted using the solibao plasmid extraction kit.

1. Centrifuging 5mL of bacterial solution at 12000rpm for 1min, and removing supernatant as much as possible

2. To the centrifuge tube where the bacterial pellet was left, 250. Mu.L of solution I (RNaseA was added in advance) was added, and the bacterial cell pellet was thoroughly suspended using a pipette or a vortex shaker.

3. 250 μ L of solution II was added to the centrifuge tube and gently turned over 6-8 times to lyse the cells thoroughly.

4. Adding 350 mu L of the solution III into a centrifuge tube, immediately and gently turning up and down for 6-8 times, and fully mixing, wherein white flocculent precipitates appear. Centrifuge at 12000rpm for 10min, and carefully transfer the supernatant to another clean centrifuge tube with a pipette, trying not to aspirate the pellet.

5. Adding the supernatant obtained in the previous step into an adsorption column (the adsorption column is added into a collection tube), standing at room temperature for 2min, centrifuging at 12000rpm for 1min, pouring off the waste liquid in the collection tube, and replacing the adsorption column into the collection tube again.

6. Adding 600 μ L of rinsing solution I (anhydrous ethanol is added in advance) into adsorption column, centrifuging at 12000rpm for 1min, discarding waste liquid, and placing adsorption column into collection tube.

7. Adding 700 μ L of rinsing liquid II (adding anhydrous ethanol in advance) into adsorption column, centrifuging at 12000rpm for 1min, discarding waste liquid, and placing adsorption column into collection tube.

8. Adding 500 μ L of rinsing liquid II into adsorption column, centrifuging at 12000rpm for 1min, discarding waste liquid, and placing adsorption column into collection tube.

Centrifuging at 9.12000rpm for 2min, and placing the adsorption column in an open air at room temperature or 50 deg.C incubator for several minutes to remove residual rinsing liquid in the adsorption column

10. Placing the adsorption column in a clean centrifuge tube, suspending and dripping 50-200 μ L of eluate preheated by 65 deg.C water bath into the center of the adsorption membrane, standing at room temperature for 2min, and centrifuging at 12000rpm for 1min.

Subjecting the extracted plasmid to Jinzhi sequencing to obtain a mutated gene sequence (SEQ ID NO: 2):

after translation, the corresponding amino acid sequence is obtained as follows (SEQ ID NO: 3):

through comparison with the amino acid sequences of natural human Cu and Zn-SOD, the sequencing confirms that the valine at the 15 th position is mutated into the glycine, and the phenylalanine at the 51 st position is mutated into the valine.

EXAMPLE 8 high expression mutant shake flask amplification culture

Activating 8-F11 mutant strain with high enzyme activity, inoculating into 30ml LB culture medium at a ratio of 1 ‰, and culturing at 37 deg.C under 180rpm overnight. Then, the cells were inoculated into 100ml of LB medium at a ratio of 10% and cultured for 3 hours, and then induced with 0.5mM IPTG for 6 hours, and the cells were collected.

Example 9 purification

1. Crushing and centrifuging

The cells obtained in example 7 were resuspended in 20mM Tris,500mM NaCl,20mM imidazole, pH8.0 at a ratio of 1. The supernatant was collected by centrifugation at 12000g for 10min and filtered through a 0.45 μm filter.

2. Affinity chromatography

Solution A: 20mM Tris,500mM NaCl,20mM imidazole, pH8.0

And B, liquid B: 20mM Tris,500mM NaCl,500mM imidazole, pH8.0

GE Ni Sepharose 6FF 5mL pre-packed column, using A liquid for balance, and loading the supernatant in the step 1 at the flow rate of 1 ml/min; then washing impurities with the solution A, wherein the volume is 25ml; eluting with 50% B solution, and collecting the elution peak to obtain the primary pure SOD.

3. Ion exchange

Solution A: 20mM Tris, pH8.0

And B, liquid B: 20mM Tris,1M NaCl, pH8.0

And (3) diluting the SOD solution obtained in the step (2) with the ion-exchanged A solution until the electric conductivity is less than 4mS/cm.

GE Q Sepharose 6FF 5mL pre-packed column, balance with A liquid, the diluted SOD solution with the flow rate of 1ml/min speed sample; then washing impurities with the solution A, wherein the volume is 25ml; eluting with 25% B solution, and collecting the elution peak to obtain purified Cu, zn-SOD.

Test example 1

The 8-F11 mutant and the natural SOD thallus with high enzyme activity were amplified and purified according to the methods of examples 7 and 8, respectively, to obtain mutant Cu, zn-SOD and natural Cu, zn-SOD.

The results of SDS-PAGE of the samples from the above steps are shown in FIG. 1. It can be seen from the figure that (1) the expression amounts of the mutant SOD and the natural SOD are not greatly different and can reach more than 60 percent of the total mycoprotein; (2) SOD is mainly expressed in a soluble form, and target protein in the precipitate accounts for a little; (2) The purity of SOD is very high, and no obvious hybrid protein band is formed, and is more than 98%.

Determination of enzyme Activity

The enzyme activities of the natural SOD and the mutant SOD obtained in step 3 were measured as in example 5. The results of the measurements are shown in the following table

	Specific activity (U/mg)
		Natural SOD	12325
Mutant SOD	23173

From the results, it can be seen that after mutation, the specific activity was increased nearly twice.

Test example 2 stability of purified SOD

The SOD purified product obtained in test example 1 was subjected to filtration sterilization, and then dispensed into 1.5ml sterile EP tubes (100. Mu.l/tube), and stored at room temperature, 4 ℃, -20 ℃ and-80 ℃ respectively, to examine the stability of the sample. The data are shown in the following table (U/mg):

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as can be seen from the data, the enzyme activity of the natural SOD at room temperature and 4 ℃ is gradually reduced along with the prolonging of the storage time; the enzyme activity of the mutant SOD has no obvious change along with time. Both of them have stable enzyme activity when stored at-20 deg.C and-80 deg.C.

Finally, it should be noted that: the above examples are only intended to illustrate the technical solution of the present invention, but not to limit it; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.

Claims

1. A recombinant human Cu, zn-SOD is characterized in that the amino acid sequence of the coding gene is shown as SEQ ID NO. 3.

2. The recombinant human Cu, zn-SOD of claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 2.

3. The method for preparing recombinant human Cu, zn-SOD as claimed in claim 1 or 2, which comprises the following steps:

(1) Carrying out codon optimization by taking amino acid sequences of human Cu and Zn-SOD as templates to obtain a nucleotide sequence SEQ ID NO 1, then carrying out whole-gene synthesis to obtain nucleic acid segments for coding the human Cu and Zn-SOD, constructing an expression vector of a gene and transforming host cells to obtain an expression strain;

(2) Selecting an upstream primer: 5' TAATACGACTCACTATAGGG-3', and 5' downstream primer 5' GCTGAGCAAATAACTAGC-3 ', wherein the gene in the step (1) is used as a template to perform error-prone PCR; constructing an expression vector of a mutant gene, converting host cells to construct a mutant library, and screening positive mutation to obtain a strain;

(3) After the strain is activated, inoculating the strain into an LB culture medium, inducing the expression of a target protein by IPTG, and collecting thalli;

(4) And (4) carrying out thallus crushing on the bacteria liquid collected in the step (3), and purifying to obtain purified recombinant human Cu and Zn-SOD.

4. The method for preparing recombinant human Cu, zn-SOD as claimed in claim 3, wherein the nucleic acid fragment encoding the human Cu, zn-SOD in step (1) is ligated to pET30a (+) plasmid via Nde I and Kpn I cleavage sites, pET30a (+) -SOD is transformed into BL21 (DE 3) competence to obtain E.coli expression strain expressing human Cu, zn-SOD.

5. The method for preparing recombinant human Cu, zn-SOD as claimed in claim 3, wherein the mutant gene in step (2) is recovered by digestion, ligated to the vector pET30a (+), and transferred to E.coli BL21 (DE 3) and spread on LB solid medium containing kanamycin to obtain the expression strain.

6. The method for preparing recombinant human Cu, zn-SOD according to claim 3, wherein the purification in step (4) is performed using Ni affinity chromatography column and ion exchange chromatography column.

7. Use of the recombinant human Cu, zn-SOD as claimed in claim 1 in medical or cosmetic fields.